WO2000036083A2 - Pkc-interacting cousin of trx (picot) polypeptides, polynucleotides, and methods of making and using them - Google Patents

Abstract

The present invention relates to the identification of novel polypeptides that bind PKCυ and PKCz, termed PICOT. The invention provides PICOT polypeptides and polynucleotides and antibodies that bind to PICOT polypeptides. PICOT also has the capability of modulating activity of various polypeptides and signaling pathways, including, for example, PKCυ, PKCz, JNK, AP-1, NFλB, Trx system, HEED/WAIT-1 and production of IL-2. Thus, PICOT polypeptides, polynucleotides and antibodies of the invention are useful in modulating the activities of these polypeptides and associated signaling pathways, including for the purpose of therapeutic treatment of PICOT associated physiological conditions or disorders in a human.

Description

PKC-INTERACTING COUSIN OF TRX (PICOT) POLYPEPTIDES, POLYNUCLEOTIDES, AND METHODS OF MAKING AND USING THEM

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to application serial no. 60/112,649, filed December 17, 1998, and is incoφorated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

This work was supported in part by NIH grant CA35299. The government may have certain rights in the invention.

BACKGROUND OF THE INVENTION Members ofthe protein kinase C (PKC) family of intracellular serine/threonine kinases play critical roles in the regulation of cellular differentiation, proliferation and response to diverse physiological stimuli, including hormones, neurotransmitters and growth factors, in many cell types (Newton (1995) J. Biol. Chem. 270, 28495-28498; Nishizuka (1995) FASEB J 9, 484-496; Jaken (1996) Curr. Opin. Cell Biol 8, 168-173). The PKC family has eleven known mammalian members that are expressed in a wide variety of tissues and cell types. Based on sequence similarities, domain structures and cofactor requirements, PKC isoenzymes can be grouped into three subfamilies: The Ca dependent conventional enzymes (cPKC) consisting of PKC-α, -βl, -βll and -γ contain three conserved domains, namely, the diacylglycerol/phorbol ester binding CI domain which contains two repeats of a cysteine-rich zinc finger, the phospholipid- and calcium-binding C2 domain, and the catalytic C3 and C4 domains. The Ca²⁺- independent enzymes (PKC-δ, -ε, -η, -θ and -μ) are termed novel PKCs (nPKCs). The C2-like N-terminal domain of these enzymes can bind acidic phosphohpids but not Ca²⁺. A third PKC subfamily, termed atypical PKCs (aPKCs) includes PKC-ζ and -ι/λ that possess a single cysteine- rich domain, lacking the ability to bind phosphohpids or phorbol esters. PKC activity is regulated by defined cofactors that interact with specific regions ofthe regulatory domain as well as transphosphorylation by serine/threonine kinases and autophosphorylation. The activation is accompanied by a conformational change that releases the basic pseudosubstrate region from the catalytic cleft ofthe kinase domain. In addition, interaction with specific proteins, termed receptors for activated PKC (RACK), that function as selective scaffolds for activated PKCs at discrete subcellular compartments, play a role in activation of PKC (Mochly-Rosen (1995) Science 268, 247-251).

PKCθ is a Ca²⁺-independent PKC isoform characterized by expression in skeletal muscle, lymphoid organs and hematopoietic cell lines, particularly in T cells (Baier et al. (1993) J. Biol. Chem. 268, 4997-5004). PKCθ plays a role in activation ofthe c-Jun N-terminal kinase

(JNK)/AP-1 pathway and the interleukin-2 (IL-2) gene in T cells (Baier-Bitterlich et al. (1996) Mol. Cell Biol. 16, 1842-1850; Ghaffari-Tabrizi et al. (1999) Eur. J. Immunol. 29, 132-142; Werlen et al. (1998) EMBOJ. 17, 3101-3111), and to colocalize with the TCR complex to the contact site between antigen-specific T cells and antigen-presenting cells (Monks (1997) Nature 385, 83-86), where it participates in the formation of a supramolecular activation cluster (Monks et al. (1998) Nature 395, 82-86). Thus, PKCθ is thought to play an important role in various aspects of T cell function, including, for example, T cell receptor-induced activation.

To gain a better understanding ofthe function and regulation of PKCθ, and its role in the various functions of T cells, a need exists to identify PKCθ-interacting proteins. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention is based on the identification and characterization of a novel polypeptide, termed "PICOT" (PKC-interacting cousin of Trx), that binds protein kinase C theta (PKCθ). PICOT is capable of modulating activity or activation of numerous polypeptides involved in various cell signaling pathways including, for example, JNK, AP-1 or NFKB activity, or production of IL-2. Thus, PICOT polypeptides are useful for various methods in which it is desired to modulate the activity or activation of these polypeptides and associated signaling pathways. Accordingly, isolated and recombinant PKC-interacting cousin of Trx (PICOT) polypeptides are provided. In one embodiment, the polypeptides have 70% or more identity to the sequence set forth in SEQ ID NO:l. In other embodiments, the polypeptides have at least 80% identity, 90% identity, or 95% identity with the sequence set forth in SEQ ID NO:l. In another embodiment, the polyeptide has the sequence set forth in SEQ ID NO: 1. In other embodiments, PICOT is characterized as having an apparent molecular weight of about 37 kDa or by expression in hematopoetic cells.

Subsequences of PICOT polypeptides, including functional subsequences, also are provided. In one embodiment, a functional subsequence comprises a subsequence having 70% or more identity to a sequence set forth in SEQ ID NO:l (e.g., 80%, 90%, 95% etc.). In another embodiment, a functional subsequence comprises a sequence set forth in SEQ ID NO:l. In yet another embodiment, a functional subsequence has one or more amino acid modifications of a sequence set forth in SEQ ID NO:l. Modifications include amino acid substitutions, deletions or insertions. Substitutions include conservative amino acid substitution. Functional subsequences ofthe invention range in size from about 7 amino acids to about 200 amino acids or more, for example, from about 5 to about 100 amino acids or about 10 to about 50 amino acids.

PICOT polypeptides and functional subsequences ofthe invention include polypeptides having various activities. In one embodiment, polypeptides and functional subsequences that bind or interact with PKCΘ are provided. In another embodiment, polypeptides and functional subsequences that bind or interact with PKCζ are provided. The interaction can occur in vitro or in intact cells, for example. In yet another embodiment, PICOT polypeptides and functional subsequences include sequences that modulate activity, or activation, of PKCΘ, PKCζ, JNK, AP- 1, NFKB, Trx system, HEED/WAIT- 1, and production of IL-2. Further included are PICOT polypeptides and subsequences having immunogenicity. Further included are PICOT polypeptides and functional subsequences having a heterologous functional domain. Heterologous domains include, for example, amino acid sequences, such as nucleic acid binding domains and transcriptional activation domains. Additional heterologous domains include tags (e.g., avidin, biotin, immunoglobulin fragment) and detectable labels (e.g., radiotisotopes). Invention polypeptides and subsequences (e.g., anitgenic subsequences) are useful for producing antibodies. Accordingly, antibodies and antibody fragments that specifically bind to PICOT polypeptides (e.g., SEQ ID NO:l), functional subsequences and immunogenic fragments are provided. Methods for producing an antibodies and antibody fragments that specifically bind to PICOT polypeptides, functional subsequences and immunogenic fragments are provided. The methods include administering a PICOT polypeptide, functional subsequence or immunogenic fragment, or a polynucleotide encoding same, to an animal in an amount sufficient to produce an antibody that specifically binds to PICOT polypeptides, functional subsequences or an immunogenic fragment. Isolated and recombinant polynucleotides encoding PICOT polypeptides and subsequences thereof also are provided. In one embodiment, the polynucleotides encode a polypeptide having the sequence set forth in SEQ ID NO:l. In another embodiment, the polynucleotides encode a functional subsequence of a sequence set forth in SEQ ID NO:l. In other embodiments, the polynucleotides encode a polypeptide having 70% or more identity to a polypeptide corresponding to amino acids 138 to 335 of SEQ ID NO:l, and subsequences thereof. In yet other embodiments, the polynucleotides encode immunogenic subsequences of PICOT polypeptides.

In additional embodiments, isolated and recombinant polynucleotides ofthe invention include a) SEQ ID NO:2; b) SEQ ID NO:2, where one or more T's are U; c) nucleic acid sequences complementary to a) or b); and subsequences of either a), b) or c) that are at least 15 base pairs long.

Isolated and recombinant polynucleotides that selectively hybridize to a sequence set forth in SEQ ID NO:2, and SEQ ID NO:2 also are provided. In one embodiment, the polynucleotides hybridize under moderately stringent conditions. In another embodiment, the polynucleotides hybridize under moderately-high stringent conditions to the sequence set forth in

SEQ ID NO:2. In another embodiment, the polynucleotides hybridize under highly stringent conditions to the sequence set forth in SEQ ID NO:2. Particular embodiments include polynucleotides that hybridize under moderately stringent conditions to the nucleotide sequence corresponding to nucleotides 421 to 1005 of SEQ ID NO:2; polynucleotides that hybridize under moderately-high stringent conditions to the nucleotide sequence corresponding to nucleotides

421 to 1005 of SEQ ID NO:2; and polynucleotides that hybridize under highly stringent conditions to the nucleotide sequence corresponding to nucleotides 421 to 1005 of SEQ ID

NO:2.

Additional isolated and recombinant polynucleotides include polynucleotides having at least 70% homology to a sequence set forth in SEQ ID NO:2, as determined using a BLAST algorithm. Such polynucleotides can have greater homology, 80% homology, 90% homology,

95% homology or more. Particular polynucleotides include polynucleotides having at least 70% homology to a sequence corresponding to nucleotides 421 to 1005 of SEQ ID NO:2, as determined using a BLAST algorithm. The polynucleotides ofthe invention are distinct from

Genbank accession numbers h59799, aa009010 and aa866363. Polynucleotides ofthe invention are not restricted in length. For example, they include polynucleotides having less than about 500 nucleotides, less than about 200 nucleotides, less than about 100 nucleotides or from about 15 to about 50 nucleotides.

Further provided are isolated and recombinant polynucleotides that include an expression cassette. In one embodiment, the expression cassette contains a nucleic acid sequence expression control element operably linked to any of the polynucleotides of the invention. In additional embodiments, the expression cassette is included in a vector (e.g., a plasmid, a viral vector).

Host cells containing invention isolated and recombinant polynucleotides also are provided. Host cells include prokaryotic and eukaryotic cells (e.g., mammalian cells). In one embodiment, the host cells contain a vector of claim 47.

Pharmaceutical compositions having invention polypeptides, antibodies and polynucleotides, including a pharmaceutically acceptable excipient, also are provided.

Methods for modulating an activity or activation of PICOT associated polypeptides and signaling pathways also are provided. In one embodiment, the polypeptide activity modulated is PKCΘ. In another embodiment, the polypeptide activity modulated is PKCζ. In various other embodiments, JNK, AP-1, NFKB, Trx system, HEED/WAIT- 1 activities, or IL-2 production are modulated. A method of the invention includes contacting a cell with a modulating amount of

PICOT polypeptide, subsequence, antibody or a nucleic acid encoding same, or an antisense thereof, sufficient to modulate activity of the relevant polypeptide in the cell. Such methods can be practiced in a subject by administering a modulating amount of PICOT polypeptide, subsequence, antibody or a nucleic acid encoding same, or an antisense thereof to a subject, sufficient to modulate JNK, AP-1, NFKB, HEED/WAIT-1, or Trx system activity, or IL- 2 production, for example, in a cell ofthe subject. Additional methods include administering an amount sufficient to ameliorate a physiological condition (e.g., stress response, an inflammatory response) associated with such polypeptides and other PICOT associated polypeptides and signaling pathways. The stress response can be a response to a pathogen, mitogen, UV or ionizing radiation, ischemia, phorbol ester or reactive oxygen species, for example.

Methods for identifying compounds that bind PICOT polypeptide also are provided. In on embodiment, a method includes incubating a test compound with a PICOT polypeptide under conditions allowing binding, and detecting whether the test compound binds the PICOT polypeptide, wherein binding ofthe test compound identifies the test compound as a compound that binds PICOT polypeptide.

Methods for identifying a compound that modulates PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide also are provided. In one embodiment, a method includes incubating a test compound with a PICOT polypeptide under conditions allowing binding and determining PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide in the presence ofthe test compound. An increase or decrease in PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide, identifies the test compound as a compound that modulates PICOT polypeptide activity, or expression of a nucleic acid encoding PICOT polypeptide. Methods for identifying a compound that modulates binding of PICOT with a binding polypeptide also are provided. In one embodiment, a method includes incubating PICOT with a binding polypeptide under conditions allowing binding; contacting a test compound with the bound complex; and determining whether the test compound increases or decreases binding between PICOT and the polypeptide. An increase or decrease in binding identifies the test compound as a compound that modulates binding of PICOT with a binding polypeptide.

The methods ofthe invention include contacting the test compound before incubating PICOT and the interacting polypeptide. The methods further include methods where the binding polypeptide is PKCΘ or PKCζ, where the test compound comprises a library of compounds, a polypeptide sequence (e.g., a chimera), a nucleic acid (e.g., encoding a polypeptide or an antisense). The methods ofthe invention are performed (i.e., incubated) in solution, in solid phase, in vitro or in an intact cell. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A-1D show A) the amino acid sequence of PICOT deduced from the cDNA (SEQ ID NO:l) illustrating the two repeats ofthe evolutionary conserved "PIH" domain (underlined; see, also FIG. 2B); B) the mRNA expression of PICOT in Jurkat T cells using PICOT plasmid isolated from the yeast two-hybrid screen as a probe; C) expression of PICOT (top panel), PKCθ

(middle panel) and loading control GAPDH (bottom panel) in several human tissues by RT- PCR; and D) the cDNA sequence of PICOT (SEQ ID NO:2).

FIG. 2A-2C is an analysis ofthe amino acid sequence of PICOT (SEQ ID NO:l). A) Schematic representation of PICOT domain structure. B) Alignment of an N-terminal portion of human PICOT with human Trx. Identical residues are indicated in black, conserved substitutions in grey, and the catalytic center of Trx and corresponding PICOT sequence are boxed. The corresponding amino acids are numbered. C) The C-terminal region of PICOT has a novel, evolutionary conserved domain; two repeats of this PICOT-homology domain, pihl (residues 145-228 of SEQ ID NO:l) and ρih2 (residues 247-330 of SEQ ID NO:l), are shown in the two upper lines and are compared to sequences from mouse, C. elegans, yeast, E. coli, H. influenza, and Arabidopsis.

FIG. 3A-3C show the association of PICOT with PKCθ in intact T cells and in vitro. A) An anti-HA monoclonal Antibody (mAb) was used to immunoprecipitate PICOT from Jurkat-TAg cells cotransfected with a chimeric HA-PICOT and PKCθ expression vectors; immunoblotting with an anti-PKCθ antibody (top panel) and anti-HA mAb (bottom panel) are shown. Whole cell lysates (WCL) or a normal mouse immunoglobulin (mlg) immunoprecipitation were used as positive and negative controls, respectively. B) A GST- PICOT chimeric protein, or a control GST protein, were used to precipitate lysates from Jurkat- TAg cells transfected with PKCθ, PKCα or PKCζ expression vectors. C) As in B) above, except using GST chimeric protein encoding either N-terminal (GST-PICOT-N) or C-terminal (GST-

PICOT-C), in addition to the full-length PICOT protein (GST-PICOT); precipitates were immunoblotted with anti-PKCθ antibody.

FIG. 4 is an analysis of PICOT expression in subcellular fractions. Whole cell lysate (WCL), membrane, detergent-insoluble and cytosolic fractions of Jurkat cells were immunoblotted with a PICOT-specific rabbit antiserum. FIG. 5 is an analysis of PICOT and PKCθ localization in T cells by confocal microscopy. Jurkat-TAg cells were cotransfected with PICOT and PKCθ expression vectors (panels a-f), or were not transfected (panels g-i). Transfected cells that were stimulated for the final 10 minutes of culture with PMA are indicated as "+" (panels d-f). The right column (panels c, f and i) is an overlay ofthe PKCθ (left column; panels a, d and g) and the PICOT (middle column; panels b, e and h) images.

FIG. 6A-C show the effect of PICOT overexpression on MAP kinase activities in Jurkat T cells. A) PICOT inhibits PKCθ-induced JNK activation. Jurkat-TAg cells were transfected with the indicated combinations of expression vectors plus an HA-tagged JNK1 plasmid. JNK1 activity was determined in in vitro immune complex (anti-HA) kinase assays

(two top panels). The same immunoprecipitates were immunoblotted with an anti-JNK antibody (third panel from top), and aliquots of cell lysates were immunoblotted with anti-PKCθ, -PICOT or -HA antibodies to reveal the proper overexpression ofthe transfected proteins. B) The activation of ERK2 was assessed in a similar way in anti-c-Myc (9E10 mAb) immunoprecipitates from cells transfected with the indicated plasmid combinations plus a c-Myc epitope-tagged ERK2 expression vector. Control immunoblots ofthe immunoprecipitates (with anti-ERK2 antibodies) or cell lysates (with anti-PKCθ, -PKCα or -PICOT antibodies) are shown in the four lower panels C) Cells were transfected with empty vector, PICOT, or dominant- negative (K/R) PKCθ expression vectors and were left unstimulated or stimulated with the indicated stimuli for the final 5 min (anti-CD3/CD28 or PMA plus ionomycin) or 1 min (UV) of culture. JNK1 was immunprecipitated and tested for in vitro kinase activity as in A) above. FIG. 7 shows inhibition of PMA plus ionomycin (P/I) stimulated JNK activation by PICOT. Assays were performed as in FIG. 6A.

FIG. 8A-C show that PICOT overexpression inhibits AP-1 and NF-κB activation. A) Jurkat-TAg cells were cotransfected with an AP-1 :luciferase reporter plasmid plus the indicated expression vectors. One group was additionally stimulated with PMA for the final 6 hrs of culture. B) Cells were transfected with the indicated combinations of empty vector, constitutively active (A/E) PKCθ, and/or full-length (wt), N-terminal (N) or C-terminal (C) subsequences of PICOT. C) PICOT inhibits NF-κB activation. Jurkat-TAg cells were cotransfected with an NF-κB:luciferase reporter plasmid plus the indicated amounts of a PICOT expression vector. The cells were either left unstimulated, or stimulated with a combination of anti-CD3 plus anti-CD28 antibodies, or with PMA, for the final 6 hrs of culture. The inset shows the expression level of transfected PICOT determined by anti-HA immunoblotting.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the isolation and characterization of a novel polypeptide that specifically binds with protein kinase C theta (PKCθ). This polypeptide, termed

"PICOT" (PKC-interacting cousin of thioredoxin polypeptide), when expressed in T cells, inhibits activation of c-Jun N-terminal kinase (JNK), and the transcription factors AP-1 and NF-KB. Thus, as PICOT modulates the function of PKCθ and several proteins that interact with PKCθ or represent targets of PKCθ, PICOT and subsequences thereof can be useful for modulating the activity of these polypeptides and associated cell signaling pathways that regulate function of T cells or other cells in which PICOT is expressed. Such pathways include, for example, stress response, response to environmental insult, cell stimuli and T-cell activation, as well as other cell responses in which JNK, AP-1, NF-KB, and associated cell signaling pathways participate (e.g., Trx system regulation, IL-2 production). Thus, the present invention provides isolated or recombinant PICOT polypeptides, and functional subsequences thereof. In one embodiment, PICOT is characterized as having an apparent molecular weight of about 37.5-kDa. In another embodiment, PICOT is characterized as specifically binding or interacting with protein kinase C theta (PKCθ). In yet another embodiment, PICOT is expressed in various tissues, including, for example, in hematopoetic (T cells) and in muscle (skeletal). In various aspects, functional PICOT subsequences include fragments that specifically bind or interact with PKCθ or protein kinase C zeta (PKCζ), modulate the activity of JNK, AP-1 and NF-κB, and IL-2 production. In another aspect, immunogenic PICOT fragments are provided.

The exemplary PICOT polynucleotide encodes a 335-amino acid protein (SEQ ID NOS:l and 2, respectively; FIG. 1) which binds to and colocalizes with PKCθ in cells (see, e.g., FIG. 5).

In addition, PICOT binds to PKCζ (see, e.g., FIG. 3). PICOT contains several domains, including an N-terminal thioredoxin (Trx)-homology domain (e.g., FIG. 2), indicating that PICOT likely plays a role in regulating the function ofthe Trx system. In addition, two tandem repeats of a novel domain ("PICOT homology domains," PIH) are present in the C-terminal region of PICOT (see, e.g., FIG. 2C; residues 145-228 and residues 247-330 of SEQ ID NO:l).

As used herein, the terms "polypeptide," "protein" and "peptide" are used interchangeably to denote two or more amino acids covalently linked by an amide bond or equivalent. The polypeptides ofthe invention are of unlimited length and include L- and D- isomers and combinations thereof. Such polypeptides can include modifications typically associated with post-translational processing of proteins, for example, cyclization (e.g., disulfide bond), phosphorylation, glycosylation, carboxylation, ubiquitination, or lipidation. Polypeptides ofthe invention further can include compounds having amino acid structural and functional analogues, for example, peptidominetics having synthetic or non-natural amino acids or amino acid analogues, so long as the mimetic has one or more functions or activities of PICOT as set forth herein. Non-natural and non-amide chemical bonds, and other coupling means can also be included, for example, glutaraldehyde, N-hydoxysuccinimide esters, bifunctional maleimides, or N,N'-dicyclohexylcarbodiimide (DCC). Non-amide bonds can include, for example, ketomethylene aminomethylene, olefin, ether, thioether and the like (see, e.g., Spatola (1983) in

Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide and Backbone Modifications," Marcel Decker, NY).

As used herein, the term "isolated," when used as a modifier of invention polypeptides, antibodies and polynucleotides described herein, means that the compositions are made by the hand of man and are separated from their naturally occurring in vivo environment. Generally, the compositions so separated are substantially free of one or more other proteins, polynucleotides, lipids, carbohydrates, or other materials with which they may normally associate with in nature. An "isolated" polypeptide, antibody or polynucleotide can also be "substantially pure" when free of most or all ofthe materials with which they may normally associate with in nature. Thus, an isolated compound that also is substantially pure does not include polypeptides or polynucleotides present among millions of other sequences, such as nucleic acids in a genomic or cDNA library, for example. Typically, the purity can be at least about 60% or more by mass. The purity can also be about 70% or 80% or more, and can be greater, for example, 90% or more. Purity can be determined by any appropriate method, including, for example, UV spectroscopy, chromatography (e.g., HPLC, gas phase), gel electrophoresis and sequence analysis (nucleic acid and peptide). As used herein, the term "recombinant," when used as a modifier of invention polypeptides, antibodies and polynucleotides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature (e.g., in vitro). A particular example of a recombinant polypeptide would be where PICOT is expressed by a cell transfected with a polynucleotide encoding the polypeptide. A particular example of a recombinant polynucleotide would be where a nucleic acid (e.g., genomic or cDNA) encoding PICOT is cloned into a plasmid, with or without 5' and 3' regions with which the gene is normally contiguous with in the genome of an organism. Another example of a recombinant polynucleotide or polypeptide hybrid or fusion sequence, such as a chimeric sequence comprising PICOT and a second sequence, such as a heterologous functional domain.

In another embodiment, the invention provides isolated or recombinant PICOT polypeptides having 70% or more identity to the sequence set forth in SEQ ID NO:l. The invention also provides isolated or recombinant PICOT polypeptides having 80% or more, 90% or more, and 95% or more identity to the polypeptide sequence set forth in SEQ ID NO:l. In yet another embodiment, the invention provides functional subsequences of PICOT, including PICOT polypeptide fragments. Functional subsequences include portions of PICOT polypeptide having 70% or more, 80% or more, 90% or more, and 95% or more identity to a sequence set forth in SEQ ID NO:l, as well as functional subsequences of SEQ ID NO:l.

As used herein, the term "subsequence" means a sequence region or portion of PICOT polypeptide or polynucleotide (e.g., SEQ ID NO:l or SEQ ID NO:2). "Functional subsequence" means a subsequence that has one or more functions or activities of exemplary PICOT polypeptide, as described herein. For example, PICOT polypeptides binding to PKCθ or PKCζ, can modulate activity of JNK, AP-1 or NF-κB, or production of IL-2. Another example of a functional PICOT subsequence is a sequence having the N-terminal, Trx-homologous domain. Thus, functional subsequences include polypeptide regions or fragments that interact with or bind to PKCθ or PKCζ, for example, as well as polypeptide regions or fragments that modulate activation of JNK, AP-1 or NF-κB, production of IL-2, or the associated signaling pathways. In addition, as PICOT polypeptide has a Trx homology domain in the N-terminal region, functional PICOT subsequences include polypeptide regions or fragments that modulate activation ofthe Trx system. Because, PKCθ co-localizes with the T cell receptor (TCR) complex to the contact site between antigen-specific T-cells and antigen presenting cells, functional subsequences also include PICOT polypeptide regions or fragments that modulate TCR complex formation.

Functional subsequences can be of any length up to full length PICOT (e.g., 335 amino acid sequences of SEQ ID NO:l). Such subsequences can be at least about seven amino acids, but less than about 100 amino acids. Other functional subsequences can be from about 10 to about 100 amino acids, or from about 10 to about 50 amino acids. A particular example of a functional subsequence is a PICOT sequence including a Trx-like domain, for example, residues 1-146 of SEQ ID NO:l, that binds to PKCθ (Example V).

PICOT subsequences (e.g., peptides) include immunogenic fragments capable of inducing an immune response (e.g., antibodies) when administered to an appropriate animal.

Immunogenic subsequences can be as few as 5 amino acids, such as an epitope capable of binding an antibody. However, immunogenic sequences can also be larger, for example, as exemplified herein, an amino acid sequence including amino acid residues 90 to 108 of PICOT polypeptide (SEQ ID NO:l) is a subsequence having immunogenic activity (see Example I). Immunogenic subsequences can be identified as those producing an immune response when administered to an animal, as determined by ELISA, for example.

Functional assays as described herein or known in the art can be used to identify the PICOT polypeptides, functional subsequences and immunogenic subsequences ofthe invention, having one or more ofthe functions or activities associated with PICOT polypeptide set forth as SEQ ID NO: 1. For example, specific binding or interaction between PICOT and PKCθ can be detected by precipitation or by using an in vivo two-hybrid assay (yeast or mammalian), as disclosed herein. Assays for detecting modulation of JNK, AP-1 or NF-κB activity, production of IL-2, or for identifying functional subsequences having one or more ofthe activities described herein, include expression analysis of a responsive reporter gene (e.g., AP-1 or NF-κB driven reporter) and Northern analysis. Functional PICOT subsequences also can be identified by expression of particular fragments. Homology with other polypeptide domains whose function has been characterized also can be used to identify functional subsequences of PICOT. For example, comparison ofthe exemplary PICOT polynucleotide set forth as SEQ ID NO:2 to a sequence database using a BLAST search identified the presence of a Trx homology domain and two tandem repeats of a novel domain (termed "PICOT homology domain" PIH). As used herein, the term "interact" includes physical contact (i.e., "binding"), indirect binding (e.g., as part of a complex composed of multiple polypeptides which may not physically contact each other), and indirect interaction. A particular example of an indirect interaction would be a situation in which a PICOT polypeptide binds PKCθ and modulates IL-2 production, but does not physically contact IL-2 protein or a regulatory region of a gene encoding IL-2.

Although the invention is not bound by any particular mechanism of action, it appears that PICOT's ability to modulate IL-2 production is conferred "indirectly" through PICOT binding to PKCθ. Thus, it is specifically intended that the term "interact" includes physical contact, indirect binding and indirect interaction. Indirect interaction can be detected using expression studies, such as PICOT expression in T cells disclosed herein (see, e.g., Examples VII and VIII) and co- precipitation analysis.

As used herein, the term "bind" or "binding" means that the compositions referred to physically contact each other. "Specific binding" is where the binding is selective. Typically, specific binding between polypeptides is where the dissociation constant (K_D) is less than 10^"5, or less than 10^"6 or 10^"7. A particular example of specific binding is that which occurs between an antibody and an antigen. Specific binding can be detected, for example, by coprecipitation analysis, with or without chemical crosslinking, two-hybrid assays and the like. Appropriate controls can be used to distinguish between "specific" from "non-specific" binding.

In another embodiment, the invention provides PICOT polypeptides and subsequences thereof having one or more modifications of a sequence set forth in SEQ ID NO: 1. The term

"modification" denotes an alteration of PICOT polypeptide sequence set forth in SEQ ID NO:l that does not significantly change or destroy an activity ofthe modified polypeptide. Modifications include but are not limited to amino acid additions, insertions, deletions and substitutions, for example. An example of an addition is one or more amino acids added to the N- or C-terminal end of PICOT. An example of an insertion is a heterologous domain comprising an amino acid sequence, such as a nucleic acid binding or a transcriptional activation domain (e.g., VP16, GAL4) fused to PICOT (i.e., a "chimera"). An example of a deletion is where one or more amino acids are deleted from the N- or C-terminal end of, or internally within PICOT. Substitutions include non-conservative and conservative amino acid substitutions, so long as the substituted polypeptide retains substantial activity associated with PICOT as defined herein. As used herein, the term "conservative substitution" means the replacement of one amino acid by a biologically or chemically similar residue. Particular examples of conservative substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative variation" includes polypeptides having a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also bind to the unsubstituted polypeptide.

Modifications also include derivatized polypeptides, for example, amino acids in which free amino groups form amine hydrochlorides, p-toluene sulfonyl groups, carobenzoxy groups; the free carboxy groups form salts, methyl and ethyl esters; free hydroxl groups that form O-acyl or O-alkyl derivatives, as well as naturally occurring amino acid derivatives, for example, 4-hydroxyproline, for proline, 5-hydroxylysine for lysine, homoserine for serine, ornithine for lysine, etc. Also included are modifications that confer covalent bonding, for example, a disulfide linkage forming between two cysteine residues thereby producing a cyclic polypeptide.

PICOT modifications can be produced using any of a variety of methods well known in the art (e.g., PCR based sited-directed, deletion and insertion mutagenesis, chemical modification and mutagenesis, etc.).

Modified PICOT polypeptide or functional subsequences thereof as set forth herein retain an amount of one or more activities of unmodified polypeptide. For example, a modified PICOT polypeptide subsequence that specifically binds PKCθ can retain substantial PKCθ binding activity relative to its unmodified counterpart. Likewise, a modified PICOT polypeptide or subsequence can retain substantial AP-1 inhibiting activity. Such modified PICOT polypeptides and functional subsequences thereof, which retain one or more activities of unmodified PICOT, can be identified using the assays disclosed herein or otherwise known in the art.

PICOT polypeptides and functional subsequences thereof can also include polypeptides having the ability to modulate one or more activities of one or more polypeptides or associated signaling pathways, or expression of a nucleic acid. For example, two important elements in the TCR/CD28 signaling cascade leading to IL-2 production (JNK and AP-1), both of which are selectively activated by PKCθ, and NF-κB, are modulated by PICOT. In particular, as disclosed herein, PICOT (e.g., SEQ ID NO:l) modulates activation or expression ofthe aforementioned polypeptides. In addition, PICOT can bind PKCζ, which in turn binds ZIP (Puls et al. (1997) Proc. Nat 'I. Acad. Sci. USA, 94:619). Thus, PICOT likely modulates PKCζ or ZIP activity or the signaling pathways in which PKCζ or ZIP participates. Moreover, a two-hybrid assay (Example IX) revealed that PICOT can interact with HEED (aka "WAIT-1"; Peytavi et al. (1999) J. Biol. Chem. 3:274; Rietzler et al (1998) J. Biol. Chem. 42:273). Thus, PICOT is also likely to modulate the activity of HEED/WAIT-1.

As used herein, the phrase "PICOT associated polypeptides and signaling pathways" includes the polypeptides and cell signaling pathways which PICOT specifically binds to or interacts with, or whose activity is modulated by expression or an activity of PICOT, as described herein (e.g., activity of PKCθ, JNK, AP-1, Trx system, IL-2 production, binding to

PKCζ, HEED/WAIT-1). The phrase also includes polypeptides and cell signaling pathways known to bind to, interact with, or modulate the activity or expression ofthe polypeptides and signaling pathways described herein, as is known in the art (e.g., HEED/WAIT-1 binds to matrix polypeptide of HIV- 1). As used herein, the term "modulate" means a measurable or detectable change in activity. Such changes in activity include, for example, the ability of PICOT to modulate activity or expression of associated polypeptides and signaling pathways (e.g., PKCθ, PKCζ , JNK, AP-1, etc.), or expression of a gene responsive to PICOT associated polypeptide and signaling pathways.

PICOT polypeptides and functional subsequences ofthe invention can be isolated with standard protein purification techniques, for example, by chromatography (e.g., ion-exchange, size-exclusion, reverse-phase, immunoaffinity, etc.) of T cell or muscle cell (tissue) lysate. Any protein purification method known in the art can be used (see, e.g., Deutscher et al, In: Guide to Protein Purification: Methods in Enzymology, Academic Press, ed., 1990). Isolated PICOT polypeptide and functional subsequences thereof also can be obtained using recombinant nucleic acid expression methods as disclosed herein. For example, polynucleotides encoding PICOT polypeptide can be produced, inserted into expression cassettes and transformed into host cells using well known techniques described herein and further known in the art (Sambrook et al, In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, ed., 1989). Following transformation, protein may be isolated and purified in accordance with conventional methods. PICOT functional subsequences also can be obtained by chemical synthesis, e.g., using a peptide synthesizer (e.g., Applied Biosystems, Inc., Foster City, CA; Model 430A or the like).

PICOT polypeptides and polynucleotides can also be isolated from organisms and identified using a binding or functional assay. For example, as exemplified herein with a human library, by screening other libraries in a two-hybrid assay, PICOT sequences from other organisms can be identified. Such libraries from a wide variety of organisms are easily manufactured or available commercially (plant, animal and microorganism). In addition, homology or expression screening also can be used to identify or isolate PICOT polypeptides from various organisms and tissues (nucleic acid or expression libraries). In this way, PICOT from primates (ape, gibbon, orangutan, monkey), canine, feline, porcine, equine, bovine can readily be identified and, as such, are included.

In another embodiment, the invention provides PICOT polypeptides and functional subsequences thereof, comprising heterologous domain. In one aspect, the heterologous functional domain is an amino acid sequence. As used herein, the term "heterologous functional domain" means a molecular entity that imparts an additional or distinct functionality upon the polypeptide. Such molecular entities include small molecules and macromolecules and combinations thereof. The functions conferred include, for example, transcriptional activation or nucleic acid binding, as exemplified herein, targeting (e.g., an antibody, cell surface receptor ligand, viral coat protein polypeptide) and to increase or decrease polypeptide activity (e.g., a derepressible or activatable moiety, such as a hormone receptor binding domain). Such heterologous functional domains also include detectable labels and tags for visualization (gold particles, fluroescein and other stains, radioactive isotopes, etc.) or purification (e.g., bitoin, avidin, Ig heavy chain, T7 tag, polyhistidine, large molecule beads, such as agarose, Sepharose, Sephadex and the like). Heterologous functional domains therefore include chimeric polypeptides comprising a

PICOT polypeptide sequence and a heterologous sequence from another protein (e.g., GAL4, lex A, VP16 DNA binding domain or activation domains). Such chimeras are useful for identifying PICOT binding compounds (e.g., polypeptides) or compounds that modulate PICOT polypeptide activity. For example, as shown in Example IX, PICOT fused with a LexA DNA- binding domain in a chimera was used as bait to identify HEED/WAIT-1 as a PICOT interacting polypeptide. Chimeras having particular PICOT subsequences also are useful for identifying an activity or function conferred by the subsequence, including a domain that participates in protein interactions (e.g., residues 1 to 146 of PICOT (SEQ ID NO:l), which includes the Trx homology domain). The skilled artisan will know or can readily ascertain other appropriate heterologous functional domains and their use depending on the application and function desired.

PICOT polypeptides and subsequences ofthe invention can be used to generate additional reagents, such as antibodies. Thus, antibodies that bind to a PICOT polypeptide, functional subsequences thereof and immunogenic fragments thereof are provided. Polyclonal antibodies, pooled monoclonal antibodies with different epitopic specificities, and distinct monoclonal antibody preparations, also are provided.

The term "antibody" includes intact antibody molecules as well as fragments thereof. Such fragments include, for example, Fab, F(ab')2, and Fv, which are capable of binding to an antigenic determinant present in a PICOT or a functional subsequence thereof. Generally, antigenic determinants have at least five contiguous amino acids. Antibodies that bind to invention polypeptides can be prepared using intact PICOT polypeptide, functional subsequences thereof or small peptide fragments, as the immunizing antigen. For example, as shown in Example I, a subsequence corresponding to amino acid 90- 108 of SEQ ID NO:l was used as the immunizing antigen to produce PICOT antibodies. To produce antibodies that specifically bind to the N- or C-terminal domains of PICOT, or internal PICOT subsequences, appropriate regions can be used as immunizing antigen. The immunizing antigen can be conjugated to a carrier protein, if desired. Commonly used carriers, which are chemically coupled to the immunizing peptide include, for example, keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.

Monoclonal antibodies can be made by methods well known to those skilled in the art (Kohler et al (1975) Nature 256:495; and Harlow et al. (1988) In: Antibodies: A Laboratory

Manual, page 726, Cold Spring Harbor Pub.). Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques which include, for example, affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, e.g., Coligan et al. (1994) In: Current Protocols in Immunology, Wiley; and Barnes et al (1992) In: Methods in Molecular Biology, Vol. 10, pages

79-104, Humana Press). The preparation of polyclonal antibodies, and their purification, also is well known to those skilled in the art (see, e.g., Green et al. (1992) In: Immunochemical Protocols, pages 1-5, Manson, ed., Humana Press; Harlow et al. (1988), supra; and Coligan et al. (1992), supra, section 2.4.1).

Thus, in accordance with the present invention, methods for producing antibodies that specifically bind to PICOT polypeptide, functional subsequences thereof and immunogenic fragments thereof are provided. A method ofthe invention includes administering a PICOT polypeptide or an immunogenic fragment, or a nucleic acid encoding such a polypeptide, to an animal in an amount sufficient to produce an antibody that specifically binds to PICOT, a functional subsequence thereof or an immunogenic fragment. A therapeutically useful anti-PICOT antibody can also be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions (CDR's) from heavy and light variable chains into a human variable domain, and then substituting human residues in the framework regions ofthe murine counterparts. The use of such humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of non-human constant regions. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al. (1986) Nature 321 :522; Riechmann et al. (1988) Nature 332:323; Verhoeyen et al. (1988) Science 239:1534; Carter et al. (1992) Proc. Natl. Acad. Sci. USA 89:4285; Sandhu (1992) Crit. Rev. Biotech. 12:437; and Singer et al. (1993) J. Immunol. 150:2844. In addition, antibodies of the present invention may be derived from a human antibody produced in a transgenic mouse "engineered" to contain human immunoglobulin genes and produce specific human antibodies in response to antigenic challenge. These transgenic mice can synthesize human antibodies specific for human antigens and can be used to produce human antibody-secreting hybridomas. See, e.g., Green et al. (1994) Nature Genet. 7: 13; Lonberg et al. (1994) Nature 368:856; and Taylor et al. (1994) Int. Immunol. 6:579.

Invention polyclonal and monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology art for purification and/or concentration of polyclonal and monoclonal antibodies and antibody fragments (see, e.g., Coligan et al (1994) supra). Antibody fragments (e.g., Fab, F(ab')2, and Fv) ofthe present invention can be prepared by proteolytic hydrolysis ofthe antibody, for example, by pepsin or papain digestion of whole antibodies. In particular, antibody fragments produced by enzymatic cleavage with pepsin provide a 5S fragment denoted F(ab')₂. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Antibodies ofthe present invention are useful for detecting or purifying polypeptides of the invention (e.g., PICOT and functional subsequences thereof including antigenic subsequences). Such methods include contacting a sample suspected of containing a PICOT polypeptide (in solution, in solid phase, in vitro or in vivo; in an intact cell or organism) with an antibody under conditions allowing binding and detecting the presence ofthe bound polypeptide, or purifying the bound antibody-polypeptide. The polypeptide can be detected by methods well known in the art, for example, ELISA, immunohistochemical staining, flow cytometry, immunoprecipitation, etc.

Invention antibodies also are contemplated for use in modulating PICOT, PKCθ or PKCζ and other polypeptides and signaling pathways with which these polypeptides are associated, including a polypeptide or nucleic acid whose activity or expression is modulated by PICOT or PKCθ , as disclosed herein. For example, an antibody that binds PICOT can inhibit PICOT interaction with other compounds, thereby modulating (as an antagonist or agonist) PICOT or the activity ofthe compounds with which PICOT interacts. Thus, an antibody or antibody fragment that inhibits PICOT binding to PKCθ can increase PKCθ activity, for example. Antibodies and binding fragments that modulate a biological activity or function of a PICOT polypeptide or functional subsequence thereof can be useful for modulating PKCθ or PKCζ activity, as well as JNK, AP-1 and NF-κB activity, and IL-2 production, as described herein.

The invention also provides isolated or recombinant polynucleotides encoding PICOT polypeptides and functional and immunogenic subsequences thereof , as described herein. In one embodiment, the polynucleotides encode a polypeptide having 70% or more identity to the amino acid sequence set forth in SEQ ID NO:l, or a subsequence thereof, with the proviso that the polynucleotide is distinct from GenBank Accession Nos. h59799 and aa009010. In another embodiment, the polynucleotides encode a polypeptide having 70% or more identity to a polypeptide corresponding to amino acids 138 to 335 of SEQ ID NO:l, or a subsequence thereof. In various additional aspects, the polynucleotides encode a polypeptide having 80% or more, 90% or more and 95% or more identity to the amino acid sequence set forth in SEQ ID NO:l, a polypeptide corresponding to amino acids 38 to 335 of SEQ ID NO:l, or subsequences thereof. As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to all forms of nucleic acid, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The polynucleotides ofthe invention can be double, single strand, or triplex, linear or circular. Nucleic acids of the invention include genomic DNA, cDNA, and antisense DNA. RNA can be spliced or unspliced mRNA, rRNA, tRNA or antisense (e.g., RNAi). Polynucleotides also refer to oligonucleotides, primers and probes. Polynucleotides of the invention include naturally occurring, synthetic, as well as nucleotide analogues and derivatives. Such altered or modified polynucleotides include analogues that provide nuclease resistance, for example, which are particularly useful in practicing the methods ofthe invention described herein. Polynucleotides ofthe invention can be of any length, e.g., the 1005 base pair long encoding PICOT. Polynucleotide lengths also can range from about 1 Kb or less, to about 500 base pairs in length. Polynucleotides can also be 100 to about 200 base pairs, or from about 12 or 15 to about 30 to 50 base pairs.

As a result ofthe degeneracy ofthe genetic code, the polynucleotides of the invention include polynucleotides that are degenerate with respect to a sequence set forth in SEQ ID NO: l. Thus, the invention includes polynucleotides having alterations from a nucleic acid sequence set forth in SEQ ID NO:2, which still encode SEQ ID NO: l, and subsequences thereof.

Polynucleotides of the invention also include sequences complementary to a sequence set forth in SEQ ID NO:2, and subsequences thereof. Such polynucleotides are useful for hybridization to detect the presence or an amount of PICOT in a sample (in vitro, cell, tissue, serum, etc.). Complementary sequences also are useful as antisense. Also included are single and double stranded RNA sequences from a PICOT coding region. The use of double stranded RNA sequences (known as "RNAi") for inhibiting gene expression is known in the art (Kennerdell et al. (1998) Cell 95: 1017-1026; Fire et al. (1998) Nature 391:806-811). Antisense and RNAi sequences can interfere with PICOT activity or expression and, therefore, be useful in modulating PKCθ or PKCζ activity, as well as JNK, AP-1, NF-κB, ZIP or HEED/WAIT-1 activity, or IL-2 production, as described herein. Thus, in another embodiment, the invention provides isolated or recombinant polynucleotides including the nucleotide sequence set forth in SEQ ID NO:2 where one or more T's are U; and nucleic acid sequences complementary thereto. Further provided are subsequences ofthe aformentioned polynucleotides that are at least about 15 base pairs in length. In various aspects, polynucleotide subsequences (e.g., primers, probes useful for hybridization, antisense polynucleotides, etc.) greater than about 15 base pairs will be from about 15 to 25 or from about 20 to 50 base pairs, but can be larger (e.g., up tolOO, 200 or more base pairs).

Polynucleotides ofthe invention can be altered intentionally by site-directed mutagenesis. For example, portions of an mRNA sequence may be altered to alternate RNA splicing patterns or to use alternate promoters for RNA transcription. Alterations of PICOT polynucleotides include but are not limited to intragenic mutations (e.g., point mutation, splice site and frameshift) and heterozygous or homozygous deletions occuring in nature, or by intention (e.g., EMS mutagenesis). Termination signals or mutations that produce a stop codon leading to a truncated PICOT translation product may retain an activity in vivo depending on the length ofthe terminated product, product stability, etc. Detection of PICOT sequences having one or more altered nucleotides can be determined by standard methods known to those of skill in the art which include, for example, sequence analysis, Southern blot analysis, PCR based analyses (e.g., multiplex PCR, sequence tagged sites (STSs) and in situ hybridization).

Polynucleotides, including subsequences, which selectively hybridize to the sequence set forth in SEQ ID NO:2 also are provided. Hybridizing polynucleotides are useful for detecting

PICOT nucleic acid and for identifying other PICOT sequences and PICOT-related genes, including sequences of various organisms. Such polynucleotides include sequences complementary to a sequence set forth in SEQ ID NO:2. Thus, in accordance with the present invention, there are provided isolated or recombinant polynucleotides that selectively hybridize to the sequence set forth in SEQ ID NO:2. In one embodiment, an invention polynucleotide hybridizes under moderately stringent conditions. In another embodiment, an invention polynucleotide hybridizes under moderately high stringent conditions. In yet another embodiment, an invention polynucleotide hybridizes under highly stringent conditions. In various aspects, polynucleotides ofthe invention hybridize under moderately stringent, moderately high, or highly stringent conditions to the nucleotide sequence set forth in SEQ ID

NO:2. As used herein, the term "hybridization" refers to the binding between complementary nucleic acids. "Selective hybridization" refers to hybridization that distinguishes PICOT related sequences from unrelated sequences. Related sequences can be more than about 50% homology to a sequence set forth in SEQ ID NO:2. PICOT sequences within the scope ofthe invention can also have about 60%, 70%, 80%, 90%, 95% or more sequence identity to SEQ ID NO:2. The region between related sequences can extend over at least about 30 base pairs, or about 50 base pairs, or about 100 to 200 or more residues.

As is understood by those skilled in the art, the T_M (melting temperature) refers to the temperature at which binding between complementary sequences is no longer stable. For two sequences to bind, the temperature of a hybridization reaction must be less than the calculated

TM for the sequences. The T_M is influenced by the amount of sequence complementarity, length, composition (%GC), type of nucleic acid (RNA vs. DNA), and the amount of salt, detergent and other components in the reaction (e.g., formamide). All of these factors are considered in establishing appropriate hybridization conditions (see, e.g., the hybridization techniques and formula for calculating TM described in Sambrook et al, 1989, supra).

Typically, wash conditions are adjusted so as to attain the desired degree of hybridization stringency. Thus, hybridization stringency can be determined empirically, for example, by washing under particular conditions, e.g., at low stringency conditions or high stringency conditions. Optimal conditions for selective hybridization will vary depending on the particular hybridization reaction involved.

An example of moderately stringent hybridization conditions is as follows: 2X SSC/0.1% SDS at about 37°C or 42°C (hybridization conditions); 0.5X SSC/0.1% SDS at about room temperature (low stringency wash); 0.5X SSC/0.1% SDS at about 42 °C (moderate stringency wash). An example of moderately-high stringency hybridization conditions is as follows: 2X SSC/0.1 % SDS at about 37 °C or 42 °C (hybridization conditions); 0.5X SSC/0.1 %

SDS at about room temperature (low stringency wash); 0.5X SSC/0.1% SDS at about 42 °C (moderate stringency wash); and 0.1 X SSC/0.1% SDS at about 52 °C (moderately-high stringency wash). An example of high stringency hybridization conditions is as follows: 2X SSC/0.1% SDS at about 37°C or 42°C (hybridization conditions); 0.5X SSC/0.1% SDS at about room temperature (low stringency wash); 0.5X SSC/0.1% SDS at about 42 °C (moderate stringency wash); and 0.1 X SSC/0.1% SDS at about 65 °C (high stringency wash).

Sequence identities can be determined using any algorithm, e.g., a BLAST search algorithm. In one embodiment, a polynucleotide ofthe invention comprises a nucleic acid sequence having at least 70% homology to a sequence set forth in SEQ ID NO:2, as determined using a BLAST search algorithm. In another embodiment, a polynucleotide ofthe invention comprises a nucleic acid sequence having at least 70% homology to a sequence corresponding to nucleotides 421 to 1005 of SEQ ID NO:2, as determined using a BLAST search algorithm. In various additional embodiments, a polynucleotide ofthe invention can have at least 80%, 90%, or 95% sequence identity.

The extent of sequence identity between two sequences can be ascertained using various computer programs and mathematical algorithms known in the art. Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the region of similarity. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10, which is publicly available through NCBI at http:/www.ncbi.nlm.nih.gov) has exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2. For polypeptide sequence comparisons, the BLAST algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 and the like. Polynucleotides ofthe invention can be obtained using various standard cloning and chemical synthesis techniques. Purity of polynucleotides can be determined through sequencing, gel electrophoresis and the like. Such techniques also include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences which can then cloned into a plasmid, propagated amplified and purified; 2) antibody screening to detect polypeptides having shared structural features, for example, using an expression library; 3) amplification, e.g., polymerase chain reaction (PCR), with genomic DNA or cDNA targets using primers (e.g., a degenerate primer mixture) capable of annealing to a PICOT nucleotide; 4) computer searches of sequence databases for related sequences; and 5) differential screening of a subtracted nucleic acid library. In another embodiment, the invention provides expression cassettes. As used herein, the term "expression cassette" refers to expression control element operably linked to a nucleic acid whose transcription is controllable by the expression control element (e.g., promoter). As used herein, the term "expression control element" refers to one or more nucleic acid sequence elements that regulate the expression of a nucleic acid sequence to which it is operatively linked. An expression control element operatively linked to a nucleic acid sequence controls transcription and, as appropriate, translation ofthe nucleic acid sequence. An expression control element can include, as appropriate, promoters, enhancers, transcription terminators, a start codon (e.g., ATG) in front of a protein-encoding gene, etc. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. Typically such elements are juxtaposed at the 5' or the 3' ends ofthe genes but can also be intronic. A particular example is a polynucleotide sequence encoding a PICOT polypeptide or functional subsequence thereof operably linked to an expression control element such that expression of a sequence contiguous thereto is under the control ofthe element.

Expression control elements can include elements that activate transcription constitutively, as well as elements that are inducible (i.e., require an external signal for activation), and derepressible (i.e., require a signal to turn transcription off; when the signal is no longer present, transcription is activated or "derepressed"). Also included in the expression cassettes ofthe invention are control elements sufficient to render gene expression controllable for specific cell-types, tissues or physiological conditions. Typically, such elements are located upstream or downstream (i.e., 5' and 3') ofthe coding sequence. Promoters are generally 5' of the coding sequence. Promoters, produced by recombinant DNA or synthetic techniques, can be used to provide for transcription ofthe polynucleotides ofthe invention. A "promoter" is meant a minimal sequence element sufficient to direct transcription.

In one embodiment, an expression cassette drives expression of polynucleotides encoding a PICOT polypeptide or subsequence thereof. In another embodiment, an expression cassette includes an expression control element (e.g., promoter) operably linked to a PICOT antisense polynucleotide.

For expression in cells, invention polynucleotides, if desired, may be inserted into a vector. The term "vector," e.g., a plasmid, virus or other vehicle known in the art that can be manipulated by insertion or incorporation of a polynucleotide can be used for genetic manipulation (i.e., "cloning vectors") or can be used to transcribe or translate the inserted polynucleotide (i.e., "expression vectors"). Such vectors are therefore useful for producing PICOT polypeptides or antisense, for example.

A vector generally contains at least an origin of replication for propagation in a cell and a promoter. Control elements, including expression control elements as set forth herein, present within a vector are included to facilitate proper transcription and translation (e.g., splicing signal for introns, maintenance ofthe correct reading frame ofthe gene to permit in-frame translation of mRNA and, stop codons, etc.). The term "control element" is intended to include, at a minimum, one or more components whose presence can influence expression, and can also include additional components, for example, leader sequences and fusion partner sequences. When cloning in bacterial systems, constitutive promoters such as T7 and the like, as well as inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) may be used, as well as inducible promoters (e.g., tetracycline responsive). When cloning in insect cell systems, constitutive or inducible promoters (e.g., ecdysone) may be used. When cloning in mammalian cell systems, constitutive promoters such as SV40, RSV and the like, or inducible promoters derived from the genome of mammalian cells (e.g., metallothionein IIA promoter; heat shock promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the inducible mouse mammary tumor virus long terminal repeat) may be used. Vectors based on bovine papilloma virus (BPV) which have the ability to replicate as extrachromosomal elements (Sarver et al, Mol. Cell. Biol. (1981) 1:486) also may be used. Vectors can be used for stable expression by including a selectable marker, such as the neo or hygromycin gene, for example.

Alternatively, a retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the PICOT polynucleotide in host cells (Cone et al, Proc. Natl. Acad. Sci. USA 81:6349-6353).

Mammalian expression systems further include vectors specifically designed for "gene therapy" including adenoviral vectors (U.S. Patent Nos. 5,700,470 and 5,731 , 172), adeno- associated vectors (U.S. Patent No. 5,604,090), herpes simplex virus vectors (U.S. Patent No. 5,501,979) and retroviral vectors (U.S. Patent Nos. 5,624,820, 5,693,508 and 5,674,703 and WIPO publications WO92/05266 and WO92/14829). Bovine papilloma virus (BPV) has also been employed in gene therapy (U.S. Patent No. 5,719,054). Such gene therapy vectors also include CMV based vectors (U.S. Patent No. 5,561,063). In yeast, a number of vectors containing constitutive or inducible promoters may be used (see, e.g., Ausubel et al, In: Current Protocols in Molecular Biology, Vol. 2, Ch. 13, ed., Greene Publish. Assoc. & Wiley Interscience, 1988; Grant et al. (1987) In: Methods in Enzymology, 153, 516-544, eds. Wu & Grossman, 31987, Acad. Press, NY.; Glover, DNA Cloning, Vol. II, Ch. 3, IRL Press, Wash., D.C., 1986; Bitter (1987) In: Methods in Enzymology, 152, 673-684, eds. Berger & Kimmel, Acad. Press, NY.; and, Strathern et al, The Molecular Biology of the Yeast Saccharomyces (1982) eds. Cold Spring Harbor Press, Vols. I and II). A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (R. Rothstein In: DNA Cloning, A Practical Approach, Vol.l 1, Ch. 3, ed. D.M. Glover, IRL Press, Wash., D.C., 1986). Alternatively, vectors that facilitate integration of foreign nucleic acid sequences into a yeast chromosome, via homologous recombination for example, are known in the art and can be used. Yeast artificial chromosomes (YAC) are typically used when the inserted polynucleotides are too large for more conventional vectors (e.g., greater than about 12 kb). The invention also provides polynucleotides (e.g., vectors) inserted into host cells. In one embodiment, the host cell is a prokaryotic cell. In another embodiment, the host cell is a eukaryotic cell. In various aspects, the eukaryotic cell is a yeast or mammalian (e.g., human, primate, etc.) cell.

As used herein, a "host cell" is a cell into which a polynucleotide is introduced that can be propagated, transcribed, or encoded polypeptide expressed. The term also includes any progeny ofthe subject host cell. Host cells include progeny cells which may not be identical to the parental cell since there may be mutations that occur during replication. Nevertheless, such cells are considered to be transformed host cells ofthe invention.

Host cells include but are not limited to microorganisms such as bacteria, yeast, plant, insect and mammalian cells. For example, bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors; yeast transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid); insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus), or transformed animal cell systems engineered for stable expression, are provided by the invention.

For long-term expression of invention polypeptides, stable expression is preferred. Although the invention is not bound or so limited by any particular theory, stable maintenance of expression vectors in mammalian cells is believed to occur by integration ofthe vector into a chromosome of the host cell. The expression vector also can contain a nucleic acid encoding a selectable marker conferring resistance to a selective pressure or an identifiable marker (e.g., β- galactosidse), thereby allowing cells having the vector to be identified, grown and expanded. Alternatively, a selectable marker can be on a second vector which is cotransfected into a host cell with a first vector containing an invention polynucleotide.

A number of selection systems may be used, including, but not limited to the herpes simplex virus thymidine kinase gene (Wigler et al. (1977) Cell 11 :223), hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al. (1962) Proc. Natl. Acad. Sci. USA 48:2026), and the adenine phosphoribosyltransferase (Lowy et al. (1980) Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells respectively. Additionally, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al. (1981) Proc. Natl. Acad. Sci. USA 78:1527); the gpt gene, which confers resistance to mycophenolic acid (Mulligan et al. (1981) Proc. Natl. Acad. Sci. USA 78:2072); the neomycin gene, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al. (1981) J. Mol. Biol. 150: 1); and the hygromycin gene, which confers resistance to hygromycin (Santerre et al. (1984) Gene 30: 147). Recently, additional selectable genes have been described, namely tipB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman et al. (1988) Proc. Natl Acad. Sci. USA 85:8047); and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine,

DFMO (McConlogue (1987) In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, ed.).

As used herein, the term "transformation" means a genetic change in a cell following incoφoration of DNA (e.g., a transgene) exogenous to the cell. Thus, a "transformed cell" is a cell into which, or a progeny of which a DNA molecule (e.g. , PICOT polynucleotide) has been introduced by means of recombinant DNA techniques. Transformation of a host cell with nucleic acid may be carried out by conventional techniques known to those skilled in the art.

The polypeptides, antibodies and polynucleotides ofthe invention are useful as reagents in various assays, including, for example, in detecting the presence of PICOT polypeptides and polynucleotides and related sequences (e.g., nucleotides or related immunogenic fragments).

The polypeptides, antibodies and polynucleotides ofthe invention also are useful to screen for or isolate PICOT and related genes in other organisms as set forth herein. Thus, the present invention provides polypeptides, antibodies and polynucleotides as probes for screening assays are provided. Further provided are antibodies and polynucleotides having a heterologous functional domains, e.g., a detectable label, or a tag.

As used herein, the term "probe" refers to an invention polypeptide, antibody or polynucleotide having a label or detectable moiety which provides a signal. Such detectable moieties included, for example, radioactive isotopes, and fluorescent or chemiluminescent agents, or chemically reactive moieties. As the polypeptides, antibodies and polynucleotides ofthe invention are useful for modulating PKCθ activity, and polypeptides and signaling pathways with which PICOT is associated (e.g., via direct binding or indirect interaction), the invention further provides methods for modulating activity of polypeptides and signaling pathways associated with PICOT in cells. Such polypeptides and associated signaling pathways include, for example, PKCθ, PKCζ, JNK, AP-1 , NF-κB, Trx system, TCR/CD28 signaling cascade, IL-2, ZIP and HEED/WAIT-1. A method ofthe invention includes contacting a cell with an amount of invention polypeptide (PICOT or functional subsequence thereof), antibody or polynucleotide (e.g., PICOT antisense) sufficient to modulate activity or expression ofthe polypeptides or signaling pathways. In one embodiment, the activity is inhibited or prevented. In another embodiment, the activity is increased or promoted.

As the polypeptides, antibodies and polynucleotides ofthe invention are useful for modulating activity of PKCθ, PKCζ, JNK, AP-1, NF-κB, Trx system, TCR/CD28 signaling cascade, IL-2 production in cells, ZIP or HEED/WAIT-1 activities, thereby modulating one or more cellular functions (e.g., T cell activation) associated with these polypeptides and signaling pathways, physiological conditions associated with these polypeptides and signaling pathways can likewise be modulated. Thus, the present invention also provides pharmaceutical formulations comprising PICOT polypeptides, antibodies and polynucleotides, useful for treating a physiological condition or disorder associated with PICOT.

The pharmaceutical compositions ofthe invention will be in a "pharmaceutically acceptable" or "physiologically acceptable" formulation. As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to carriers, diluents, excipients and the like that can be administered to a subject, preferably without excessive adverse side effects (e.g., nausea, headaches, etc.). Such preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) and the like. Preservatives and other additives may also be present such as, for example, antimicrobial, anti-oxidants, chelating agents, and inert gases and the like. Various pharmaceutical formulations appropriate for administration known in the art are applicable in the methods ofthe invention (e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; and The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ). Controlling the duration of action or controlled delivery of an administered composition can be achieved by incoφorating the composition into particles or a polymeric substance such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers. The rate of release ofthe composition may be controlled by altering the concentration or composition of such macromolecules. For example, it is possible to entrap PICOT polypeptide in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drug delivery system. Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The compositions administered ofthe invention can be administered parenterally by injection or by gradual perfusion over time. The composition can be administered via inhalation, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally, and preferably is administered intravascularly. The compositions can be administered in a single dose, or multiple doses. The doses needed for treating a subject having or at risk of having physiological condition or disorder associated with PICOT, or associated polypeptides and signaling pathways, will be sufficient to ameliorate some or all ofthe symptoms ofthe condition or disorder. Appropriate dosages can readily be determined by those skilled in the art (see, e.g., Ansel et al Pharmaceutical Drug Delivery Systems (1990) 5th ed. Lea and Febiger, Gennaro ed.).

Such pharmaceutical formulations are useful in treating a subject having or at risk of having a PICOT associated physiological condition, including, for example, an undesirable stress response. As used herein, the phrase "physiological condition or disorder associated with PICOT" is any undesirable physiological condition or state (acute or chronic) associated with activity or expression of PICOT, or associated polypeptides or signaling pathways (e.g., PKCθ,

PKCζ, JNK, AP-1, NF-κB, polypeptides, Trx system, TCR CD28 signaling cascade, and IL-2, etc., as set forth herein). Physiological conditions or disorders associated with PICOT that can be treated in a method ofthe invention include, for example, undesirable or excessive stress response, and inflammation, for example. PICOT polypeptides and subsequences thereof are useful for identifying a compound that binds or interacts with PICOT polypeptide. Thus, in accordance with the present invention, there are provided methods for identifying a compound that binds or interacts with PICOT polypeptide. In one embodiment, a method ofthe invention includes incubating a test compound with a PICOT polypeptide under conditions allowing binding; and detecting whether the test compound binds the PICOT polypeptide. Binding ofthe test compound identifies the test compound as a compound that binds or interacts with PICOT polypeptide.

In accordance with the present invention, there are also provided methods for identifying a compound that modulates PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide. In one embodiment, a method of the invention includes incubating a test compound with a PICOT polypeptide under conditions allowing binding; and determining

PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide in the presence ofthe test compound. An increase or decrease in PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide identifies the test compound as a compound that modulates PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide. In accordance with the present invention, there are additionally provided methods for identifying a compound that modulates binding or interaction of PICOT polypeptide with a binding or interacting polypeptide. In one embodiment, a method ofthe invention includes incubating PICOT polypeptide with PKCΘ or PKCζ under conditions allowing binding; contacting a test compound with the bound PICOT polypeptide and PKCΘ or PKCζ; and determining whether the test compound increases or decreases binding between PICOT polypeptide and PKCΘ or PKCζ. An increase or decrease in binding identifies the test compound as a compound that modulates binding of PICOT polypeptide with PKCΘ or PKCζ. In one aspect, the test compound is contacted before incubating PICOT polypeptide with the binding polypeptide. In various other aspects, a test compound comprises a library of compounds, a polypeptide sequence (e.g., a chimeric polypeptide or an antibody), a nucleic acid sequence

(e.g., a nucleic acid encoding a polypeptide) or an antisense.

As used herein, the term "incubating" refers to conditions that allow specific binding or interaction. For example, in a method for identifying a compound that binds PICOT, incubating denotes conditions appropriate for binding between PICOT and the test compound. The term "contacting" refers to direct or indirect binding or interaction as set forth herein. Incubating, contacting and specific binding as used herein include in solution, in solid phase, in vitro, in a cell and in vivo.

PICOT binding compounds can be identified or isolated using conventional biochemical methods. For example, a protein that binds PICOT can be identified by incubating PICOT with a partially purified protein or peptide expression library, fractions of cell extracts, whole cell extracts, or mixtures of naturally occurring substances. The bound complex can be separated from uncomplexed PICOT by conventional means well known to one of skill in the art. The presence of a protein bound to PICOT can be detected by size separation or other standard methods, such as non-denaturing gel electrophoresis. The binding protein(s) can then be isolated from the gel, sequenced and, if desired, identified using the methods disclosed herein and further known in the art. Protein(s) that bind or interact weakly with PICOT can be isolated by chemical cross-linking the agent prior to isolating the complex. For example, subjecting cells to an agent that selectively cross-links proteins in close proximity prior to lysis or precipitation can be used to isolate complexes containing weakly interacting proteins. Such cross-linking agents are known in the art and can be chosen in order to minimize non-specific cross-linking. If desired, the proteins so isolated can be identified using methods disclosed herein or known in the art.

Various detection methods can be employed in the methods ofthe invention. For example, to detect an increase in activity or expression, precipitation analysis or confocal microscopy (immunostaining with a PICOT specific antibody) as exemplified herein can be employed. Likewise, precipitation analysis can be used to detect specific binding between

PICOT and PKCΘ or PKCζ and, therefore, is useful for identifying a compound that binds PICOT. As the two-hybrid assay detects interaction between PICOT and PKCΘ, this assay also is useful for detecting PICOT binding or interaction with other polypeptides.

In cells, a compound that modulates PICOT polypeptide activity, or binding of PICOT with PKCΘ or PKCζ, can be identified by treating a cell that expresses, or is made to express, a

PICOT polypeptide with a test compound, and then performing an appropriate activity assay. For example, a two-hybrid assay can be used to detect compounds that modulate binding or interaction between PICOT and PKCΘ or PKCζ, or any other polypeptide whose interaction with PICOT is detectable with this assay. A compound that enhances PICOT and PKCΘ binding or interaction will increase expression of a reporter gene operatively linked to an expression control element responsive to the binding or interaction, whereas a compound that decreases binding or interaction will reduce reporter gene expression. The reporter provides a detection signal (e.g., the amount of transcript or protein product produced by the reporter gene) that corresponds to PICOT interaction with PKCΘ or PKCζ. The signal provided by the reporter gene can be, for example, RNA, protein, an enzymatic activity and the like and can be detected by a variety of methods known in the art, including northern analysis, RNA dot blots, nuclear run-off assays, ELISA or RIA, Western blots, SDS-PAGE alone, or in combination with antibodies that immunoprecipitate the reporter gene product. Expressed products that provide an enzymatic activity or detection signal are preferred and include, for example, β-galactosidase (β-gal), alkaline phosphotase, horseradish peroxidase, luciferase, green fluorescent protein (GFP) and chloramphenicol acetyl transferase (CAT). A yeast two-hybrid assay is a particular example of an assay useful in practicing the methods ofthe invention. Mammalian two-hybrid cell systems can also be used and are commercially available.

Additional methods useful for determining whether there is a change in activity or expression include the PICOT expression assays exemplified herein for JNK, AP-1, NF-κB

(Examples VII and VIII), as well as those known in the art for detecting changes in activity of the Trx system, TCR/CD28 signaling cascade, and IL-2 production. Thus, assays useful in practicing the methods ofthe invention therefore include assays that detect changes in activity of PKCΘ or PKCζ polypeptide, and the polypeptides and signaling pathways with which PKCΘ and PKCζ polypeptides interact, as disclosed herein and known in the art (e.g., JNK, AP-1, NF-κB,

ZIP, HEED/WAIT-1).

Generally a test compound will be found among biomolecules including, but not limited to: polypeptides, peptidomimetics, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Test compounds further include chemical compounds (e.g., small organic molecules having a molecular weight of more than 50 and less than 5,000 Daltons, such as hormones). Candidate organic test compounds comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two ofthe functional chemical groups. The candidate organic compounds often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more ofthe above functional groups. Known pharmacological agents are test compounds and may further include agents subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs, for example. Test compounds can additionally be contained in libraries, for example, synthetic or natural compounds in a combinatorial library. Numerous libraries are commercially available or can be readily produced; means for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides, are known. Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or can be readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Such libraries are useful for the screening of a large number of different test compounds. Moreover, such test compounds additionally can be modified so as to facilitate their identification or purification. Such modifications are well known to the skilled artisan (e.g. , biotin and streptavidin conjugated compounds, polyhistidine and T7 tags).

A compound that modulates PICOT polypeptide activity or expression of a polynucleotide encoding a PICOT polypeptide, or an interaction of PICOT polypeptide with PKCΘ or PKCζ , includes "agonists," which are compounds that stimulate or activate PICOT activity or expression of a polynucleotide encoding a PICOT polypeptide , and "antagonists," which are compounds that inhibit or interfere with PICOT activity or expression of a polynucleotide encoding a PICOT polypeptide . An example of an agonist would be a compound that binds to PICOT and promotes or enhances interaction between PICOT and PKCΘ. An example of an antagonist is a compound that inhibits or prevents interaction between PICOT and PKCΘ . As used herein, the term antagonist also includes compounds that inhibit or prevent PICOT modulation of PKCΘ or PKCζ activity, even without inhibiting PICOT binding to PKCΘ or PKCζ.

The methods ofthe invention for identifying a compound that modulates PICOT polypeptide activity, or PICOT binding to PKCΘ or PKCζ, also are applicable for identifying therapeutic agents useful for treating a physiological condition or disorder associated with PICOT. Such compounds can be formulated into pharmaceutical compositions and used in the therapeutic methods, as described herein.

In accordance with the present invention, there are provided kits useful for practicing the methods ofthe invention. In one embodiment, a kit ofthe invention contains one or more PICOT polypeptides, functional subsequences thereof, antibodies, or PICOT polynucleotides, and a label or packaging insert for treating a physiological condition or disorder associated with

PICOT, in suitable packaging material. As used herein, the term "packaging material" refers to a physical structure housing the components ofthe kit, such as invention polypeptides, antibodies and polynucleotides. The packaging material can maintain the components sterilely, and can be made of material commonly used for such puφoses (e.g., paper, corrugated fiber, glass, plastic, foil, etc.). The label or packaging insert can indicate that the kit is to be used in a method ofthe invention, for example, for treating a physiological condition or disorder associated with PICOT.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, suitable methods and materials are described herein.

All publications, patents, other references, GenBank citations and ATCC citations mentioned herein are incoφorated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages ofthe invention will be apparent from the following detailed description, and from the claims. The invention is further described in the following examples, which do not limit the scope ofthe invention(s) described in the claims.

EXAMPLES

Example I

This example describes the materials and methods used in the Examples described herein. Antibodies and Expression Plasmids- he anti-CD3 monoclonal antibody (mAb) was affinity-purified from culture supematants of the OKT3 hybridoma as described (Liu (1997) J.

Biol. Chem. 272, 168-173). An anti-human CD28 mAb was obtained from Pharmingen. The PKCθ-specific mAb and rabbit polyclonal antibody were from Transduction Laboratories (Lexington, KY) and Santa Cmz Biotechnology, Inc. (Santa Cruz, CA), respectively. The anti-c- Myc mAb (9E10) was purified from culture supematants of the corresponding hybridoma by protein A-Sepharose chromatography, and the anti- hemagglutinin (HA) mAb (12CA5) was from

Boehringer Mannheim (Indianapolis, IN)- Polyclonal goat anti-JNK and rabbit anti-ERK2 antibodies were obtained from Santa Cmz Biotechnology, and phospho-c-Jun- or phospho- ERK2-specific antibodies were from New England Biolabs (Beverly, MA). Fluorescein isothiocyanate (FITC)-coupled secondary antibodies were from Pierce (Rockford, IL). A polyclonal rabbit anti-PICOT antisemm was generated using standard protocols. In brief, a peptide comprising amino acids 90-108 of the deduced human PICOT sequence (SEQ ID NO:l) was coupled to KLH and injected into 2 rabbits. Both antisera recognized a protein band with the predicted electrophoretic mobility of PICOT (38 kDa) in immunoblots of cell lysates. For immunohistochemistry, a portion of the antisemm was affinity-purified on a Sepharose-coupled synthetic peptide column.

The full-length PKCθ cDNA with a C-terminal hexahistidine tag in the pEF mammalian expression vector has been described previously (Meller et al. (1996) Mol. Cell Biol. 16, 5782- 5791). An HA-tagged constitutively active calcineurin mutant consisting of the catalytic subunit of calcineurin (CnA), from which the calmodulin-binding and the autoinhibitory domains were deleted (CnAΔCaM-AI) was a generous gift from Dr. M. Karin (UCSD), and was described

(Werlen et al (1998) EMBO J. 17, 3101-3111). To construct human PICOT expression vectors, the corresponding full-length cDNA or fragments encoding the N- (residues 1-146) or C- (residues 133-335) terminal regions of PICOT were subcloned into pEF, and an N-terminal hexahistidine tag followed by a subsequent HA tag was added at the 5' end of the cDNA. HA- tagged JNKl and c-Myc-tagged ERK2 were cloned in pcDNA3 (Villalba et al. (1998) Submitted or publication). The AP-1 and NF-kB luciferase reporter plasmids were obtained from M. Karin.

Yeast Two-Hybrid Screen—The, yeast two-hybrid system used in this study was provided by E. A. Golemis (Fox Chase Cancer Center, Philadelphia, PA), and has been described previously (Golemis et al. (1997) In: Current Protocols in Molecular Biology, Ausubel et al. eds)

3, 20.1.1-20.1.8, Wiley Interscience, New York). cDNAs encoding full-length PKCθ or fragments including its regulatory (amino acids 1-378) or catalytic (amino acids 379-706) domains were subcloned into pGilda (Witte et al. (1997) J. Biol. Chem. 272, 22243-22247) to generate in-frame fusion proteins with the LexA DNA-binding domain. These baits were used to screen a Jurkat T cell cDNA library (Witte et al. (1997) supra). To map the PKCθ-binding domain of human PICOT, the cDNAs encoding the N- or C-terminal fragments of PICOT were subcloned into the activation domain fusion vector pJG4-5 (Golemis et al. (1997) supra).

Cell Culture and Transfection— Simian vims 40 large T antigen (TAg)-transfected human leukemic Jurkat T Jurkat-TAg cells were grown in RPMI 1640 medium (Life Technologies, Inc., Rockville, MD) supplemented with 10 mM HEPES, pH 7.5, 10 mM MEM non-essential amino acids, 1 mM sodium pymvate, 10% fetal bovine semm and antibiotics. For expression of recombinant proteins, cells were transfected for 48 h with appropriate amounts of plasmids (usually 3-20 μg total) by electroporation as described (Liu (1997) supra; Meller et al. (1996) supra). In each experiment, cells in different groups were transfected with the same total amount of plasmid DNA by supplementing expression vector DNA with the proper amounts of the corresponding empty vector. Where indicated, the cells were stimulated with anti-CD3 and/or anti-CD28 antibodies, phorbol myristate acetate (PMA; 100 ng/ml) and/or ionomycin (1 μg/ml), or irradiated with UV (312 nm) for one min at room temperature using a transilluminator (FBTI- 88; Fisher Scientific) and cultured for an additional h. Cells were lysed in 1 x NP-40 Lysis buffer (1% NP-40/20 mM Tris-HCl, pH 7.5/100 mM NaCl/5 mM NaPiP/5 mM NaF/5 mM Na₃VO₄) supplemented with protease inhibitors (Boehringer Mannheim) for 10 min on ice. The insoluble material was removed by centrifugation.

Glutathione-S-transferase (GST) Fusion Proteins and In Vitro Binding Assays— The cDNAs encoding full-length human PICOT or its N-terminal or C-terminal fragments were subcloned into the bacterial GST fusion vector pGEX-5X-l (Pharmacia, Kalamazoo, MI).

Expression and purification of the GST fusion proteins was performed as described (Liu et al. (1996) J. Biol. Chem. 271, 14591-14595). Cell lysates (about 1 x 10⁷ cells) were incubated with 10 μg GST fusion proteins coupled to 40 μl glutathione-agarose for 2 h at 4°C. The binding mixtures were washed in 1 x NP-40 lysis buffer and analyzed by SDS-PAGE and Western blotting. RT-PCR and Northern Blotting-Multi Tissue cDNA Panels (Clontech, Palo Alto, CA) were screened by RT-PCR with a pair of PICOT-specific primers according to the instmctions of the manufacturer. For amplification of a 1-kb PICOT fragment, 35 cycles were used, and the GAPDH control was amplified using 25 PCR cycles. 20 μg of total RNA from Jurkat cells was prepared using standard procedures. The PICOT plasmid isolated from the yeast two-hybrid screen or a full-length GAPDH cDNA were used for generation of ³²P-labeled probes using a

Rediprime II kit (Amersham, Piscataway, NJ)

Subcellular Fractionation —Jurkat cells were lysed and separated into a membrane, cytosol, and detergent-insoluble fractions as described (Meller et al (1996) supra).

Immunofluorescence— Transfected or nontransfected Jurkat-TAg cells were left unstimulated, or stimulated with 100 ng/ml PMA for 10 min at 37 C. Cells were then spun down, washed with cold PBS, fixed with 3.7% paraformaldehyde, and permeabilized in 0.05% saponin. Transfected Cells were then stained with a polyclonal rabbit anti-PICOT antibody and an anti- PKCθ mAb. Nontransfected cells were stained with a polyclonal anti-PKCθ antibody and Alexa 488 (Molecular Probes, Eugene, OR)-conjugated, affinity-purified anti-PICOT antibody. Samples were then incubated with FITC-conjugated secondary antibodies (Pierce) or Alexa 594

(Molecular Probes), respectively, and were subsequently washed 4 times with 1% BS A in PBS. After the final wash, the cells were mounted on glass slides using a drop of Aqua-Poly/mount (Polysciences, Inc., Warrington, PA). Samples were viewed with a Plan-Apochromat 63X lens on a Nikon microscope. Images were taken using a Bio Rad MRC 1024 laser scanning confocal imaging system.

Immunoprecipitαtion and Immunoblotting— Lysates (1-2 x 10 cells) were mixed with antibodies (1-2 μg) for 2 h, followed by addition of 40 μl protein A/G Plus-Sepharose beads (Santa Cmz Biotechnology) for an additional h at 4°C. Immunoprecipitates were washed 2x with lx NP-40 lysis buffer and 2x with phosphate-buffered saline (PBS; pH 7.2). After boiling in 20 μl 2x Laemmeli sample buffer, samples were subjected to SDS-PAGE and electrotransferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were immunoblotted with the indicated primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. Bands were visualized by chemiluminescence (Amersham). When necessary, membranes were stripped by incubation in 62.5 mM Tris-HCl, pH 6.7/100 mM 2- mercaptoethanol/2% SDS for 1 h at 65°C, washed and then reprobed with other antibodies as indicated.

In vitro Kinase Assays— In vitro kinase assays of immunoprecipitated JNK or ERK2 were conducted as described (Villalba et al. (1998) supra). Briefly, washed JNKl or ERK2 immunoprecipitates were assayed using 2 μg GST-c-Jun fusion protein or myelin basic protein (MBP) as substrates, respectively, in 20 μl of JNK or ERK2 kinase buffers containing 3 μCi [γ- ³²P]ATP (30 Ci/mmol, Amersham). Kinase reactions were incubated for 20 min at 30°C with gentle shaking, and were stopped by addition of 20 μl 2x Laemmli buffer. Proteins were resolved by SDS/13% PAGE, transferred to nitrocellulose and subjected to autoradiography. Substrate phosphorylation was quantified by phosphoimaging (Storm 860; Molecular Dynamics, Sunnyvale, CA) analysis. The nitrocellulose membranes were routinely reprobed with anti-JNK or -ERK2 antibodies to confirm equal expression levels ofthe immunoprecipitated kinases.

Reporter Assays— Transfected Jurkat-TAg cells were harvested, washed twice with PBS and lysed in 100 μl of lysis buffer (100 mM KPO₄, pH 7.8/1 mM dithiothreitol/0.5% Triton X- 100) for 10 min at room temperature. The lysates were then centrifuged (15,000 x g, 5 min at 4 C). Fifty μl of the supernatant were mixed with 100 μl of assay buffer (17.5 mM glycilglycine, pH 7.8; 10 mM MgCl₂/5 mM ATP/0.135 mM coenzyme A 0.235 mM luciferin), and the luciferase activity determined in a luminometer (Monolight 2010; Analytical Luminescence Laboratory, Sparks, MD). The protein content was determined using the BioRad Protein assay (Bio Rad). The final results were expressed as arbitrary relative luciferase units (RLU) per μg protein.

Example II

This example describes the identification of a polypeptide that interacts with PKCθ in cells.

To identify proteins which interact with PKCθ, three independent yeast two-hybrid screens were performed using as bait 1) full-length PKCθ, 2) PKCθ regulatory domain, and 3) PKCθ catalytic domain. The baits were screened for binding to polypeptides encoded by a Jurkat T lymphoma cDNA library, which had been fused to a cDNA encoding a transcription activation domain (Golemis et al. (1997) supra). Since expression of catalytically active forms of PKCθ as LexA fusion proteins was toxic to yeast (data not shown), a point-mutated cDNA encoding a catalytically inactive PKCθ (K409R) was used in the two-hybrid screening. Approximately 5 x 10⁷ independent clones were screened with each of the three baits.

No clones growing on selective medium were obtained with the isolated regulatory or catalytic domains of PKCθ, respectively (Table 1). Screening with the catalytically inactive, full-length PKCθ bait resulted in 17 primary positive colonies which grew on selective medium and were β- galactosidase-positive when tested in a filter lift assay (Table 1). Thus, the observed interaction in yeast requires full-length PKCθ, but does not appear to depend on its intact catalytic activity.

TABLE I

Specific interactions between PKCθ and PICOT in the yeast two-hybrid system"

DNA-binding domain Activation domain Growth on Ura^" His^" Colony color hybrid "bait" hybrid "prey" Tφ^" Leu^" medium

PKCΘ K409R _ _ white

PKCΘ reg. dom. (1-378) - - white

PKCθ cat. dom. (379-706) - - white

PKCθ K409R PICOT +++ blue

PKCθ K409R PICOT-N (1-146) - white

PKCθ K409R PICOT-C (133-335) - white

PKCθ reg. dom. (1-378) PICOT - white

PKCθ reg. dom. (1-378) PICOT-N (1-146) - white

PKCθ reg. dom. (1-378) PICOT-C (133-335) - white

PKCθ cat. dom. (379-706) PICOT - white

PKCΘ cat. dom. (379-706) PICOT-N (1-146) - white

PKCθ cat. dom. (379-706) PICOT-C (133-335) - white

Bicoid PICOT - white c-Myc PICOT - white

" EGY48 yeast cells were cotransformed with expression vectors encoding various LexA DNA-binding domain and activation domain chimeric proteins. Activation of the leu2 reporter gene was monitored by growth on leucine-deficient medium, and the activity of the lacZ reporter gene was monitored using a filter assay.

Ten of the positive, PKCθ-interacting clones were found to contain cDNAs which specifically interacted with the bait. Partial sequence analysis revealed that these clones encoded identical cDNAs. The two longest cDNAs (-1,250 base pairs in length) were sequenced, and found to contain an open reading frame (ORF) encoding a putative protein of 335 amino acids (FIG. 1A). Since the putative polypeptide encoded by the open reading frame contained a Trx- homologous domain (see below), it was named PKC-interacting cousin of Trx (PICOT). Northern blot analysis using one of the yeast two-hybrid clones as a probe indicated the presence of a 1.5 kb mRNA expressed in a preparation of total RNA from Jurkat T cells

(FIG. IB), suggesting the isolated clones contained the complete ORF. A rapid amplification of cDNA ends (RACE) PCR of a human thymus cDNA library failed to reveal additional upstream sequences, thus confirming that the isolated cDNAs were full-length.

A search of the Genbank database of expressed sequence tags (ESTs) for sequences homologous to PICOT identified putative partial ORFs; human (accession no. h59799), mouse (accession no. aa009010) and rat (accession no. aa866363). Using the BLAST algorithm, homologous sequences were also identified in S. cerevisiae (Swissprot ye04_yeast), E. coli (Swissprot ydhd_ecoli), H. influenzae (Swissprot ydhd_haein), C. elegans (Genbank g3217992) and Arabidopsis thaliana (Genbank g3335374) (see FIG. 2).

Example III

This example describes the structure, expression and tissue distribution of PICOT. The ORF of the isolated PICOT cDNA encodes a putative protein with a predicted molecular weight of about 37.5 kDa (FIG. 1A). The codon for the first methionine is surrounded by a consensus Kozak sequence, but is not preceded by an in-frame stop codon.

To confirm the expression of the putative protein encoded by the cDNA, rabbit antisera were generated against a synthetic peptide corresponding to amino acids 90-108 of PICOT, a hydrophilic sequence with a high surface probability. Immunoblotting of lysates from E. coli expressing full-length PICOT as a GST fusion protein (GST-PICOT), or from untransfected Jurkat cells, revealed expression of an immunoreactive ~74-kDa protein in E. coli, and a -38- kDa protein in Jurkat cells, consistent with the predicted size of PICOT based on the cDNA. This antisemm was used in subsequent expression studies. Tissue distribution of PICOT was analyzed by RT-PCR using a commercial cDNA panel derived from different human tissues. Our findings revealed that the expression of PICOT mRNA was more ubiquitous than that of PKCθ but, nevertheless, it was not expressed in all tissues. In particular, PICOT was abundant in heart, spleen, and testis, with low but detectable expression in the other tissues, including the thymus and peripheral blood leukocytes (PBL). Expression of PICOT mRNA in the lung, placenta, colon and small intestine was very low

(FIG. ID). Example IV

This example describes the sequence identity analysis of PICOT.

A search of the GenBank nucleotide database revealed a 29% amino acid sequence identity and an additional 11% similarity between the N-terminal region of PICOT (residues 12-

143) and Trx family of proteins (FIGS. 2A, B). It is noted that the Trx-homology domain of PICOT lacks the conserved Cys-Gly-Pro-Cys motif which is important for catalytic activity (Nakamura et al. (1997) Annu. Rev. Immunol. 15, 351-369), and contains instead an Ala-Pro- Gln-Cys motif. Since the Trx system, highly conserved throughout evolution, plays an important role in regulating the intracellular redox state which is critical for both cell viability and proliferation (Nakamura et al. (1997) supra; Powis et al. (1994) Oncol. Res. 6, 539-544; Holmgren et al. (1995) Methods Enzymol 252, 199-208), actions that are mediated in part by regulation of the transcription factors NF-κB and AP-1 Holmgren et al. (1995) supra), PICOT can have a role in modulating the Trx system. Analysis ofthe protein sequence of PICOT against sequences of putative proteins (amino acid sequences derived from genome sequencing projects) revealed a highly conserved sequence motif of 84 amino acids. This motif, termed PICOT-homology (PIH) domain, has not previously been recognized and is highly conserved in evolution from plants to mammals and represents a novel, previously unknown domain found in all organisms searched, including nematodes, yeast, bacteria, vimses and plants (FIG. 2C). The human PICOT and putative derived mouse and rat proteins display two tandem repeats of this domain (FIGS. 2 A, C), whereas homologous proteins present in lower organisms contain only a single repeat. The high degree of conservation of this domain suggests that it plays an important, yet to be identified, role in cellular functions. Thus, PICOT shows a discrete domain stmcture, consisting of an N-terminal Trx-like domain followed by two novel PICOT-homology domains (FIG. 2A).

Example V

This example describes studies demonstrating specific binding of PKCθ with PICOT in intact T cells and in vitro. This example also describes identification of a PICOT subsequence that binds with PKCθ. To confirm the two-hybrid system observations that PICOT binds with PKCθ, and to analyze its specificity for PKCs, it was first ascertained whether these two proteins bind in intact T cells. Jurkat-TAg cells were cotransfected with PKCθ plus HA epitope-tagged PICOT expression vectors. When lysates from these cells were immunoprecipitated with an anti-HA mAb, PKCθ was found to coimmunoprecipitate with PICOT (FIG. 3A). An unrelated control antibody did not precipitate either of these two proteins. This interaction was confirmed by precipitating T cell lysates with a GST-PICOT fusion protein in vitro. Incubation of the recombinant protein, but not the control GST protein, with lysates from Jurkat cells transfected with PKCθ, PKCα or PKCζ, followed by immunoblotting with antibodies against the respective PKC isoforms, revealed that PICOT bound PKCθ and, to a lesser extent, PKCζ (FIG. 3B). Under the same conditions, no interaction with PKCα was detected. These results show that PICOT displays selectivity with regard to interaction with PKC isoforms.

To further define the stmctural basis for interaction of PICOT with PKCθ, GST fusion proteins having 1) a PICOT N-terminal fragment having its Trx-like domain (residues 1-146) and 2) a PICOT C-terminal fragment having the two tandem PICOT-homology domains

(residues 133-335) were expressed as recombinant proteins and precipitated Jurkat cell lysates from PKCθ-transfected cells and analyzed for co-precipitating protein. As shown in FIG. 3C, N- terminal, Trx-like domain of PICOT was sufficient for PKCθ binding, although it appears that its affinity is slightly less than full length PICOT. In contrast, the C-terminal region of PICOT did not display any detectable binding to PKCθ. In yeast, only full-length PICOT associated with

PKCθ (Table 1), suggesting that the interaction affinity may be less in yeast. This difference may reflect a favorable conformation of PICOT for this interaction in vitro, where it was expressed as a GST fusion protein, compared to yeast, where PICOT was expressed as a chimeric protein with a transcription activation domain. These results establish the association of PICOT with PKCθ in intact T cells and in vitro, which is conferred by the N-terminal, Trx-homology domain of PICOT. Since PICOT interacts with kinase-inactive PKCθ and is not phosphorylated by PKCθ in vitro, PICOT therefore does not appear to represent a PKCθ substrate. Furthermore, PICOT associated not only with PKCθ, but also with PKCζ. PICOT's interaction with PKCs is not promiscuous since it did not associate with PKCα. Coimmunoprecipitation of some endogenous proteins was not always reproducible, which may reflect some conditions unfavorable for the maintenance of association, or the requirement of other cellular factors (e.g., lipids or adaptor proteins) for optimal interaction between these two proteins. Alternatively, the association may take place in specific sites within the cell, and only under specific conditions.

Example VI

This example describes studies showing the intracellular localization of PICOT and PKCΘ. To determine the subcellular localization of PICOT, Jurkat T lymphoma cells were fractionated and analyzed the cellular distribution of the protein. As shown in FIG. 4, PICOT was almost exclusively localized in the cytosol, with additional, very low expression in the membrane fraction, but no detectable expression in the detergent-insoluble cellular fraction. This pattern was not appreciably altered following stimulation ofthe cells with phorbol ester plus Ca²⁺ ionophore.

In order to determine the relative localization of PICOT vs. PKCθ in situ, transfected and untransfected Jurkat cells were stained with PICOT- and PKCθ-specific antibodies, and analyzed by confocal microscopy (FIG. 5). The images are representative of about 50 cells for each group. In cells transfected with both PICOT and PKCθ the proteins colocalized to a distinct cytoplasmic area under the plasma membrane (panel a and b), and their colocalization was evident when the two individual images were overlaid (panel c). PMA stimulation caused translocation of both PICOT and PKCθ to a more extended membrane (or sub-membrane) area, with colocalization ofthe two proteins still evident (panel d, e and f). The localization of the relevant endogenous protein in untransfected cells was also analyzed. Due to lower protein expression it was necessary to use a polyclonal anti-PKCθ antibody, which produces a less satisfactory staining than that obtained with the monoclonal antibody used in the transfected cells. Nevertheless, endogenous PICOT and PKCθ appeared to colocalize in the same area of the cell, with an overall staining pattern similar to the one observed in the transfected cells (panel g, h and i). In sum, analysis by confocal microscopy revealed overlap between the intracellular localization of PICOT and PKCθ, even in untransfected cells.

Example VII This example describes studies showing that PICOT inhibits JNK activation.

As PICOT was identified on the basis of its interaction with PKCθ and the stress- activated protein kinase JNK represents a target of PKCθ in the TCR/CD28 signaling pathway (Ghaffari-Tabrizi et al. (1999) supra; Werlen et al. (1998) supra), the effect of transient PICOT overexpression on the activation of JNK was studied. Jurkat-TAg cells were cotransfected with different combinations of expression plasmids encoding PICOT, PKCθ and/or constitutively active calcineurin (CnAΔCaM-AI). The latter plasmid was used since calcineurin cooperates with PKCθ in the activation of JNK and the IL-2 promoter (Werlen et al. (1998) supra).

Transient overexpression of PKCθ caused activation of the cotransfected epitope-tagged JNK reporter, and this effect was somewhat augmented by CnA coexpression (FIG. 6A, lanes 3 and 5 vs. lane 1 in the two upper panels). When the cells were additionally cotransfected with a

PICOT expression vector, the PKCθ- or PKCθ/CnA-induced JNK activation was significantly reduced (FIG. 6A, lanes 4 and 6). Overexpression of PICOT alone also seemed to reduce basal JNK activity (lane 2). Densitometric analysis of the phospho-c-Jun bands revealed that PICOT reduced the PKCθ- or PKCθ/CnA-induced JNK activation by 70% and 55%, respectively. These studies were repeated two additional times with consistent results. Immunobloting of immunoprecipitated JNK with a specific antibody (anti-JNK) confirmed that all groups expressed a similar level of transfected JNK (FIG. 6A, third panel from the top). Similarly, immunoblottting of cell lysates from the same cells with antibodies specific for PKCθ, PICOT or HA-CnA confirmed overexpression of the corresponding proteins in the cells (FIG. 6A, three bottom panels).

In order to study selectivity of this inhibitory effect, the effect of transient PICOT overexpression on the activation of another MAP kinase, ERK2 was assessed, using similar in vitro immune complex kinase assays. ERK2 can be non-selectively activated by both PKCθ and PKCα (FIG. 6B, lanes 3 and 5 vs. lane 1 in the upper panel), consistent with previous reports. Coexpression of PICOT alone did not reduce ERK2 activity and, in some experiments, appeared to enhance it. Similarly, coexpression of PICOT with PKCθ (lane 4) or PKCα (lane 6) did not inhibit the PKC-induced ERK2 activity. Immunoblotting with an ERK2-specific antibody confirmed equivalent expression levels of ERK2 in most groups, with the exception of the two PKCθ-transfected groups, which displayed lower ERK2 expression, thereby making the PKCθ- induced ERK2 activation even more pronounced than the apparent level. In these studies, PKC and/or PICOT were properly overexpressed in the transfected cells. Thus, these findings indicate that PICOT inhibition of PKC-induced MAP kinase activation is selective for JNK and, furthermore, PICOT alone can induce ERK, but not JNK, activation.

Next, the effect of PICOT overexpression on JNK activation induced by several stimuli, including a physiological stimulus provided by anti-CD3/CD28 antibodies was determined. Stimulation of Jurkat cells with anti-CD3/CD28 antibodies, a combination of PMA plus ionomycin, or UV irradiation, all induced marked activation of the cotransfected JNKl reporter when compared to the unstimulated cells (FIG. 6C, lane 1; see also FIG. 7). Coexpression of PICOT in the same cells reduced the basal or anti-CD3/CD28-induced JNK activity by 80%, but had a much smaller effect on the PMA/ionomycin- or UV-induced kinase activity (FIG. 6C, lane 2; see also FIG. 7). Furthermore, the inhibitory effect of PICOT overexpression was very similar to that caused by overexpressing a dominant-negative PKCθ (Θ-K/R) mutant (FIG. 6C, lane 3), supporting the notion that PICOT exerts its inhibitory activity by interfering with the cellular function of PKCθ.

This data indicating that PICOT regulates cellular functions mediated by PKCθ or physiological stimuli in T cells indicates that the interaction between PICOT and PKC is physiologically relevant. Thus, the activation of two important elements in the TCR/CD28 signaling cascade leading to IL-2 production, i.e., JNK and AP-1 (in the Example that follows), both of which are selectively activated by PKCθ (Baier-Bitterlich et al. (1996) supra; Ghaffari- Tabrizi et al. (1999) supra; Werlen et al. (1998) supra), was inhibited by PICOT. This effect was selective and did not reflect a general inhibition of cellular functions, since the activation of another MAP kinase, ERK2, was not inhibited by PICOT. Example VIII

This example describes studies showing that PICOT inhibits activation of AP-1 and NFKB in T Cells.

Among several PKC isoforms, PKCθ functions as a selective AP-1 activator via a Ras- dependent pathway (Baier-Bitterlich et al. (1996) supra). Since JNK positively regulates AP-1 activity by phosphorylating two regulatory serine residues in the activation domain of c-Jun (Su et al. (1994) Cell 11, 727-736), it was assessed whether PKCθ-mediated activation of AP-1 was also inhibited by PICOT.

The effect of transient PICOT overexpression on the activation of an AP-1 reporter plasmid was studied. PMA (100 ng/ml) stimulation or transient overexpression of PKCθ caused a marked increase of AP-1 activity, and the constitutively active plasmid (PKCθ-A/E) was more active than the wild-type kinase in that regard (FIG. 8A). When the cells were cotransfected with increasing amounts of the PICOT expression plasmid (verified by immunoblotting), a dose- dependent inhibition of the basal or PKCθ-induced AP-1 activity was observed. Five μg of the transfected PICOT plasmid reduced the basal activity of AP-1 by about 90%. The activities induced by wild-type or constitutively active PKCθ (in the absence of PMA stimulation) was reduced by about 95 and 60%, respectively. Activation of AP-1 by other endogenous PKC isoforms that are not sensitive to the inhibitory effect of PICOT, e.g., PKCα (see FIG. 3B), may account for the observation that PICOT does not appreciably inhibit AP-1 activity in PMA- stimulated cells.

Further analysis of the stmctural requirements for PICOT inhibition of AP-1 activation revealed that while native PICOT was capable of inhibiting AP-1 activation induced by constitutively active PKCθ by about 60%, particular N- and C-terminal fragments of PICOT displayed minimal inhibitory activity (FIG. 8B). In order to determine the effect of transient PICOT overexpression on the activity of another transcription factor induced by stress signals, the activation of the NF-κB transcription factor by the combined stimulation of anti-CD3 plus anti-CD28 antibodies was studied (FIG. 8C). In the absence of PICOT coexpression, this antibody combination induced an about 5-fold activation of NF-κB. Transient expression of a lower dose of PICOT (5 μg) caused a minimal reduction of activity, but at the higher dose (10 μg plasmid DNA), NF-κB activity was reduced by about 63% (FIG. 8C). Selectivity of this inhibitory effect is indicated by the fact that the PMA-induced activation of NF-κB was not inhibited by PICOT. As with AP-1, the observed inhibition appears to require full-length PICOT. These studies were repeated four additional times with consistent results.

These results indicate that the JNK/ AP-1 pathway is not the only target for inhibition by PICOT. The JNK/ AP-1 and NF-κB pathways are commonly activated in response to stress signals and inflammatory stimuli (Verma et al. (1995) Gene Dev. 9, 2723-2735; Baeuerle et al.

(1996) Cell 87, 13-20; Karin (1997) N. Engl J. Med. 336, 1066-1071; Karin et al. (1997) Curr. Opin. Cell Biol 9, 240-246; Muller et al (1997) Methods 11, 301-312; O'Neill et al. (19898) J. Leuk. Biol. 63, 650-657). Thus, PICOT regulates stress-induced signaling pathways in other cell types and organisms.

Although the exact mechanism by which PICOT inhibits the activation of the JNK/ AP-1 pathway and NF-κB remains to be elucidated, the homology of PICOT to Trx is of particular interest. The evolutionary conserved Trx system has evolved to protect cells from damage mediated by reactive oxygen species (ROS) generated as part of a cellular defense mechanism against invading pathogens (Nakamura et al. (1997) supra; Powis et al. (1994) supra; Holmgren et al. (1995) supra). Various cellular insults, i.e., mitogens, inflammatory stimuli, UV or ionizing radiation, ischemia, phorbol ester and hydrogen peroxide upregulate the expression of Trx and induce its translocation to the nucleus. Trx exerts both extracellular and intracellular functions, including its extracellular ability to protect cells from tumor necrosis factor (TNF)- or Fas-mediated apoptosis (Nakamura et al. (1997) supra). Trx is known to promote the DNA binding and transcriptional activities of AP-1 and NF-κB as well as the activity of the estrogen receptor (Nakamura et al. (1997) supra; Hayashi et al. (1997) Nucleic Acids Res. 25, 4035-4040; Hirota et al. (1997) Proc. Natl Acad. Sci. USA 94, 3633-3638). It mediates these effects by reducing cysteine residues in the p50 subunit of NF-κB, the two components of AP-1, i.e., c-Jun and c-Fos, and Ref-1, an endonuclease which participates in AP-1 activation (Nakamura et al.

(1997) supra; Hayashi et al. (1997) supra; Hirota et al. (1997) supra). These modifications are necessary for the binding of these transcription factors to their cognate DNA sequences in the promoter regions of various genes (Nakamura et al. (1997) supra).

The production of reactive oxygen species (Muller et al. (1997) Methods 11, 301-312) and the concommitant induction of genetic programs which mediate defense mechanisms against pathogenic agents are highly conserved in evolution, including in plants (Fearon et al. (1996) Science 272, 50-53; Medzhitov et al. (1997) Cell 91, 295-298; Whitham et al. (1994) Cell 78, 1101-1115). The conservation of Trx system (Nakamura et al. (1997) supra; Powis et al. (1994) supra; Holmgren et al. (1995) supra) and the PKC superfamily (Kruse et al. (1996) J. Mol Evol 43, 374-383; Mellor et al. (1998) Biochem. J. 332 (Pt. 2), 281-292) during evolution, and the findings that PKC homologues play a role in plant defense mechanisms against viral pathogens

(Sokolova et al. (1997) FEBS Lett. 400, 201-205; Subramaniam et al. (1997) Plant Cell 9, 653- 664) shows that an axis consisting of PICOT/PKC/Trx homologues plays a general and well conserved important regulatory role in cellular functions. Thus, because PICOT overexpression inhibits the activation of AP-1 and one of its upstream activators (JNK), as well as NF-κB, PICOT likely can modulate Trx system activity and, therefore, the biological significance of

PICOT and its putative homologues extends well beyond its role in T cell activation.

Example IX

This example describes the identification of a polypeptide that interacts with PICOT. Using a two-hybrid yeast system in which full-length PICOT was fused with the LexA DNA- binding domain in the pGilda vector and used as a "bait," a human Jurkat T cell library was screened for interacting proteins. Twenty million clones were screened, and 20 positive clones were selected for further characterization. Partial sequencing indicated that 90% of these clones encoded the same sequence. Sequence analysis demonstrated that the isolated cDNA encoded the HEED/WAIT-1 polypeptide, which has been shown to bind the matrix protein of HIV-1.

Thus, as with PKCΘ and PKCζ, PICOT likely modulates activity of HEED/WAIT-1 polypeptide and associated signaling pathways, including proliferation or pathology of AIDS.

Claims

What is claimed is:

1. An isolated or recombinant PKC-interacting cousin of Trx (PICOT) polypeptide, said polypeptide having 70% or more identity to the sequence set forth in SEQ ID NO:l.

2. The polypeptide of claim 1 having at least 80% identity with the sequence set forth in SEQ ID NO: 1.

3. The polypeptide of claim 1 having at least 90% identity with the sequence set forth in SEQ ID NO: 1.

4. The polypeptide of claim 1 having at least 95% identity with the sequence set forth in SEQ ID NO: 1.

5. The polypeptide of claim 1 comprising the sequence set forth in SEQ ID NO.T.

6. A functional subsequence ofthe polypeptide claim 1.

7. The functional subsequence of claim 6 having one or more amino acid modifications of SEQ ID NO: 1.

8. The functional subsequence of claim 7, wherein said modification comprises an amino acid substitution, deletion or insertion.

9. The functional subsequence of claim 8, wherein said substitution comprises a conservative amino acid substitution.

10. The functional subsequence of claim 6, said subsequence having at least about 7 amino acids.

11. The functional subsequence of claim 6, said subsequence having from about 5 to about 100 amino acids.

12. The functional subsequence of claim 6, said subsequence having from about 10 to about 50 amino acids.

13. The polypeptide of claim 1 characterized as having an apparent molecular weight of about 37 kDa.

14. The polypeptide or a functional subsequence of claim 1 that specifically binds to PKCΘ or PKCζ polypeptide.

15. The polypeptide of claim 1 or a functional subsequence thereof that inhibits JNK, AP-1 or NFKB activity, or production of IL-2.

16. The polypeptide of claim 1 or a functional subsequence thereof further comprising a heterologous functional domain.

17. The polypeptide of claim 16, wherein said heterologous domain comprises an amino acid sequence.

18. The polypeptide of claim 16, wherein said heterologous domain comprises a nucleic acid binding domain.

19. The polypeptide of claim 16, wherein said heterologous domain comprises a transcriptional activation domain.

20. The polypeptide of claim 16, wherein said heterologous domain comprises a tag.

21. The polypeptide of claim 16, wherein said heterologous domain comprises a detectable label.

22. An antibody or fragment thereof that specifically binds to the polypeptide of claim 1 or an immunogenic fragment thereof.

23. The antibody of claim 22, wherein the polypeptide comprises the sequence set forth in SEQ ID NO: 1.

24. A method for producing an antibody that specifically binds to the polypeptide of claim 1, comprising administering a polypeptide of claim 1, or a polynucleotide encoding same, to an animal in an amount sufficient to produce an antibody that specifically binds to the polypeptide of claim 1.

25. An isolated or recombinant polynucleotide encoding a polypeptide of claim 1 or a subsequence thereof, with the proviso that the polynucleotide is distinct from Genbank accession numbers h59799, aa009010 and aa866363.

26. The isolated polynucleotide of claim 25, wherein the polypeptide has the sequence set forth in SEQ ID NO: 1.

27. An isolated or recombinant polynucleotide encoding a polypeptide having 70% or more identity to a polypeptide corresponding to amino acids 138 to 335 of SEQ ID NO:l, or a subsequence thereof.

28. An isolated or recombinant polynucleotide selected from: a) SEQ ID NO:2; b) SEQ ID NO:2, wherein one or more T's are U; c) nucleic acid sequences complementary to a) or b); and d) subsequences of either a), b) or c) that are at least 15 base pairs long.

29. An isolated or recombinant polynucleotide that hybridizes under moderately stringent conditions to the sequence set forth in SEQ ID NO:2, with the proviso that the polynucleotide is distinct from Genbank accession numbers h59799, aa009010 and aa866363.

30. An isolated or recombinant polynucleotide that hybridizes under highly stringent conditions to the sequence set forth in SEQ ID NO:2, with the proviso that the polynucleotide is distinct from Genbank accession numbers h59799, aa009010 and aa866363.

31. An isolated or recombinant polynucleotide that hybridizes under moderately stringent conditions to the nucleotide sequence corresponding to nucleotides 421 to 1005 of SEQ ID NO:2.

32. An isolated or recombinant polynucleotide that hybridizes under highly stringent conditions to the nucleotide sequence corresponding to nucleotides 421 to 1005 of SEQ ID NO:2.

33. The polynucleotide of any of claims 28 to 32, wherein the polynucleotide has less than about 500 nucleotides.

34. An isolated or recombinant polynucleotide comprising a nucleic acid having at least 70% homology to a sequence set forth in SEQ ID NO:2, as determined using a BLAST algorithm, with the proviso that the polynucleotide is distinct from Genbank accession numbers h59799, aa009010 and aa866363.

35. An isolated or recombinant polynucleotide comprising a nucleic acid having at least 70% homology to a sequence corresponding to nucleotides 421 to 1005 of SEQ ID NO:2, as determined using a BLAST algorithm.

36. The polynucleotide of claims 34 or 35, said polynucleotide having at least 80% homology to a sequence of SEQ ID NO:2.

37. The polynucleotide of claims 34 or 35, said polynucleotide having at least 90% homology to a sequence of SEQ ID NO:2.

38. An expression cassette comprising a nucleic acid sequence expression control element operably linked to the polynucleotide of any of claims 25 or 27.

39. A vector comprising the expression cassette of claim 38.

40. A host cell containing the vector of claim 39.

41. A method for modulating Jun N-terminal kinase (JNK) activity in a cell comprising contacting a cell with an amount of polypeptide of claim 1, a JNK modulating subsequence thereof, a nucleic acid encoding same, or an antisense thereof, sufficient to modulate JNK activity in the cell.

42. A method for modulating AP-1 activity in a cell comprising contacting a cell with an amount of polypeptide of claim 1, an AP-1 modulating subsequence thereof, a nucleic acid encoding same, or an antisense thereof, sufficient to modulate AP-1 activity in the cell.

43. A method for modulating NFKB activity in a cell comprising contacting a cell with an amount of polypeptide of claim 1, an NFKB modulating subsequence thereof, a nucleic acid encoding same, or an antisense thereof, sufficient to modulate NFKB activity in the cell.

44. A method for modulating IL-2 production in a cell comprising contacting a cell with an amount ofthe polypeptide of claim 1, an IL-2 modulating subsequence thereof, a nucleic acid encoding same, or an antisense thereof, sufficient to modulate IL-2 production in the cell.

45. A method for modulating Trx system activity in a cell comprising contacting a cell with an amount of polypeptide of claim 1, a Trx system modulating subsequence thereof, a nucleic acid encoding same, or an antisense thereof, sufficient to modulate Trx system activity in the cell.

46. A method for identifying a compound that binds PICOT polypeptide, comprising: a) incubating a test compound with a PICOT polypeptide under conditions allowing binding; and b) detecting whether the test compound binds the PICOT polypeptide, wherein binding ofthe test compound identifies the test compound as a compound that binds PICOT polypeptide.

47. A method for identifying a compound that modulates PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide, comprising: a) incubating a test compound with a PICOT polypeptide under conditions allowing binding; and b) determining PICOT polypeptide activity or expression of a nucleic acid encoding

PICOT polypeptide in the presence ofthe test compound, wherein an increase or decrease in PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide identifies the test compound as a compound that modulates

PICOT polypeptide activity or expression of a nucleic acid encoding PICOT polypeptide.

48. A method for identifying a compound that modulates binding of PICOT with a binding polypeptide, comprising: a) incubating PICOT with a binding polypeptide under conditions allowing binding; b) contacting a test compound with the bound complex; and c) determining whether the test compound increases or decreases binding between

PICOT and the polypeptide, wherein an increase or decrease in binding identifies the test compound as a compound that modulates binding of PICOT with a binding polypeptide.

49. The method of claim 48, wherein the binding polypeptide is PKCΘ or PKCζ.