WO2001051509A2 - Proteine 140 associee a shc (sap-140) - Google Patents

Proteine 140 associee a shc (sap-140) Download PDF

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WO2001051509A2
WO2001051509A2 PCT/CA2001/000023 CA0100023W WO0151509A2 WO 2001051509 A2 WO2001051509 A2 WO 2001051509A2 CA 0100023 W CA0100023 W CA 0100023W WO 0151509 A2 WO0151509 A2 WO 0151509A2
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sap
protein
nucleic acid
expression
cell
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PCT/CA2001/000023
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WO2001051509A3 (fr
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Chaim M. Roifman
Nigel Sharfe
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The Hospital For Sick Children
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Publication of WO2001051509A3 publication Critical patent/WO2001051509A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the fields of immunology and hematology and is concerned with a novel She associated protein (SAP), a nucleic acid sequence encoding the protein, and with its function in cells of the hematopoietic system and methods of its manipulation for the modulation of cellular processes.
  • SAP She associated protein
  • a wide range of growth factors coordinate cell proliferation and differentiation. Malignant cells arise as a result of a stepwise progression of events that include the unregulated expression of growth factors or components of their signaling pathways. Tyrosine phosphorylation events initiated by receptor and cytoplasmic kinases, and regulated by phosphatases, are central to these processes (Hunter 1991, Sun and Tonks 1994). Tyrosine kinases were originally believed to control tyrosine phosphorylation, with a small number of phosphatases playing largely housekeeping roles. However, the structural and functional diversity of the growing number of phosphatases suggests they have important roles in the regulation of growth and differentiation in normal and neoplastic cells (Li and Dixon 2000, Neel and Tonks 1997).
  • Tyrosine phosphatases are divided broadly into two groups, membrane-bound receptors and intracellular phosphatases.
  • the receptor phosphatases generally contain tandem intracellular phosphatase domains, while non-receptor phosphatases typically have a single catalytic domain; some also possess SH2 domains (SH-PTPI and SH-PTP2), allowing them to interact with a variety of tyrosine phosphorylated proteins.
  • All tyrosine phosphatases have a highly conserved catalytic core, but outside the catalytic domain, cytoplasmic phosphatases display remarkable diversity in structure, typically with a few easily identifiable functional domains, such as the SH2 domain.
  • tyrosine phosphatases appear to be essential for lymphocyte development and activation (Li and Dixon 2000, Dahia PL 2000).
  • CD45 a transmembrane phosphatase expressed exclusively in hematopoietic cells, is required for antigenic activation of both B and T lymphocytes (Byth et al 1996, Conroy et al 1996, Stone et al 1997).
  • SH2- containing cytoplasmic phosphatase, SHP-1 negatively regulates signaling through the B cell receptor, FcyR and the IL-3 receptor ⁇ chain.
  • SHPI also participates in T cell signaling, through dephosphorylation of the T cell receptor, pS61ck and Zap70. Mutations in the murine motheaten locus, encoding SHP-1, result in severe immunodeficiency and systemic autoimmunity, and other hematopoietic abnormalities (Shultz et al 1997, Nakayama 1997). All these studies suggest a critical role for phosphatases, not only in regulating signaling in mature cells, but also in cell differentiation.
  • Lyp plays a role in signal transduction from the TCR, binding to and possibly modulating the phosphorylation of the proto-oncogene Cbl (Cohen et al 1999).
  • Cbl undergoes selective tyrosine phosphorylation by both receptor tyrosine kinases and cytoplasmic kinases and down-regulates receptor kinase activity by inducing ubiquitination of the activated receptor, targeting it for internalization and degradation.
  • De-phosphorylation of Cbl by Lyp may significantly affect its activity, as tyrosine phosphorylation deficient Cbl is ineffective at modulating signaling. Decreased phosphorylation as the result of interaction with Lyp could alter Cbl associations with other proteins, its ability to cause ubiquitination, or to correctly target kinase receptors for degradation (Fournel et al 1996, Lee PS et al, 1999, Levkowitz et al 1996& 1998). She and Grb2 Signal Transducing Adaptor Proteins.
  • the adapter protein She has three isoforms that have been implicated as mediators of signal transduction from growth factor receptors and nonreceptor tyrosine kinases to ras (Baldari and Telford 1999, Clements and Koretzky 1999, Ravichandran KS et al 1995). She proteins contain a carboxy terminal SH2 domain (Zhou et al 1995), a proline rich region and a novel non-SH2 phosphotyrosine-binding (PTB) domain that specifically recognizes phosphorylated tyrosine motifs (Zhou et al 1995, van de Greer and Pawson, 1995) in target proteins such as the EGF receptor.
  • PTB phosphotyrosine-binding
  • Activated She is bound by the small adapter protein Grb2, through the Grb2 SH2 domain. She then serves to localize Grb2 and its associated proteins, eg Sosl and Sos2, to the plasma membrane through binding to activated receptor molecules (Ravichandran et al 1995a, 1995b).
  • a complex of She and Grb2 has been found associated with the CD3 ⁇ chain of the TCR/CD3 complex subsequent to TCR activation (Ravichandran et al 1993, Zhou et al 1995). She can also bind directly to the cytoplasmic tyrosine kinase Zap-70 in response to TCR stimulus.
  • Ras is a widely expressed membrane associated GTPase that functions as a molecular switch in response to signals from a variety of growth and differentiation factors (Henning and Cantrell 1998, Downward J 1996, Medema and Bos 1993).
  • Grb2 is a small adapter protein composed of three modular protein-protein interaction domains (SH3-SH2-SH3) (Downward J, 1994). As well as binding receptors through She, Grb2 may also interact directly with certain activated receptors through its SH2 domain, such as the linker for activation of T cells LAT (Cantrell 1998). Grb2 can also associate with the proto-oncogene c-Cbl.
  • the present inventors have isolated a novel non-transmembrane protein isolated by binding to the human cytoplasmic phosphatase Lypl. On the basis of its functional behaviour this novel protein is designated herein as 140kD She Associated Protein or SAP140.
  • an isolated nucleic acid molecule comprising a nucleotide sequence encoding a She associated protein (SAP140).
  • an isolated She associated protein SAP 140.
  • SAP 140 is only expressed in hematopoietic cells and that in lymphocytes it is an early component of the antigen receptor mediated signal transduction pathway.
  • SAP 140 is tyrosine phosphorylated and inducibly forms complexes with a number of signal transduction proteins, preferably Crk-1, Cbl, Grb2 and She.
  • Manipulation of SAP 140 may provide a method of manipulating antigen receptor signaling in both B and T lymphocytes and their precursors. Accordingly, in its broad aspect, the present invention provides a method of modulating the activity of a hematopoietic cell or system of an animal comprising administering an effective amount of an agent capable of modulating SAP 140 expression and/or activity to an animal or cell in need thereof.
  • the method involves modulating the activity of a B cell or a T cell.
  • SAP 140 and the She adapter protein are constitutively associated and phosphorylated in acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the present invention provides a method of modulating the proliferation of a cancer cell, preferably an acute myeloid leukemic cell or an acute lymphoblastic leukemic cell (ALL) comprising administering an effective amount of an agent capable of modulating SAP 140 expression and/or activity to a cell or animal in need thereof.
  • ALL acute lymphoblastic leukemic cell
  • the agent inhibits the proliferation of a cancer cell and can be used to treat cancer.
  • a method of determining compounds useful for diagnosis or treatment of an abnormal condition, such as AML or ALL, in an organism wherein said condition is associated with an aberration in a signal transduction pathway characterized by an interaction between SAP and its binding proteins, preferably She or Grb2, or Crk-L or Cbl or Lyp comprising the steps of adding a compound to cells and detecting whether the compound promotes or disrupts the interaction.
  • a method of diagnosis of a disease as described above which comprises detecting the level of interaction between SAP and its binding proteins, preferably She or Grb2 or Crk-L or Cbl or Lyp.
  • a method of diagnosis of a disease as described above which comprises detecting changes in the gene encoding SAP or in the DNA regulatory sequence controlling its expression.
  • a method of treating a disease which comprises either promoting or disrupting the interaction between SAP and its binding proteins, preferably She or Grb2 or Crk-L or
  • Figure 1 shows the predicted amino acid coding of the cDNA fragment isolated by two hybrid screening with the C-terminus of the Lypl phosphatase.
  • the ORF of the isolated fragment was determined by reference to the yeast activator domain fusion partner. Translation was stopped at the first stop codon. A possible Six domain can be identified in the sequence.
  • Figure 2 is an immunoblot showing Lyp binding to a SAP GST-fusion protein.
  • T7 tagged Lyp was transfected into 293 cells as indicated (i.e. lanes 1, 2, and 3). The samples were precipitated with the indicated GST-fusion proteins. The precipitates were blotted with anti-T7 to detect Lyp. Only GST-SAP precipitated the T7-Ly ⁇ protein as indicated in lane 3. GST-SAP did not precipitate anything from the control cells as shown in lane 4.
  • Figure 3 A and B shows the nucleic acid and amino acid sequence for SAP 140. The complete coding sequence was obtained from a single DNA fragment isolated by PCR amplification. PCR primer design was based upon the composite sequence first obtained by screening cDNA libraries. A semi-nested PCR approach was used in the library screening.
  • a pair of adjacent ohgonucleotide primers were designed from the original SAP sequence and used in sequential PCR reactions. Human spleen and thymocyte ⁇ gtl 1 cDNA libraries were used for screening. The cDNA sequence is shown in A. and the predicted amino acids sequence in B. Minor variants of both sequences were obtained upon sequencing of full length PCR products amplified from different sources, possibly representing allellic variants.
  • FIG 4 illustrates the genomic structure of the SAP 140 gene. Comparison of public genomic DNA sequence databases with the SAP cDNA sequence resulted in the identification most of the intron-exon boundaries of SAP as well as identification of its 5' upstream promoter sequences. A schematic of the genomic structure is presented in A. The SAP mRNA is encoded by at least 19 exons of which 18 probably encode the open reading frame of the protein. Most of the SAP exon-intron boundaries conform to the recognized conserved donor and acceptor sequences (B). Exon and intron sizes vary considerably (C), such that the gene spans at least 36 kilobases of genomic DNA.
  • Figure 5 A is a hydrophobicity plot of the predicted SAP 140 amino acid sequence. The sequence appears to code for a non-transmembrane protein as there are no large regions with a high hydrophobicity index typical of a transmembrane domain.
  • Figure 5B is an immunoblot showing the identification of the product of the
  • SAP 140 cDNA by transfection.
  • the cDNA for FLAG tagged SAP 140 was transfected into 293-T cells allowed to express for 24 hours and then immunoprecipitated and western blotted with antibodies to the FLAG epitope.
  • a single protein band was revealed of approximately 135kD (lane 2-T). An untransfected control is also shown
  • Figure 6A and B are immunoblots illustrating the testing of polyclonal antibodies raised to SAP 140.
  • RT-PCR results indicating that SAP 140 was expressed in Jurkat cells, the ability of the antibody to precipitate the endogenous protein was assessed as shown.
  • a single 140kDa band was precipitated and western blotted by anti-sera 99 (A).
  • the specificity of recognition of the precipitated protein was confirmed by the ability of the immunizing peptide to prevent recognition of the protein upon inclusion in the Western blotting buffer.
  • Anti-sera 601 could not precipitate any protein, but recognized the 140kDa protein precipitated by anti-sera 99 in immunoblot assays (B).
  • FIG. 7 is an immunoblot illustrating the expression pattern of the SAP 140
  • Figure 8 is an immunoblot illustrating the induction of SAP 140 expression by PHA.
  • SAP 140 expression was low in human peripheral blood T lymphocytes, but mcreased greatly after 24 hours activation with the strong mitogen phytohemaglutinin
  • Figure 9 is an immunoblot illustrating the binding of transfected FLAG- SAP to GST-SH3 domain fusion proteins.
  • FLAG-SAP did not bind any GST-SH2 domain examined, but demonstrated selective association with the C-terminal SH3 domain of the linker protein Grb2 (lane 2) and the SH3 domain of Phospholipase C ⁇ -1 (lane 5) was observed.
  • the N-terminal SH3 domain of Grb2 did not bind SAP, nor did SH3 domains from the src-like kinases Lck (lane 3) or Fyn (lane 4), or from the rasGAP protein (lane 6). Binding to Grb2 may link SAP to many signaling pathways.
  • FIGS. 10A and B are immunoblots illustrating the binding of endogenous SAP 140 from T cells to GST-SH2 and GST-SH3 domain fusion proteins.
  • a similar pattern to FLAG-SAP 140 binding was observed when GST fusion proteins of SH2 and SH3 domains were used as bait with lysates from T cell lines.
  • SAP binding was detected by blotting PAGE resolved GST-fusion complexes with affinity purified anti- sera 99. Strong association with the C-terminal SH3 domain of the Grb2 adapter molecule was seen. Little if any SAP binding to the N-terminal Grb2 SH3 domain occurred.
  • SAP also bound to the SH3 domain of PLC ⁇ -1 when isolated from T cell lysates.
  • Figure 11 are immunoblots illustrating the binding of endogenous SAP 140 from B cells to GST-SH3 domain fusion proteins. A similar pattern of SAP 140 binding was observed when GST fusion proteins of SH2 and SH3 domains were used as bait with lysates from B cell lines. Curiously SAP did not bind the SH3 domain of PLC ⁇ -1 when isolated from B cell lysates.
  • Figure 12 is an immunoblot illustrating the conformation of SAP 140 binding to GST-Grb2-SH3(C) and not GST-Grb2-(N) by blotting with two independent aanti- SAP140 antibodies.
  • the selective binding of SAP to the C-terminal but not N- terminal SH3 domain of Grb2 was observed when Western blotting with both anti-sera 601 and 99.
  • Figure 13 is an immunoblot illustrating the co-transfection of SAP 140 with tyrosine kinases.
  • Co-transfection of the SAP 140 with cytoplasmic tyrosine kinases in 293-T cells revealed that it was a substrate of several src-family tyrosine kinases.
  • Co- transfection with the cDNA for Src (c-Src and v-Src) or Lyn resulted in SAP phosphorylation (lanes marked by asterisk), but not with the related Fyn or Lck kinases, or with Jak3 or Emt.
  • co-transfection with Emt did not result in SAP phosphorylation, the kinase co-immunoprecipiated with the protein.
  • Figure 14 is an immunoblot illustrating the sub-cellular localization of SAP 140 in Jurtkat cells. Specific sub-cellular fractionation of Jurkat T cells revealed that SAP 140 was cytoplasmic and present in neither plasma-membrane nor mitochondrial fractions. Non-SAP 140 expressing E6.1 cells were used as a control. Cells were swelled in hypotonic buffer and the suspension passed through a 30 gauge needle to break open the cells. Remaining whole cells and nuclei were removed by low speed centrifugation. Plasma membrane fraction was separated from the cytoplasmic fraction by high-speed centrifugation. The detergent soluble component of the crude membrane preparation and the cytoplasmic fraction were then analyzed by immunoprecipitation of SAP140 and LAT as a marker of the plasma membrane.
  • Figure 15 is an immunoblot illustrating the TCR Induced tyrosine phosphorylation of SAP 140.
  • Stimulation of Jurkat T cells with anti-CD3 and immunoprecipitation of pi 40 revealed that it underwent rapid and transient tyrosine phosphorylation.
  • pi 40 demonstrated strongly phosphorylation, which reached a peak after two minutes and slowly subsided thereafter. Although the peak in phosphorylation was rapidly attained, substantially higher than basal levels of phosphorylation were still observed after 15 minutes anti-CD3 treatment.
  • a highly phosphorylated protein of approximately 1 lOkD was observed to associate with pl40. Minor bands of 130, 60, 36, and 32 kD were also.
  • Pre-bleed control and no-antibody controls did not precipitate the above mentioned phosphoproteins.
  • a simpler pattern was observed in human thymocytes where anti-CD3 inducible phosphorylation of SAP 140 itself only was visible.
  • Figure 16 is an immunoblot illustrating the BCR stimulation Induces SAP 140 tyrosine phosphorylation.
  • the stimulation of B cells with F(ab)' 2 antibody fragment to the B cell antigen receptor also resulted in rapid tyrosine phosphorylation of SAP 140 with a pattern of associated phosphorylated proteins extremely similar to those observed in the Jurkat T cell line.
  • FIG 17 is an immunoblot illustrating the SAP 140 binding to Grb2-SH3(C) is constitutive.
  • SAP 140 binding was determined to be constitutive and relatively unaltered by anti-CD3 stimulation. Blotting the Grb2 complexes with anti-phosphotyrosine revealed that the associated SAP 140 protein did undergo TCR induced phosphorylation. This did not alter its interaction with Grb2-SH3(C).
  • SAP140 binding was similar to that observed between Grb2-SH3(C) and a known binding partner Sosl, whose interaction with the Grb2-SH3 (C) domain was also observed to be constitutive.
  • FIG 18 is an immunoblot illustrating the SAP 140 does not bind the Grb2- SH2 domain. Examination of the Grb2-SAP140 association revealed that SAP 140 did not bind the isolated SH2 domain of Grb2, even after induction of its phosphorylation by anti-CD3. SAP 140 did, however, bind a GST fusion protein of the entire Grb2 protein (SH3(n)-SH2-SH3(c)). E6.1 cells (no SAP140 expression) were used as controls.
  • FIG 19 is an immunoblot illustrating the comparison of Grb2 and SAP 140 immunoprecipitates.
  • Grb2 and SAP 140 were immunoprecipitated from anti-CD3 stimulated cells and western blotted with anti-phosphotyrosine. Association between Grb2 and ppl40-SAP was only observed in the parental Jurkat T cell and not in the E6.1 subclone. In E6.1, ppl40 SAP is absent from Grb2 precipitates, although the Grb2 protein clearly undergoes TCR induced association with several other phosphorylated proteins, most notably a 36kD protein, previously identified as the transmembrane protein LAT.
  • Figure 20 is an immunoblot illustrating the pre-clearing stimulated T cell lysates with anti-SAP 140 removes the ppl40 band from Grb2 immunoprecipitates.
  • Pre-clearing of T cell lysates (anti-CD3 stimulated) with anti-SAP 140 prior to Grb2 immunoprecipitation significantly and specifically reduced the ppl40 band observed in Grb2 immunoprecipitates, in comparison to pre-clearing with non-specific anti-sera (Ig).
  • the material immunoprecipitated by anti-SAP and non-specific antisera during the pre-clearing step is shown in lanes 3&4, 7&8.
  • FIG 21 is an immunoblot illustrating the SAP140-Grb2 association in human thymocytes. Human thymocytes were purified and stimulated with anti-CD3. A complex of Grb2 and SAP 140 could be directly identified in human thymocytes by Western blotting of Grb2 immunoprecipitates with anti-SAP. Grb2-SAP140 association appeared to be constitutive, as observed in Jurkat T cells.
  • Figure 22 is an immunoblot comparing Grb2 and She immunoprecipitates.
  • FIG 23 A and B are immunoblots illustrating the inducible association of She and SAP140 in Jurkat T cells. Comparison of She and SAP140 immunoprecipitates revealed exceedingly similar patterns upon phosphotyrosine blotting, with a dominant ppl40 band and a minor HOkD band (A). ppl40 was not present in She immunoprecipitates from anti-CD3 stimulated E6.1 cells. Western blotting of She immunoprecipitates with anti-SAP 140 demonstrated the association between the proteins (B). SAP binding to She was only induced after TCR stimulation. Association between She and SAP 140 paralleled SAP 140 phosphorylation, being maximal after two minutes TCR stimulation and declining by ten minutes. Control E6.1 cells did not show SAP 140 in She immunoprecipitates.
  • FIG 24 is an immunoblot illustrating that SAP 140 is constitutively phosphorylated in AML and bound to She.
  • a 52kD phosphoprotein, the same size as a She isoform was clearly visible in SAP 140 immunoprecipitates while ppl40-SAP was apparent in She immunoprecipitates.
  • She in AML cells has previously been shown to be highly tyrosine phosphorylated, consistent with an ongoing proliferative signal transduction cascade, either as the consequence of an autocrine loop, or a mutation in regulatory element. She-SAP 140 association may therefore be a central event in the proliferative capacity of certain AML and ALL malignancies.
  • Figure 25A and B are immunoblots illustrating the inducible association of SAP 140 with Crk-L and c-Cbl.
  • Western blotting of SAP 140 immunoprecipitates with anti-Cbl revealed that the two proteins inducibly associate upon TCR stimulation (A, lane 2).
  • Pre-bleed anti-sera controls did not co-precipitate Cbl (not shown).
  • SAP immunoprecipitates from the parental Jurkat line and the E6.1 clone, which does not express SAP 140 were compared (A).
  • SAP immunoprecipitates from E6.1 Jurkat cells did not contain Cbl suggesting that the pl40 anti-sera did not directly cross-react with the Cbl protein (lane3&4).
  • the present inventors have isolated a novel non-transmembrane protein isolated by binding to the human cytoplasmic phosphatase Lypl. On the basis of its functional behaviour this novel protein is designated as 140kD She Associated Protein, SAP or SAP140.
  • SAP 140 is a cytoplasmic signal transduction molecule characterized by the possession of interactive domains, called Vietnamese domains, which may form the basis for protein-protein interaction.
  • SAP 140 is a component of the antigen receptor mediated signal transduction pathway. Tyrosine phosphorylation of SAP 140 occurs rapidly upon activation through the antigen receptors. There is also inducible formation of complexes with a number of signal transduction proteins, preferably Crk-L, Cbl, and She, as well as a constitutive association with Grb2. Manipulation of SAP 140 may provide a method of manipulating antigen receptor signaling in both B and T lymphocytes and their precursors.
  • SAP 140 is highly tyrosine phosphorylated and constitutively associated with the She adapter protein in acute myeloid leukemia.
  • Manipulation of SAP 140 expression and / or activity is likely to provide a method of altering the proliferative capacity of AML.
  • the phosphorylation status of SAP 140 and the status of association with other signaling transducing molecules, preferably She may permit diagnosis of certain cancerous disease conditions.
  • the inventors cloned and sequenced the human cDNA sequence for SAP, which encodes a protein of 1098 amino acids.
  • the non-catalytic portion of Lypl was used to screen a human B lymphocyte yeast two-hybrid library and a partial cDNA for a novel protein ( Figure 1 and SEQ.ID.NO.:l) was isolated, which passed all tests for false-positive interaction.
  • This sequence was used to isolate a complete cDNA fragment encoding the human SAP protein.
  • the entire coding cDNA sequence of SAP is shown in Figure 3A and SEQ.ID.NO.:2.
  • the corresponding amino acid sequence is shown in Figure 3B and SEQ.ID.NO.:3.
  • the complete cDNA was isolated from ⁇ gtlO human thymus and spleen libraries.
  • an intact SAP cDNA was isolated by RT-PCR from several types of human cells and each PCR product was sequenced for conformation.
  • the cDNA isolated for the human SAP 140 gene predicts a novel non-transmembrane protein of molecular weight 124 kDa, but the actual protein has an apparent molecular weight of 140kD. Although novel, areas of the protein display significant homology with a conserved fold known as the Informo. This domain was first described as a repeated element in the product of the Drosophila locus tudor, which is required during oogenesis for formation of primordial germ cells and segmentation (Bardsley et al.
  • the fragment of SAP isolated by two-hybrid screening with the C-terminal tail of Lyp contains at least one Tale domain, and the C-terminal 20 amino acids of Lyp contains and unusually high number of potentially positively charged arginine or lysine residues.
  • SAP 140- Lyp association may occur as the result of a similar interaction.
  • SAP 140 expression was low in human peripheral blood T lymphocytes, but increased greatly after 24 hours activation with the strong mitogen phytohemaglutinin. This suggests that SAP 140 may have an important function in either antigen induced clonal expansion of T cells or the function of the mature activated T cell.
  • the inventors demonstrate that SAP 140 is a predominantly cytoplasmic protein and is a potential target for several cytoplasmic tyrosine kinases. SAP 140 undergoes rapid tyrosine phosphorylation following antigen receptor stimulation in both B and T lymphocytes and in thymocytes. The rapidity of SAP 140 phosphorylation suggests a role in the immediate receptor proximal signaling pathway.
  • the inventors demonstrate that both transfected and endogenous SAP 140 proteins bind selectively to SH3 domains from a number of signal transduction proteins, in particular the adapter Grb2, the Phospholipase C ⁇ -1 enzyme and the tyrosine kinases Lyn and Btk.
  • binding to Grb2 may serve to link SAP 140 to many signaling pathways as this small adapter protein serves to link effector molecules, most notably the ras activation pathway, to numerous receptors.
  • binding to the PLC ⁇ -1 SH3 domain suggests that SAP may also be involved in inositol tri-phosphate and diacylglycerol secondary messenger production.
  • SAP 140 may be a target of certain src-family tyrosine kinases, resulting in its modification through phosphorylation, and with subsequent alterations in its properties and associations, while other kinases may form a complex with SAP 140, although it is not a substrate for their catalytic activity.
  • SAP 140 may function to organize the interactions of certain signal transduction molecules.
  • SAP 140 may be recognized by certain SH2- domain-containing proteins, serving to further organize protein-protein interactions. For these reasons, SAP 140 may be central to the signal transduction pathway.
  • Grb2 immunoprecipitated from E6.1 Jurkat T cells which do not express SAP 140
  • a phosphoprotein of 36kD presumably the well characterized lymphocyte activator of transcription LAT (Cantrell 1998)
  • binding of a strongly phosphorylated 75-85 kD protein, found in Grb2 precipitates from the parental line is absent.
  • the 75- 85 kD phosphoprotein does not appear to be directly bound to SAP 140 as pre-clearing SAP 140 protein from activated cell lysates does not reduced the appearance of this band in subsequent Grb2 immunoprecipitates, only the ppl40 SAP band.
  • the absence of SAP 140 expression while not affecting LAT phosphorylation of its association with Grb2, may either prevent phosphorylation of the 75-85KD protein or its recruitment to Grb2, with undoubted repercussions for signal transduction and ultimately gene transcription and physiological response.
  • Grb2 forms a well characterized complex with the adapter protein She (Ravichandran et al. 1995, Clements et al. 1999).
  • the inventors demonstrate that both Grb2 and She immunoprecipitated from anti-CD3 stimulated Jurkat cells co- precipitates ppl40, She possibly more strongly that Grb2.
  • Western blotting of She immunoprecipitates with anti-SAP 140 clearly demonstrates the association between the proteins. Unlike SAP 140 association with Grb2, which is constitutive, SAP binding to She is only induced after stimulation.
  • the inventors compare She and SAP 140 immunoprecipitates from an AML cell line (OCI-AML3) revealing that SAP140 does indeed demonstrate significant tyrosine phosphorylation and does appear to be constitutively complexed with She.
  • the She in ppl40 complexes has previously been shown to be highly tyrosine phosphorylated, without wishing to be bound by any particular theory, this is consistent with an ongoing proliferative signal transduction cascade, either as the consequence of an autocrine loop, or a mutation in regulatory element. She-SAP 140 association may therefore be a central event in the proliferative capacity of some AML and ALL malignancies.
  • SAP 140 has many implications for its role in the hematopoietic cell. She is plays an important role in signaling not only from the antigen receptors of B and T lymphocytes, but also in transduction of the signals from a variety of cytokine receptors, including, IL-2, IL-3, IL-5, GM-CSF, G-CSF, IL-6 (Baldari and Telford 1999, Hibi and Hirano 1998).
  • She activation and recruitment to the membrane is essential in most hematopoietic cells for the activation of a variety of signaling pathways, in particular, the ras activation pathway, through its recruitment of the Sosl and Sos2 proteins to the membrane and into the proximity of the ras protein. She is also known to bind directly to the cytoplasmic tail of a number of activated receptors, including the CD3 ⁇ chain component of the TCR complex (Zhou et al. 1995, Ravichandran et al. 1993).
  • SAP140 may play a role in cytokine signal transduction in all hematopoietic cells and SAP 140 may also be recruited to the plasma membrane as a consequence of its association with She and /or Grb2 and form a part of the complexes formed with receptor chains. Like She, SAP 140 is likely to have multiple roles.
  • one of the biological functions of SAP 140 may be to permit the clonal expansion of antigen activated T-cells through transduction of either the TCR signal or the IL-2R signals required for proliferation. In the absence of SAP 140 expression or activity, either the TCR or IL-2R signal may not be properly transmitted, resulting a failure of clonal expansion and subsequently reduced immunological response to pathogen.
  • SAP 140 may potentiate TCR or cytokine receptor signaling and may lead to uncontrolled activation or undesirable activation upon very low affinity interaction with antigen.
  • the consequences of these events may be multiple, but include autoimmune reactions, as low affinity self-self interactions are improperly regulated, or inadvertent activation of bystander cells due to cytokine overproduction by uncontrolled activated cells.
  • the inventors demonstrate the association of SAP 140 with the proto- oncogene c-Cbl and the Crk-L adapter protein in T cells.
  • Cbl is responsible for the physical downregulation of many tyrosine kinase receptors through induction of receptor ubiquitination (Levkowitz et al. (1998), Lee et al (1999)).
  • Addition of ubiquitin moieties to the lysine residues of a protein targets it for degradation, either in cytoplasmic proteasomes or in lysosomes.
  • the ubiquitination ability of Cbl is derived from its ring finger domain, which appears to be an E3 ubiquitin-ligase (Joazeiro et al.
  • the present invention provides an isolated nucleic acid molecule comprising a sequence encoding a She associated protein having a molecular weight of approximately 140Kd or SAP140.
  • isolated refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
  • nucleic acid is intended to include DNA and RNA and can be either double stranded or single stranded.
  • an isolated nucleic acid molecule having a sequence which encodes a SAP 140 protein having the amino acid sequence as shown in Figure 3B and SEQ.ID.NO.:3.
  • the invention provides an isolated nucleic acid sequence comprising:
  • nucleic acid sequence that is complimentary to a nucleic acid sequence of (a);
  • nucleic acid sequence that has substantial sequence homology to a nucleic acid sequence of (a) or (b);
  • nucleic acid sequence that is an analog of a nucleic acid sequence of (a), (b) or (c); or (e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of
  • sequence that has substantial sequence homology means those nucleic acid sequences which have slight or inconsequential sequence variations from the sequences in (a) or (b), i.e., the sequences function in substantially the same manner and can be used to modulate a hematopoietic cell. The variations may be attributable to local mutations or structural modifications. Nucleic acid sequences having substantial homology include nucleic acid sequences having at least 65%, more preferably at least 85%, and most preferably 90-95% identity with the nucleic acid sequences as shown in Figure 3 A.
  • sequence that hybridizes means a nucleic acid sequence that can hybridize to a sequence of (a), (b), (c) or (d) under stringent hybridization conditions.
  • Appropriate "stringent hybridization conditions" which promote DNA hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, NN. (1989), 6.3.1-6.3.6. For example, the following may be employed: 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C; 0.2 x SSC at 50°C to 65°C; or 2.0 x SSC at 44°C to 50°C.
  • SSC sodium chloride/sodium citrate
  • the stringency may be selected based on the conditions used in the wash step.
  • the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be at high stringency conditions, at about 65°C.
  • a nucleic acid sequence which is an analog means a nucleic acid sequence which has been modified as compared to the sequence of (a), (b) or (c) wherein the modification does not alter the utility of the sequence as described herein.
  • the modified sequence or analog may have improved properties over the sequence shown in (a), (b) or (c).
  • One example of a modification to prepare an analog is to replace one of the naturally occurring bases (i.e.
  • adenine, guanine, cytosine or thymidine of the sequence shown in Figure 3 A, with a modified base such as such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza ura
  • a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclie intersugar linkages in the nucleic acid molecule shown in Figure 3A.
  • the nucleic acid sequences may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.
  • a further example of an analog of a nucleic acid molecule of the invention is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254, 1497).
  • PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand.
  • nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506).
  • the analogs may also contain groups such as reporter groups, a group for improving the pharmacokinetic or pharmacodynamic properties of nucleic acid sequence.
  • the invention includes nucleic acid molecules encoding truncations of proteins of the invention, and analogs and homologs of proteins of the invention and truncations thereof, as described below. It will further be appreciated that variant forms of nucleic acid molecules of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention. Isolated and purified nucleic acid molecules having sequences which differ from the nucleic acid sequence of the invention due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode functionally equivalent proteins but differ in sequence from the above mentioned sequences due to degeneracy in the genetic code.
  • An isolated nucleic acid molecule of the invention which comprises DNA can be isolated by preparing a labelled nucleic acid probe based on all or part of the nucleic acid sequences of the invention and using this labelled nucleic acid probe to screen an appropriate DNA library (e.g. a cDNA or genomic DNA library).
  • a genomic library isolated can be used to isolate a DNA encoding a novel protein of the invention by screening the library with the labelled probe using standard techniques.
  • Nucleic acids isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques.
  • An isolated nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid encoding a novel protein of the invention using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleic acid sequence of the invention for use in PCR.
  • a nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL.
  • An isolated nucleic acid molecule of the invention which is RNA can be isolated by cloning a cDNA encoding a novel protein of the invention into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a protein of the invention.
  • a cDNA can be cloned downstream of a bacteriophage promoter, (e.g., a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by standard techniques.
  • a nucleic acid molecule of the invention may also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).
  • Determination of whether a particular nucleic acid molecule encodes a novel protein of the invention may be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the activity of the protein using the methods as described herein.
  • a cDNA having the activity of a novel protein of the invention so isolated can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein.
  • initiation codon and untranslated sequences of nucleic acid molecules of the invention may be determined using currently available computer software designed for the purpose, such as PC/Gene (IntelliGenetics Inc., Calif). Regulatory elements can be identified using conventional techniques. The function of the elements can be confirmed by using these elements to express a reporter gene which is operatively linked to the elements. These constructs may be introduced into cultured cells using standard procedures. In addition to identifying regulatory elements in DNA, such constructs may also be used to identify proteins interacting with the elements, using techniques known in the art.
  • sequence of a nucleic acid molecule of the invention may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule which are more fully described herein.
  • an antisense sequence is constructed by inverting a region preceding the initiation codon or an unconserved region.
  • the nucleic acid sequences contained in the nucleic acid molecules of the invention or a fragment thereof may be inverted relative to its normal presentation for transcription to produce antisense nucleic acid molecules.
  • the invention also provides nucleic acids encoding fusion proteins comprising a novel protein of the invention and a selected protein, or a selectable marker protein (see below).
  • portions of the nucleic acid sequence encoding fragments, functional domains or antigenic determinants of the SAP protein.
  • the present invention also provides for the use of portions of the sequence as probes and PCR primers for SAP and related proteins and well as for determining functional aspects of the sequence.
  • SAP 140 protein includes all homo logs, analogs, fragments or derivatives of the SAP 140 protein which can modulate a hematopoietic cell.
  • the isolated SAP 140 has an amino acid sequence as shown in Figure 3B (SEQ.ID.NO.:3).
  • a protein of the invention may include various structural forms of the primary protein which retain biological activity.
  • a protein of the invention may be in the form of acidic or basic salts or in neutral form.
  • individual amino acid residues may be modified by oxidation or reduction.
  • the protein of the present invention may also include truncations of the protein, and analogs, and homologs of the protein and truncations thereof as described herein.
  • Truncated proteins or fragments may comprise peptides of at least 5, preferably 10 and more preferably 15 amino acid residues of the sequence shown in Figure 3B.
  • the invention further provides polypeptides comprising at least one functional domain or at least one antigenic determinant of a SAP 140 protein.
  • Analogs of the protein of the invention and/or truncations thereof as described herein may include, but are not limited to an amino acid sequence containing one or more amino acid substitutions, insertions, and/or deletions.
  • Amino acid substitutions may be of a conserved or non-conserved nature. conserveed amino acid substitutions involve replacing one or more amino acids of the proteins of the invention with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting analog should be functionally equivalent.
  • Non-conserved substitutions involve replacing one or more amino acids of the amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics.
  • amino acid insertions may be introduced into the amino acid sequences of the invention.
  • Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length.
  • amino acid insertions may be used to destroy target sequences so that the protein is no longer active. This procedure may be used in vivo to inhibit the activity of a protein of the invention.
  • Deletions may consist of the removal of one or more amino acids, or discrete portions from the amino acid sequence of the SAP 140.
  • the deleted amino acids may or may not be contiguous.
  • the lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 100 amino acids.
  • Analogs of a protein of the invention may be prepared by introducing mutations in the nucleotide sequence encoding the protein. Mutations in nucleotide sequences constructed for expression of analogs of a protein of the invention must preserve the reading frame of the coding sequences. Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which could adversely affect translation of the receptor mRNA.
  • Mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide-directed site specific mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required.
  • Deletion or truncation of a protein of the invention may also be constructed by utilizing convenient restriction endonuclease sites adjacent to the desired deletion. Subsequent to restriction, overhangs may be filled in, and the DNA religated. Exemplary methods of making the alterations set forth above are disclosed by Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989).
  • the proteins of the invention also include homologs of the amino acid sequence of the SAP 140 protein and/or truncations thereof as described herein.
  • Such homologs are proteins whose amino acid sequences are comprised of amino acid sequences that hybridize under stringent hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain a protein of the invention.
  • Homologs of a protein of the invention will have the same regions which are characteristic of the protein.
  • a homologous protein includes a protein with an amino acid sequence having at least 70%, preferably 80-95%) identity with the amino acid sequence of the SAP 140 protein.
  • the invention also contemplates isoforms of the proteins of the invention.
  • An isoform contains the same number and kinds of amino acids as a protein of the invention, but the isoform has a different molecular structure.
  • the isoforms contemplated by the present invention are those having the same properties as a protein of the invention as described herein.
  • the present invention also includes a protein of the invention conjugated with a selected protein, or a selectable marker protein to produce fusion proteins.
  • the SAP cDNA sequence is inserted into a vector that contains a nucleotide sequence encoding another peptide (e.g. GST-glutathione succinyl transferase).
  • the fusion protein is expressed and recovered from prokaryotic (e.g. bacterial or baculovirus) or eukaryotic cells.
  • the fusion protein can then be purified by affinity chromatography based upon the fusion vector sequence and the SAP protein obtained by enzymatic cleavage of the fusion protein.
  • the proteins of the invention may be prepared using recombinant DNA methods. Accordingly, nucleic acid molecules of the present invention having a sequence which encodes a protein of the invention may be incorporated according to procedures known in the art into an appropriate expression vector which ensures good expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used.
  • cosmids plasmids
  • modified viruses e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • vectors suitable for transformation of a host cell means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences, selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. "Operatively linked” is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.
  • the invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence.
  • Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, or viral genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art.
  • regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the native protein and/or its flanking regions.
  • the invention further provides a recombinant expression vector comprising a
  • DNA nucleic acid molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to a nucleotide sequence of the invention. Regulatory sequences operatively linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule.
  • the recombinant expression vectors of the invention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
  • selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die.
  • the recombinant expression vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of a target recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Recombinant expression vectors can be introduced into host cells to produce a transformed host cell.
  • the invention includes a host cell comprising a recombinant expression vector of the invention.
  • the term "transformed host cell” is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention.
  • the terms "transformed with”, “transfected with”, “transformation” and “transfection” are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art.
  • Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation.
  • Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofectin, electroporation or microinjection.
  • Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other such laboratory textbooks.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • the proteins of the invention may be expressed in bacterial cells such as E. coli, Pseudomonas, Bacillus subtillus, insect cells (using baculovirus), yeast cells or mammalian cells.
  • Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991).
  • E. coli can be used using the T7 RNA polymerase/promoter system using two plasmids or by labeling of plasmid-encoded proteins, or by expression by infection with Ml 3 Phage mGPI-2.
  • E. coli vectors can also be used with Phage lamba regulatory sequences, by fusion protein vectors (e.g. lacZ and trpE), by maltose-binding protein fusions, and by glutathione-S-transferase fusion proteins.
  • the SAP protein can be expressed in insect cells using baculo viral vectors, or in mammalian cells using vaccinia virus.
  • the cD A sequence may be ligated to heterologous promoters, such as the simian virus (SV40) promoter in the pSV2 vector and introduced into cells, such as COS cells to achieve transient or long-term expression.
  • SV40 simian virus
  • the stable integration of the chimeric gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin and mycophoenolic acid.
  • SAP DNA sequence can be altered using procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence alteration with the use of specific oligonucleotides together with PCR.
  • the cDNA sequence or portions thereof, or a mini gene consisting of a cDNA with an intron and its own promoter is introduced into eukaryotic expression vectors by conventional techniques. These vectors permit the transcription of the cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation.
  • the endogenous SAP gene promoter can also be used. Different promoters within vectors have different activities which alters the level of expression of the cDNA. In addition, certain promoters can also modulate function such as the glucocorticoid-responsive promoter from the mouse mammary tumor virus.
  • vectors listed contain selectable markers or neo bacterial genes that permit isolation of cells by chemical selection. Stable long-term vectors can be maintained in cells as episomal, freely replicating entities by using regulatory elements of viruses. Cell lines can also be produced which have integrated the vector into the genomic DNA. In this manner, the gene product is produced on a continuous basis.
  • Vectors are introduced into recipient cells by various methods including calcium phosphate, strontium phosphate, electroporation, lipofection, DEAE dextran, microinjection, or by protoplast fusion. Alternatively, the cDNA can be introduced by infection using viral vectors.
  • SAP proteins may also be isolated from cells or tissues, including mammalian cells or tissues, in which the protein is normally expressed.
  • the protein may be purified by conventional purification methods known to those in the art, such as chromatography methods, high performance liquid chromatography methods or precipitation.
  • an anti-SAP antibody (as described below) may be used to isolate a SAP protein, which is then purified by standard methods.
  • the proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E.
  • the present invention includes all uses of the nucleic acid molecule and SAP 140 proteins of the invention including, but not limited to, the preparation of antibodies and antisense oligonucleotides, the preparation of experimental systems to study SAP 140, the isolation of substances that modulate SAP 140 expression and/or activity as well as the use of the SAP 140 nucleic acid sequences and proteins and modulators thereof in diagnostic and therapeutic applications. Some of the uses are further described below.
  • Eukaryotic expression systems can be used for many studies of the SAP gene and gene product(s) including determination of proper expression and post- translational modifications for full biological activity, identifying regulatory elements located in the 5' region of the SAP gene and their role in tissue regulation of protein expression, production of large amounts of the normal and mutant protein for isolation and purification, to use cells expressing the SAP protein as a functional assay system for antibodies generated against the protein or to test effectiveness of pharmacological agents, or as a component of a signal transduction system, to study the function of the normal complete protein, specific portions of the protein, or of naturally occurring and artificially produced mutant proteins.
  • the expression vectors containing the SAP cDNA sequence or portions thereof can be introduced into a variety of mammalian cells from other species or into non-mammalian cells.
  • the recombinant cloning vector comprises the selected DNA of the DNA sequences of this invention for expression in a suitable host.
  • the DNA is operatively linked in the vector to an expression control sequence in the recombinant DNA molecule so that SAP protein can be expressed.
  • the expression control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.
  • the expression control sequence may be selected from the group consisting of the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of the fd coat protein, early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus, simian virus, 3 -phosphogly cerate kinase promoter, yeast acid phosphatase promoters, yeast alpha-mating factors and combinations thereof.
  • SAP gene expression in heterologous cell systems may also be used to demonstrate structure-function relationships as well as to provide cell lines for the purposes of drug screening.
  • SAP DNA sequence into a plasmid expression vector to transfect cells is a useful method to test the proteins influence on various cellular biochemical parameters including the identification of substrates as well as activators and inhibitors of the phosphatase.
  • Plasmid expression vectors containing either the entire coding sequence for SAP, or for portions thereof, can be used in in vitro mutagenesis experiments that will identify portions of the protein crucial for regulatory function.
  • the DNA sequence can be manipulated in studies to understand the expression of the gene and its product.
  • the changes in the sequence may or may not alter the expression pattern in terms of relative quantities, tissue-specificity and functional properties.
  • the invention also provides methods for examining the function of the SAP 140 protein encoded by the nucleic acid molecule of the invention.
  • Cells, tissues, and non-human animals lacking in expression or partially lacking in expression of the protein may be developed using recombinant molecules of the invention having specific deletion or insertion mutations in the nucleic acid molecule of the invention.
  • a recombinant molecule may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a deficient cell, tissue or animal.
  • Such a mutant cell, tissue or animal may be used to define specific cell populations, developmental patterns and in vivo processes, normally dependent on the protein encoded by the nucleic acid molecule of the invention.
  • a SAP 140 knockout mouse can be prepared.
  • a targeted recombination strategy may be used to inactivate the endogenous SAP 140 gene.
  • a gene which introduces stop codons in all reading frames and abolishes the biological activity of the protein may be inserted into a genomic copy of the protein.
  • the mutated fragment may be introduced into embryonic stem cells and colonies may be selected for homologous recombination with positive (neomycin)/negative (gancyclovir, thymidine kinase) resistance genes.
  • two clones carrying the disrupted gene on one allele may be injected into blastocyts of C57/B16 mice and transferred into B6/SJL foster mothers. Chimeras may be mated to C7B1/6 mice and progeny analysed to detect animals homozygous for the mutation (SAP 140 -/-). The effects of the mutation on the hematopoietic system in comparison to non-mutated controls may be determined, and the survival and histologic pattern of disease may be analyzed. (ii) Antibodies The isolation of the SAP 140 protein enables the preparation of antibodies specific for SAP 140. Accordingly, the present invention provides an antibody that binds to a SAP 140 protein.
  • Antibodies may be used advantageously to monitor the expression of SAP 140. Antibodies can be prepared which bind a distinct epitope in an unconserved region of the protein. An unconserved region of the protein is one that does not have substantial sequence homology to other proteins.
  • poly clonal antisera or monoclonal antibodies can be made using standard methods.
  • a mammal e.g., a mouse, hamster, or rabbit
  • an immunogenic form of the peptide which elicits an antibody response in the mammal.
  • Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art.
  • the protein or peptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies.
  • antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.
  • antibody producing cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
  • Such techniques are well known in the art, (e.g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256, 495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985) Allen R.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated. Therefore, the invention also contemplates hybridoma cells secreting monoclonal antibodies with specificity for SAP 140 as described herein.
  • antibody as used herein is intended to include fragments thereof which also specifically react with SAP 140, or peptide thereof, having the activity of the SAP 140.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulf ⁇ de bridges to produce Fab' fragments.
  • Chimeric antibody derivatives i.e., antibody molecules that combine a non- human animal variable region and a human constant region are also contemplated within the scope of the invention.
  • Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes the gene product of SAP 140 antigens of the invention (See, for example, Morrison et al, Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al, Nature 314, 452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No.
  • Monoclonal or chimeric antibodies specifically reactive with a protein of the invention as described herein can be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin.
  • Such immunoglobulin molecules may be made by techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and PCT Publication WO92/06193 or EP 0239400).
  • Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)
  • Specific antibodies, or antibody fragments, reactive against SAP 140 proteins may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with peptides produced from the nucleic acid molecules of SAP 140.
  • complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al., Nature 341, 544-546: (1989); Huse et al., Science 246, 1275- 1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).
  • phage expression libraries See for example Ward et al., Nature 341, 544-546: (1989); Huse et al., Science 246, 1275- 1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).
  • SCID-hu mouse for example the model developed by Genpharm, can be used to produce antibodies or fragments thereof.
  • Isolation of a nucleic acid molecule encoding SAP 140 enables the production of antisense oligonucleotides that can modulate the expression and/or activity of SAP140.
  • the present invention provides an antisense oligonucleotide that is complimentary to a nucleic acid sequence encoding SAP140.
  • antisense oligonucleotide as used herein means a nucleotide sequence that is complimentary to its target.
  • oligonucleotide refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages.
  • the term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases.
  • the term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide.
  • the antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8- thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8- hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, aden
  • antisense oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclie intersugar linkages.
  • the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.
  • phosphorothioate bonds link all the nucleotides.
  • the antisense oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents.
  • An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254, 1497). PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro.
  • PNA peptide nucleic acid
  • oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have morpholino backbone structures (U.S. Pat. Nol 5,034, 506).
  • Oligonucleotides may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide.
  • Antisense oligonucleotides may also have sugar mimetics.
  • the antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the antisense nucleic acid molecules of the invention or a fragment thereof may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides.
  • the antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
  • the antisense oligonucleotides may be introduced into tissues or cells using techniques in the art including vectors (retroviral vectors, adenoviral vectors and DNA virus vectors) or physical techniques such as microinjection.
  • the antisense oligonucleotides may be directly administered in vivo or may be used to transfect cells in vitro which are then administered in vivo.
  • the antisense oligonucleotide may be delivered to macrophages and/or endothelial cells in a liposome formulation.
  • Diagnostic Assays The finding by the present inventors that SAP140 is involved in the regulation of signal transduction and/or cell proliferation allows the detection of conditions involving an increase or decrease in SAP 140 activity or expression.
  • the present invention provides a method of detecting a condition associated with increased or decreased SAP 140 expression or activity (including an absence) comprising assaying a sample for (a) a nucleic acid molecule encoding a SAP 140 protein or a fragment thereof or (b) a SAP 140 protein or a fragment thereof.
  • the condition associated with increased SAP 140 expression or activity is a cancer of a hematopoietic cell including lymphomas, myelomas and leukemias such as ALL, AML or chronic myelogenous leukemia (CML).
  • the assay may comprise assaying for phosphorylated SAP 140 using techniques known in the art.
  • nucleic acid molecules encoding SAP 140 as described herein or fragments thereof allow those skilled in the art to construct nucleotide probes for use in the detection of nucleotide sequences encoding SAP 140 or fragments thereof in samples, preferably biological samples such as cells, tissues and bodily fluids.
  • the probes can be useful in detecting the presence of a condition associated with SAP 140 or monitoring the progress of such a condition.
  • Such conditions include neoplasia, such as AML or ALL.
  • the present invention provides a method for detecting a nucleic acid molecules encoding SAP 140 comprising contacting the sample with a nucleotide probe capable of hybridizing with the nucleic acid molecule to form a hybridization product, under conditions which permit the formation of the hybridization product, and assaying for the hybridization product.
  • Example of probes that may be used in the above method include fragments of the nucleic acid sequences shown in Figure 3 A or SEQ.ID.NO.:2.
  • a nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32P, 3H, 14C or the like.
  • detectable substances which may be used include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and chemiluminescence.
  • An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleic acid to be detected and the amount of nucleic acid available for hybridization.
  • Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleotide probes may be used to detect genes, preferably in human cells, that hybridize to the nucleic acid molecule of the present invention preferably, nucleic acid molecules which hybridize to the nucleic acid molecule of the invention under stringent hybridization conditions as described herein.
  • Nucleic acid molecules encoding a SAP 140 protein can be selectively amplified in a sample using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA.
  • PCR polymerase chain reaction
  • a nucleic acid can be amplified from cDNA or genomic DNA using oligonucleotide primers and standard PCR amplification techniques.
  • the amplified nucleic acid can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979).
  • the SAP 140 protein may be detected in a sample using antibodies that bind to the protein as described in detail above. Accordingly, the present invention provides a method for detecting a SAP 140 protein comprising contacting the sample with an antibody that binds to SAP 140 which is capable of being detected after it becomes bound to the SAP 140 in the sample.
  • reverse transcriptase for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL.
  • Antibodies specifically reactive with SAP140, or derivatives thereof, such as enzyme conjugates or labeled derivatives, may be used to detect SAP140 in various biological materials, for example they may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of SAP 140, and the antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination and histochemical tests. Thus, the antibodies may be used to detect and quantify SAP 140 in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.
  • the antibodies of the invention may be used in immuno- histochemical analyses, for example, at the cellular and sub-subcellular level, to detect SAP140, to localise it to particular cells and tissues and to specific subcellular locations, and to quantitate the level of expression.
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect SAP 140.
  • an antibody of the invention may be labelled with a detectable substance and SAP 140 may be localised in tissue based upon the presence of the detectable substance.
  • detectable substances include various enzymes, fluorescent materials, luminescent materials and radioactive materials.
  • Suitable enzymes include horseradish peroxidase, biotin, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include radioactive iodine 1-125, 1-131 or 3-H.
  • Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualised by electron microscopy.
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against SAP 140.
  • a second antibody having specificity for the antibody reactive against SAP 140.
  • the antibody having specificity against SAP 140 is a rabbit IgG antibody
  • the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.
  • SAP 140 may be localized by autoradiography.
  • the results of autoradiography may be quantitated by determining the density of particles in the autoradiographs by various optical methods, or by counting the grains.
  • Nucleic acid sequences of SAP 140 might be determined in order to assay for changes, preferably disease-causing mutations that may be used as indicators of disease prognosis or as aids to inform treatment of these diseases.
  • the knowledge of the human SAP 140 sequence provides a method for screening for diseases involving abnormally activated or inactivated SAP in which the activity defect is due to a mutant SAP 140 gene.
  • unregulated Jak 3 kinase leads to tumorigenesis (Schwaller, J. et al, (1998), EMBO J., v. 17, p. 5321-33; Lacronique et al., (1997), Science, v. 278, p. 1309-12; Peeters et al, (1997), Blood, v. 90, p. 2535-40).
  • the SAP protein may play a role in the regulation of cell proliferation and differentiation.
  • Genomic DNA used for the diagnosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen, or autopsy material.
  • the DNA may be isolated and used directly for detection of a specific sequence or may be PCR amplified prior to analysis.
  • RNA or cDNA may also be used.
  • direct DNA sequencing, restriction enzyme digest, RNase protection, chemical cleavage, and ligase-mediated detection are all methods which can be utilized.
  • Oligonucleotides specific to mutant sequences can be chemically synthesized and labelled radioactively with isotopes, or non-radioactively using biotin tags, and hybridized to individual DNA samples immobilized on membranes or other solid-supports by dot-blot or transfer from gels after electrophoresis. The presence or absence of these mutant sequences is then visualized using methods such as autoradiography, fluorometry, or colorimetric reaction. Suitable PCR primers can be generated which are useful for example in amplifying portions of the subject sequence containing identified mutations. Direct DNA sequencing reveals sequence differences between normal and mutant SAP DNA. Cloned DNA segments may be used as probes to detect specific DNA segments. PCR can be used to enhance the sensitivity of this method.
  • PCR is an enzymatic amplification directed by sequence-specific primers, and involves repeated cycles of heat denaturation of the DNA, annealing of the complementary primers and extension of the annealed primer with a DNA polymerase. This results in an exponential increase of the target DNA.
  • nucleotide sequence amplification techniques may be used, such as ligation-mediated PCR, anchored PCR and enzymatic amplification as would be understood by those skilled in the art. Sequence alterations may also generate fortuitous restriction enzyme recognition sites that are revealed by the use of appropriate enzyme digestion followed by gel-blot hybridization. DNA fragments carrying the site (normal or mutant) are detected by their increase or reduction in size, or by the increase or decrease of corresponding restriction fragment numbers. Genomic DNA samples may also be amplified by PCR prior to treatment with the appropriate restriction enzyme and the fragments of different sizes are visualized under UV light in the presence of ethidium bromide after gel electrophoresis.
  • Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels. Small sequence deletions and insertions can be visualized by high-resolution gel electrophoresis. Small deletions may also be detected as changes in the migration pattern of DNA heteroduplexes in non-denaturing gel electrophoresis. Alternatively, a single base substitution mutation may be detected based on differential primer length in PCR. The PCR products of the normal and mutant gene could be differentially detected in acrylamide gels.
  • Nuclease protection assays (SI or ligase-mediated) also reveal sequence changes at specific locations.
  • ASO to confirm or detect a polymorphism restriction mapping changes ligated PCR, ASO, REF-SSCP and SSCP may be used. Both REF-SSCP and SSCP are mobility shift assays that are based upon the change in conformation due to mutations.
  • DNA fragments may also be visualized by methods in which the individual DNA samples are not immobilized on membranes.
  • the probe and target sequences may be in solution or the probe sequence may be immobilized. Autoradiography, radioactive decay, spectrophotometry, and fluorometry may also be used to identify specific individual genotypes.
  • the portion of the DNA segment that is informative for a mutation can be amplified using PCR.
  • the DNA segment immediately surrounding a specific mutation acquired from peripheral blood or other tissue samples from an individual can be screened using constructed oligonucleotide primers. This region would then be amplified by PCR, the products separated by electrophoresis, and transferred to membrane. Labeled probes are then hybridized to the DNA fragments and autoradiography performed.
  • the present invention includes the use of the nucleic acids encoding SAP 140 and the SAP 140 protein to develop or identify substances that modulate SAP 140 expression or activity or that modulate the phosphorylation of SAP 140.
  • Substances that affect SAP 140 activity can be identified based on their ability to bind to SAP 140.
  • Substances which can bind with the SAP 140 of the invention may be identified by reacting the SAP 140 with a substance which potentially binds to SAP 140, and assaying for complexes, for free substance, or for non-complexed SAP 140, or for activation of SAP 140.
  • a yeast two hybrid assay system may be used to identify proteins which interact with SAP140 (Fields, S. and Song, O., 1989, Nature, 340:245-247).
  • Systems of analysis which also may be used include ELISA.
  • the invention provides a method of identifying substances which can bind with SAP 140, comprising the steps of:
  • the SAP 140 protein used in the assay may have the amino acid sequence shown in Figure 3B or may be a fragment, analog, derivative, homolog or mimetic thereof as described herein.
  • Conditions which permit the formation of substance and SAP 140 complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.
  • the substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
  • antibody against SAP 140 or the substance, or labelled SAP 140, or a labelled substance may be utilized.
  • the antibodies, proteins, or substances may be labelled with a detectable substance as described above.
  • Substances that bind to and activate the SAP 140 of the invention may be identified by assaying for phosphorylation of the tyrosine residues of the protein. Substances that bind to and inactivate the SAP 140 of the invention may be identified by assaying for reduction in phosphorylation of the protein.
  • SAP 140 or the substance used in the method of the invention may be insolubilized.
  • SAP 140 or substance may be bound to a suitable carrier.
  • suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
  • the insolubilized protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • proteins or substance may also be expressed on the surface of a cell using the methods described herein.
  • the invention also contemplates assaying for an antagonist or agonist of the action of SAP140.
  • the agonists and antagonists that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or allosteric sites.
  • the invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of SAP 140.
  • the invention may be used to assay for a substance that competes for the same binding site of SAP 140.
  • the methods described above may be used to identify a substance which is capable of binding to activated SAP 140, and to assay for an agonist or antagonist of the binding of activated SAP 140, with a substance which is capable of binding with activated SAP140.
  • Activated (i.e. phosphorylated) SAP140 may be prepared using the methods described (for example in Reedijk et al. The EMBO Journal, 11(4):1365, 1992) for producing a tyrosine phosphorylated protein.
  • the invention further provides a method for assaying for a substance that affects a SAP 140 regulatory pathway comprising administering to a human or animal or to a cell, or a tissue of an animal, a substance suspected of affecting a SAP 140 regulatory pathway, and quantitating the SAP 140 protein or nucleic acids encoding SAP 140, or examining the pattern and/or level of expression of SAP 140, in the human or animal or tissue, or cell.
  • SAP 140 may be quantitated and its expression may be examined using the methods described herein.
  • Peptide Mimetics The present invention also includes peptide mimetics of the SAP 140 of the invention.
  • a peptide derived from a binding domain of SAP 140 will interact directly or indirectly with an associated molecule in such a way as to mimic the native binding domain.
  • Such peptides may include competitive inhibitors, enhancers, peptide mimetics, and the like. All of these peptides as well as molecules substantially homologous, complementary or otherwise functionally or structurally equivalent to these peptides may be used for purposes of the present invention.
  • Peptide mimetics are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of a peptide, or enhancer or inhibitor of the invention. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide of the invention.
  • Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic.
  • the mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states.
  • the mimetics can also include mimics of inhibitor peptide secondary structures. These structures can model the 3- dimensional orientation of amino acid residues into the known secondary conformations of proteins.
  • Peptoids may also be used which are oligomers of N- substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.
  • Peptides of the invention may also be used to identify lead compounds for drug development.
  • the structure of the peptides described herein can be readily determined by a number of methods such as NMR and X-ray crystallography. A comparison of the structures of peptides similar in sequence, but differing in the biological activities they elicit in target molecules can provide information about the structure-activity relationship of the target. Information obtained from the examination of structure- activity relationships can be used to design either modified peptides, or other small molecules or lead compounds that can be tested for predicted properties as related to the target molecule. The activity of the lead compounds can be evaluated using assays similar to those described herein.
  • Information about structure-activity relationships may also be obtained from co-crystallization studies. In these studies, a peptide with a desired activity is crystallized in association with a target molecule, and the X-ray structure of the complex is determined. The structure can then be compared to the structure of the target molecule in its native state, and information from such a comparison may be used to design compounds expected to possess.
  • Modulation of the SAP Promoter As would be readily apparent to those skilled in the art, it is also possible to modulate SAP 140 through manipulation of its promoter. One or more alterations to a promoter sequence of SAP 140 may increase or decrease promoter activity, or increase or decrease the magnitude of the effect of a substance able to modulate the promoter activity.
  • Promoter activity is used to refer to the ability to initiate transcription.
  • the level of promoter activity is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter.
  • the amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction.
  • Substances which affect the SAP 140 promoter's activity may also be identified using the methods of the invention by comparing the pattern and level of expression of a reporter gene, in cells in the presence, and in the absence of the substance. Accordingly, a method for assaying for the presence of an agonist or antagonist of SAP 140 promoter activity is provided comprising providing a cell containing a reporter gene under the control of the promoter with a substance which is a suspected agonist or antagonist under conditions which permit interaction and assaying for the increase or decrease of reporter gene product. (d) Drug Screening Methods
  • the invention enables a method for screening candidate compounds for their ability to increase or decrease the activity of a SAP 140 protein. The method comprises providing an assay system for assaying SAP activity, assaying the activity in the presence or absence of the candidate or test compound and determining whether the compound has increased or decreased SAP activity.
  • the present invention provides a method for identifying a compound that affects SAP 140 protein activity or expression comprising:
  • the invention enables a method for screening candidate compounds for their ability to increase or decrease expression of a SAP protein.
  • the method comprises putting a cell with a candidate compound, wherein the cell includes a regulatory region of a SAP gene operably joined to a reporter gene coding region, and detecting a change in expression of the reporter gene.
  • the present invention enables culture systems in which cell lines which express the SAP gene, and thus SAP protein products, are incubated with candidate compounds to test their effects on SAP expression.
  • Such culture systems can be used to identify compounds which upregulate or downregulate SAP 140 expression or its function, through the interaction with other proteins.
  • Such compounds can be selected from protein compounds, chemicals and various drugs that are added to the culture medium. After a period of incubation in the presence of a selected test compound(s), the expression of SAP can be examined by quantifying the levels of SAP mRNA using standard Northern blotting procedure, as described in the examples included herein, to determine any changes in expression as a result of the test compound.
  • Cell lines transfected with constructs expressing SAP can also be used to test the function of compounds developed to modify the protein expression.
  • transformed cell lines expressing a normal SAP protein could be mutagenized by the use of mutagenizing agents to produce an altered phenotype in which the role of mutated SAP can be studied in order to study structure/function relationships of the protein products and their physiological effects.
  • the present invention provides a method for identifying a compound that affects the binding of an SAP 140 protein and an SAP 140 binding protein comprising:
  • the SAP 140 of the invention is likely involved in the regulation of cell signaling pathways that regulate proliferation of hematopoietic cells. Accordingly, the present invention provides a method of modulating the activity of a hematopoietic cell comprising of administering to a cell or animal in need thereof, an effective amount of agent that modulates SAP140 expression and/or activity. The invention also includes a use of an agent that modulates SAP 140 expression or activity to modulate the activity of a hematopoietic cell or to prepare a medicament to modulate the activity of a hematopoietic cell.
  • agent that modulates SAP140 expression and/or activity means any substance that can alter the expression and/or activity of SAP 140 and includes agents that can inhibit SAP 140 expression or activity and agents that can enhance SAP 140 expression or activity.
  • agents which may be used to modulate SAP 140 include nucleic acid molecules encoding SAP140; the SAP140 protein as well as fragments, analogs, derivatives or homologs thereof; antibodies; antisense nucleic acids; peptide mimetics; substances isolated using the screening methods described herein or substances that modulate the interaction of SAP 140 with SAP associating or binding proteins.
  • SAP 140 may be involved in modulating cell proliferation and stimulators and inhibitors of SAP 140 may be useful in modulating disorders involving cell proliferation such as neoplasia and autoimmunity.
  • substances that inhibit SAP 140 for example, identified using the methods of the invention may be used to prevent cell proliferation, to stimulate cell death or apoptosis, and stimulators could be used where an increase in cell proliferation would be advantageous.
  • the invention provides a method of inhibiting or reducing cell proliferation, such as in neoplasia, by administering to a cell or animal an effective amount of an agent that modulates, preferably inhibits, the expression or the biological activity of the SAP 140, such that there is an inhibition or reduction in cell proliferation.
  • an agent that modulates, preferably inhibits, the expression or the biological activity of the SAP 140 such that there is an inhibition or reduction in cell proliferation.
  • Substances that inhibit the activity of SAP 140 include peptide mimetics, SAP 140 antagonists and certain antibodies to SAP 140.
  • Substances that inhibit the expression of the SAP 140 gene include antisense oligonucleotides to a SAP 140 nucleic acid sequence.
  • the present invention provides a method of treating cancer comprising administering an effective amount of an agent that inhibits SAP 140 activity or expression to an animal in need thereof.
  • the cancer may be any cancer of a hematopoietic cell including leukemia (such as AML, ALL and CML), lymphomas and myelomas.
  • the present invention provides a method of inducing cell proliferation by administering to a cell or an animal an effective amount of a substance that modulates, preferably stimulates, the expression or the biological activity of SAP 140, such that there is an induction of cell proliferation.
  • autoimmune disorders may include cell-associated autoimmunities such as multiple sclerosis, lupus, arthritis, thyroiditis, diabetes, psoriasis and Crohn's disease and colitis.
  • Allergic disorders include asthma and hyper-IgE and eosinophilic syndromes and T-cell dependent graft-versus-host reactions.
  • the substances identified by the methods described herein may be used for modulating SAP 140 regulatory pathways and accordingly may be used in the treatment of conditions involving perturbation of SAP 140 signaling. In particular, the substances may be particularly useful in the treatment of disorders of cell proliferation.
  • This invention enables a method for modulating signaling, the method comprising administering an agent that increases SAP activity or increases SAP expression in the hematopoietic cell.
  • the invention further enables a method for reducing or preventing cell activation and/or proliferation, the method comprising administering to the cell an agent which modulates SAP activity or expression in the hematopoietic cell.
  • the invention further enables a method for treating a disorder which requires immunosuppression such as transplantation, the method comprising administering to the subject in need of treatment an immunosuppressive amount of an agent which modulates SAP activity or expression.
  • the invention enables a method for treating lymphoma in a subject, the method comprising administering to the subject an agent that regulates SAP activity or expression, in an amount effective to reduce or prevent cell proliferation.
  • the invention further provides methods for preventing or treating disorders characterized by an abnormality in signaling, comprising modulating signaling by administration of an agent which increases or decreases SAP activity or expression.
  • Signaling modulation is useful in disorders such as autoimmune diseases and in transplant situations, as discussed elsewhere herein.
  • the present invention enables gene therapy as a potential therapeutic approach, in which normal copies of the SAP gene are introduced into patients to successfully code for normal SAP protein in several different affected cell types. Mutated copies of the SAP gene, in which the protein product is inactivated, can also be introduced into patients. Retroviral vectors can be used for somatic cell gene therapy especially because of their high efficiency of infection and stable integration and expression. The targeted cells however must be able to divide and the expression of the levels of normal protein should be high.
  • the full length SAP gene can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest (such as lymphoid cells).
  • viral vectors which can be used include adeno-associated virus, vaccinia virus, bovine papilloma virus, or a herpesvirus such as Epstein-Barr virus. Gene transfer could also be achieved using non-viral means requiring infection in vitro. This would include calcium phosphate, DEAE dextran, electroporation, cationic or anionic lipid formulations (liposomes) and protoplast fusion. Although these methods are available, many of these are lower efficiency.
  • Transplantation of normal genes or mutated genes that code for an active SAP into a targeted affected area of the patient can also be useful therapy for any disorder in which SAP activity is deficient.
  • a SAP gene is transferred into a cultivatable cell type such as lymphoid cells, either exogenously or endogenously to the patient.
  • the transformed cells are then injected into the patient.
  • the invention also provides a method for reversing a transformed phenotype that results from excessive expression from the SAP human gene sequence, and/or hyper-activation of a SAP protein product.
  • Anti-sense based strategies can be employed to explore gene function, inhibit gene function and as a basis for therapeutic drug design. The principle is based on the hypothesis that sequence specific suppression of gene expression can be achieved by intracellular hybridization between mRNA and a complementary anti-sense species. It is possible to synthesize anti-sense strand nucleotides that bind the sense strand of RNA or DNA with a high degree of specificity. The formation of a hybrid RNA duplex may interfere with the processing/transport/translation and/or stability of a target mRNA.
  • Hybridization is required for an antisense effect to occur.
  • Antisense effects have been described using a variety of approaches including the use of antisense oligonucleotides, injection of antisense RNA, DNA and transfection of antisense RNA expression vectors.
  • Therapeutic antisense nucleotides can be made as oligonucleotides or expressed nucleotides. Oligonucleotides are short single strands of DNA which are usually 15 to 20 nucleic acid bases long. Expressed nucleotides are made by an expression vector such as an adenoviral, retroviral or plasmid vector. The vector is administered to the cells in culture, or to a patient, whose cells then make the antisense nucleotide.
  • Expression vectors can be designed to produce antisense RNA, which can vary in length from a few dozen bases to several thousand.
  • Antisense effects can be induced by control (sense) sequences.
  • the extent of phenotypic changes are highly variable. Phenotypic effects induced by antisense are based on changes in criteria such as biological endpoints, protein levels, protein activation measurement and target mRNA levels.
  • Multidrug resistance is a useful model for the study of molecular events associated with phenotypic changes due to antisense effects since the MDR phenotype can be established by expression of a single gene mdrl (MDR gene) encoding P- glycoprotein (a 170 kDa membrane glycoprotein, ATP-dependent efflux pump).
  • MDR gene mdrl
  • mammalian cells in which the SAP gene is overexpressed and which demonstrate an abnormal phenotype can be transfected with anti-sense SAP nucleotide DNA sequences that hybridizes to the SAP gene in order to inhibit the transcription of the gene and reverse or reduce the abnormal phenotype.
  • Expression vectors can be used as a model for anti-sense gene therapy to target the SAP which is expressed in abnormal cells. In this manner abnormal cells and tissues can be targeted while allowing healthy cells to survive. This may prove to be an effective treatment for cell abnormalities, preferably hematopoietic cells, induced by SAP. Immunotherapy is also possible for the treatment of diseases associated with excess SAP activity.
  • Antibodies can be raised to a hyperactive SAP protein (or portion thereof) and then be administered to bind or block the abnormal protein and its deleterious effects.
  • An immunogenic composition may be prepared as injectables, as liquid solutions or emulsions.
  • the SAP protein may be mixed with pharmaceutically acceptable excipients compatible with the protein. Such excipients may include water, saline, dextrose, glycerol, ethanol and combinations thereof.
  • the immunogenic composition and vaccine may further contain auxiliary substances such as emulsifying agents or adjuvants to enhance effectiveness.
  • Immunogenic compositions and vaccines may be administered by subcutaneous or intramuscular injection. The immunogenic preparations and vaccines are administered in such amount as will be therapeutically effective, protective and immunogenic. Dosage depends on the route of administration and will vary according to the size of the host.
  • the invention also makes it possible to screen for antagonists that inhibit the effects of SAP 140.
  • the invention may be used to assay for a substance that anatagonizes or blocks the action of the protein.
  • Substances identified by the methods described herein may be used for modulating SAP 140 activity or action and accordingly may be used in the treatment of conditions involving perturbation of the protein.
  • the substances may be particularly useful in the treatment of disorders of hematopoietic cell proliferation.
  • compositions for administration to subjects in a biologically compatible form suitable for administration in vivo.
  • biologically compatible form suitable for administration in vivo is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects.
  • the substances may be administered to living organisms including humans, and animals.
  • a therapeutically active amount of pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • An active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. If the active substance is a nucleic acid encoding, for example, a modified SAP 140 it may be delivered using techniques known in the art.
  • compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985) or Handbook of Pharmaceutical Additives (compiled by Michael and Irene Ash, Gower Publishing Limited, Aldershot, England (1995)).
  • the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and may be contained in buffered solutions with a suitable pH and/or be iso-osmotic with physiological fluids.
  • Example 1 Isolation of SAP by Two hybrid Screening with the non-catalytic portion of Lyp.
  • the non-catalytic domain of Lypl was used in the yeast two hybrid binding domain vector vector pBTM116 to screen a B lymphocyte MATCHMAKER cDNA library (Clontech, Palo Alto, CA) in the pACT activation domain vector. Screening was performed as per the manufacturers instructions. After isolating a positive, LacZ+, His+ clone (containing SAP) the following steps were taken to ensure this was not a false positive interaction. 1. The DNA from the positive clone was PCR-amplified using specific oligonucleotide primers to the AD/library plasmid.
  • the PCR reaction amplified a single DNA band, proving that the colony contained single bait.
  • Cycloheximide counterselection was performed to obtain an AD plasmid segregant (colony that has lost the DNA-BD and retained the AD).
  • 3. Yeast colonies transformed with AD-SAP plasmid were grown on SD ⁇ Leu plate and colony-lift filter assays for ⁇ -galactosidase activity performed. There was no- ⁇ -galactosidase activity, proving that the positive clone wasn't picked because of auto- ⁇ -galactosidase activation by AD-SAP.
  • the following yeast mating experiment was performed (Matchmaker Gal4 two-hybrid system- Invitrogen. Inc.(CA)).
  • DNA-BD/ lamin C (in Yl 87) with AD/SAP (in CG- 1945)
  • a semi-nested PCR approach was taken to library screening.
  • a pair of adjacent oligonucleotide primers were designed from the SAP sequence and used in sequential PCR reactions.
  • Human spleen and thymocyte ⁇ gtl l cDNA libraries purchased from Clontech (CA) were used for screening.
  • a reaction was performed with the downstream primer and a primer to either 5' or 3' vector sequence.
  • SAP cDNA was then isolated by RT-PCR from several types of human cells (EBV transformed B cell, Jurkat T cell, primary thymocytes and primary peripheral lymphocytes) and each PCR product sequenced for conformation.
  • the open reading frame of SAP codes for an 1098 amino acid protein that does not belong to any identified family.
  • the cDNA encodes a novel non-transmembrane protein of 124kDa. Although novel, areas of the protein display significant homology with a conserved fold known as the Vietnamese domain. This domain was first described as a repeated element in the product of the Drosophila locus tudor, which is required during oogenesis for formation of primordial germ cells and segmentation (Boswell and Mahowald 1985, Bardsley et al 1993, Golumbeski et al 1991).
  • SAP cDNA sequence Comparison of public genomic DNA sequence databases with the SAP cDNA sequence resulted in the identification of the intron-exon boundaries of SAP as well as identification of its 5' upstream promoter sequences.
  • the genomic structure of SAP is illustrated in Figure 4.
  • the SAP mRNA is encoded by 19 exons of which 18 encode the open reading frame of the protein. Most of the SAP exon-intron boundaries conform to the recognized conserved donor and acceptor sequences.
  • Analysis of the 5' untranslated sequence with a variety of predictive software programs revealed several potential TATA box sequences (sites of RNA polymerase entry) and thus several potential sites of transcriptional initiation.
  • Modified 5' RACE performed to recognize only 5' capped mRNA structures, resulted in the amplification of a single DNA fragment of 400bp (utilizing a SAP primer at +100bp in the coding sequence), suggesting the presence of about 300bp 5' untranslated sequence and co-inciding precisely with a predicted transcription initiation site, 20 bp downstream of a putative TATA box.
  • the genomic clones encoding the SAP sequences were derived from human chromosome 9.
  • the predicted SAP protein has an isoelectric point of 6.92. No potential transmembrane spans are evident in hydrophobicity plots. A representative hydrophobicity plot is shown in Figure 5 A.
  • Example 5 Example 5
  • the antibody to the C-terminus (anti-sera 601) was unable to immunoprecipitate the pi 40 protein from Jurkat lysates, it was found to also recognize the protein immunoprecipitated by anti-sera 99 in western blot assays. Matching pre-bleeds for both anti-sera were unable to recognize the 140kD protein.
  • the pi 40 protein was the only major band detected in lysates from Jurkat cells by 99 or 601 anti-sera.
  • the protein is slightly larger than the product from the transfected cDNA in COS-7 cells (at 135kD), but this may represent an increase in post-translational modification in T cells, with increased serine/threonine or tyrosine phosphorylation.
  • Example 8 the protein is slightly larger than the product from the transfected cDNA in COS-7 cells (at 135kD), but this may represent an increase in post-translational modification in T cells, with increased serine/threonine or tyrosine phosphorylation.
  • SAP 140 expression was low in human peripheral blood T lymphocytes, but increased greatly after 24 hours activation with the strong mitogen phytohemaglutinin (Figure 8).
  • pl40 was also strongly expressed in freshly isolated primary human thymocytes, without any in vitro stimulation.
  • the 99 anti-sera could also precipitate pi 40 from murine thymocytes, but did not detect any protein in murine brain lysates. In sum, pi 40 expression would appear to be restricted to hematopoietic cells.
  • FLAG-SAP did not bind any GST- SH2 domain examined but, as shown in Figure 9, selective association with the C- terminal SH3 domain of the linker protein Grb2 (lane 2) and the SH3 domain of Phospholipase C ⁇ -1 (lane 5) was observed.
  • the N-terminal SH3 domain of Grb2 which has the basic structure SH3-SH2-SH3 (lane 1), did not bind SAP, nor did SH3 domains from the src-like kinases Lck (lane 3) or Fyn (lane 4), or from the rasGAP protein (lane 6). Binding to Grb2 may link SAP to many signaling pathways as this small adapter protein serves to link effector molecules, most notably the ras activation pathway, to numerous receptors. Binding to the PLC ⁇ -1 SH3 domain suggests that SAP may also be involved in inositol tri-phosphate and diacylglycerol secondary messenger production.
  • the transfected SAP protein did not demonstrate basal tyrosine phosphorylation and minimal phosphorylation was induced by treatment of the transfected cells with sodium orthovanadate and peroxide, a reagent which greatly increases the general level of tyrosine phosphorylation activity ).
  • co- transfection of the protein with cytoplasmic tyrosine kinases revealed that it was a substrate of several src-family tyrosine kinases ( Figure 13).
  • Co-transfection with Src (c-Src and v-Src) or Lyn resulted in SAP phosphorylation, but not with the related Fyn or Lck kinases, or with Jak3 or Emt.
  • co-transfection with Emt did not result in SAP phosphorylation, the kinase co-immunoprecipiated with the protein.
  • SAP underwent tyrosine phosphorylation upon co-transfection with several members of the src-family of kinases in 293 -T cells, suggesting that it may be a target of the Fyn and Lck kinases in T cells. Both of these kinases play an important role in initiating signaling through the T cell antigen receptor. Stimulation of Jurkat T cells with anti-CD3 and immunoprecipitation of pi 40 revealed that it underwent rapid and transient tyrosine phosphorylation within 30 seconds (Figure 15). pl40 demonstrated strongly phosphorylation, which reached a peak after two minutes and slowly subsided thereafter.
  • ppl40 would indeed appear to be SAP 140 as demonstrated by its absence from Grb2 immunoprecipitates in E6.1 cells, which do not express SAP 140, and by the ability to reduce the ppl40 band by pre-clearing with anti-SAP.
  • a complex of Grb2 and SAP 140 could be directly identified in human thymocytes ( Figure 21).
  • SAP 140 does not undergo the same degree of TCR induced phosphorylation as in Jurkat, Western blotting of Grb2 immunoprecipitates with anti-SAP permitted the direct visualization of the complex, although only a small portion of the entire SAP140 pool in thymocytes appeared to be associated with Grb2.
  • Grb2 forms a well characterized complex with the adapter protein She (Ravichandran et al 1995). Comparison of the tyrosine phosphorylation patterns of Grb2 and She immunoprecipitated from anti-CD3 stimulated Jurkat cells revealed that She also co-precipitated ppl40, possibly more strongly that Grb2 ( Figure 22). Once again, the phosphorylation kinetics of ppl40 in She matched those of SAP140. Direct comparison of She and SAP 140 immunoprecipitates revealed exceedingly similar patterns upon phospho tyrosine blotting, with a dominant ppl40 band and a minor 1 lOkD band ( Figure 23). ppl40 was not present in She immunoprecipitates from E6.1 cells as expected.
  • She and SAP 140 immunoprecipitates from an AML cell line revealed that SAP 140 did indeed demonstrate significant tyrosine phosphorylation and did appear to be constitutively complexed with She ( Figure 24).
  • the She in ppl40 complexes has previously been shown to be highly tyrosine phosphorylated, without wishing to be bound by any particular theory, this is consistent with an ongoing proliferative signal transduction cascade, either as the consequence of an autocrine loop, or a mutation in regulatory element. She-SAP 140 association may therefore be a central event in the proliferative capacity of certain AML and ALL malignancies.
  • Rabbit polyclonal antibodies were raised commercially by Research Genetics Inc (Huntsville A1.35801) against three peptides, LEGDLVSKMLRAVLQ and corresponding to the N-terminal and the third Mathematics domain respectively of the SAP 140 sequence.
  • the peptides were conjugated via their 5 'ends to a carrier protein, Keyhole limpet hemacyanin, and the rabbits given a primary injection of 0.5mg peptide in Freund's complete adjuvant. This was followed by two booster injections of 0.5mg peptide in Freund's incomplete adjuvant, given at two-week intervals. Serum was collected four weeks after the first, second and third injections and assayed for specific antibodies against the immunizing peptides using an ELISA. Eucaroyotic expression.
  • the SAP cDNA was subcloned into the pcDNA3 expression vector (Invitrogen) and tagged at the 5' end by the addition of the 8 amino acid FLAG® tag. Lyp binding to GST-SAP
  • a GST fusion protein of SAP was constructed and the ability to bind T7-tagged Lyp assessed.
  • the fragment of the entire SAP sequence originally isolated was cloned in frame into the pGEX4-T-2 GST fusion procaryotic expression vector (Pharmacia Biotech, Uppsala). Expression of the protein was induced in transfected DH5 ⁇ with ImM IPTG followed by two hours growth before harvest. Bacteria were lysed by sonication and addition of Triton-XlOO to a 1% final concentration.
  • the resulting supernatant was clarified by centrifugation and the released GST-fusion proteins purified by binding to Glutathione Sepharose 4B (Pharmacia Biotech). After extensive washing the complexes were checked for purity and quantitated on SDS- PAGE by reference to standard proteins of similar molecular weight after staining with Coomassie Blue.
  • GST was prepared from bacteria containing empty vector.
  • 293 cells were transfected with Lyp, 1% NP-40 lysates made in standard lysis buffer (20mM Tris pH 7.6, 150mM NaCl, ImM Na 3 VO 4 , 1% NP-40) and incubated overnight with purified GST-SAP fusion protein on glutathione beads at 4°C with constant agitation. Complexes were washed, subjected to SDS-PAGE and transferred to nitrocellulose membrane. Western blotting was then performed with anti-T7 tag (Novagen) to detect bound Lyp. Lyp bound specifically to the GST-SAP fusion, as neither GST itself, nor the negative control GST-PLC ⁇ l -SH2 (purchased from Santa Cruz Biotechnology (Santa Cruz, CA)) demonstrated detectable Lyp binding. Binding of SAP to GST-SH3 fusion proteins of signaling proteins.
  • Adherent 293 cells were transiently transfected using the lipid reagent Lipofectamine (Life Technologies, Grand Island, NY). 5 ⁇ g FLAG-SAP cDNA in the pcDNA3 expression vector (Invitrogen) was used in each transfection. The DNA-lipid mixtures were applied to the cells for 5 hours in the absence of serum, before the addition of complete medium. Cells were given 48 hours to express the transfected proteins before harvesting. Cells were harvested by mechanical scraping, pelleted and resuspended in ice cold lysis buffer consisting of lOmM Tris pH7.6, 150mM NaCl, 5mM EDTA, ImM Na 3 VO 4 , ImM PMSF and 1% NP-40.
  • Antibodies to phosphotyrosine and FLAG were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies to Cbl, PLC ⁇ l, Vav, Grb2 and She were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti- T7 epitope tag from Novagen Inc. (Madison, WI). Rabbit polyclonal antibodies to human Lyp, were created in this laboratory as previously described (Cohen et al. 1999), except further purified by affinity chromatography on resin linked immunizing peptides. Immunoprecipitation and Western Blotting.
  • Cells were resuspended in RPMI 1640 at a density of 10xl0 6 /ml and incubated at 37°C with 2-5 ⁇ g/ml anti-CD3 for the indicated periods of time. Cells were then quickly pelleted and resuspended in ice cold lysis buffer consisting of 1 OmM Tris pH7.6, 150mM NaCl, 5mM EDTA, ImM Na 3 VO 4 , ImM PMSF and 1% NP-40. After solubilization for 15 minutes on ice, debris was removed by centrifugation at 12,000g for 10 minutes at 4°C.
  • Protein A Sepharose- CL4B or Protein-G Sepharose for monoclonal antibodies
  • Immunoprecipitates were collected by a brief centrifugation and washed 3-4 times in lysis buffer (without PMSF) before addition of SDS sample buffer. Samples were separated on SDS-polyacrylamide gels and transferred to nylon supported nitrocellulose membranes (Amersham, Arlington Heights, IL). Membranes were blocked overnight at 4°C with 5% blotting grade non-fat milk (Biorad, Richmond, CA) in PBS.
  • Immunoblotting antibodies were added at optimal dilutions in PBS-T (0.1% Tween-20) containing 1% non-fat milk and incubated either at RT for two hours or at 4°C overnight. After extensive washing with PBS-T, bound antibodies were detected using horse-radish peroxidase conjugated donkey anti-rabbit or sheep anti-mouse antibodies (Amersham) and Lumiglo chemiluminescent reagents (Kirkegaard and Perry, MA). Transfection of COS-7/293
  • the adherent COS-7 and 293 cells were routinely transiently transfected using the lipid reagent lipofectamine (Life Technologies, Grand Island, NY).
  • the DNA- lipid mixtures were applied to the cells for 5 hours in the absence of serum, before the addition of complete medium. Cells were given 24 hours to express the transfected proteins before harvesting.
  • Purification of T lymphoctyes from peripheral blood Normal peripheral blood was diluted with PBS and mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia) gradient centrifugation. T lymphocytes were isolated from the mononuclear cell population by rosetting with neuraminidase treated sheep red blood cells.
  • Rosettes were isolated on a second Ficoll-Hypaque gradient centrifugation at 4°C. The rosetted T cells were then briefly treated with ACT (ammonium chloride-Tris), in order to lyse the sheep red blood cells. T cell populations recovered by this technique are typically 95-97% CD3 + , with less than 1% B cell contamination.
  • ACT ammonium chloride-Tris
  • Boswell RE Mahowald AP., tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster, Cell. 1985 Nov;43(l):97-104.
  • CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation, J Exp Med. 1996 Apr 1 ; 183(4): 1707-18.
  • Dahia PL PTEN, a unique tumor suppressor gene, Endocr Relat Cancer. 2000 Jun;7(2): 115-29.
  • Pelicci G Lanfrancone L, Salcini AE, Romano A, Mele S, Grazia Borrello M, Segatto O, Di Fiore PP, Pelicci PG, Constitutive phosphorylation of She proteins in human tumors, Oncogene. 1995 Sep 7;ll(5):899-907.

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Abstract

La présente invention concerne la protéine SAP140, une nouvelle protéine associée à Shc, qui est isolée par liaison à la tyrosine phosphatase Lyp. La protéine SAP est exprimée dans des cellules hématopoïétiques et est impliquée dans la transduction de signal, à la fois sur des voies de récepteur d'antigène et sur des voies de récepteur de cytokine. La protéine SAP140 est préalablement activée en cas de leucémie myéloïde aiguë. La manipulation de l'activité de la protéine SAP peut donc présenter un intérêt thérapeutique en cas de leucémie myéloïde aiguë, de leucémie lymphoblastique aiguë et de maladies à cellules T incontrôlées ou de maladies du système hématopoïétique en général.
PCT/CA2001/000023 2000-01-10 2001-01-10 Proteine 140 associee a shc (sap-140) WO2001051509A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002068470A2 (fr) * 2001-02-26 2002-09-06 Pharma Pacific Pty Ltd Gene induit par l'interferon alpha
CN115177730A (zh) * 2022-08-05 2022-10-14 华中科技大学同济医学院附属协和医院 Ptpn22及其表达抑制剂的新用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIROSE : "Identification of a novel protein, TRAP, that interacts with N-terminal domain of PCTAIRE 2 in rat brain" SWALL:Q9R1R4, 1 May 2000 (2000-05-01), XP002172288 *
J]CKER ET AL: "A tyrosine-phosphorylated protein of 140 kD is constitutively associated with the phosphotyrosine binding domain of Shc and the SH3 domain of Grb2 in acute myeloid leukemia cells" BLOOD, vol. 89, no. 6, 15 March 1997 (1997-03-15), pages 2024-2035, XP002172287 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002068470A2 (fr) * 2001-02-26 2002-09-06 Pharma Pacific Pty Ltd Gene induit par l'interferon alpha
WO2002068470A3 (fr) * 2001-02-26 2002-12-05 Pharma Pacific Pty Ltd Gene induit par l'interferon alpha
CN115177730A (zh) * 2022-08-05 2022-10-14 华中科技大学同济医学院附属协和医院 Ptpn22及其表达抑制剂的新用途
CN115177730B (zh) * 2022-08-05 2024-02-27 华中科技大学同济医学院附属协和医院 Ptpn22及其表达抑制剂的新用途

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