WO1988001303A1

WO1988001303A1 - Protein kinase c enzymes

Info

Publication number: WO1988001303A1
Application number: PCT/US1987/002005
Authority: WO
Inventors: John L. Knopf
Original assignee: Genetics Institute, Inc.
Priority date: 1986-08-13
Filing date: 1987-08-13
Publication date: 1988-02-25
Also published as: EP0317574A4; EP0317574A1; JPH02500243A; AU7851387A

Abstract

A novel family of cDNA sequences which encode protein kinase C enzime. Such sequences are employed in a simple binding assay to assess the tumor promoting activity of various compounds.

Description

PROTEIN KINASE C ENZYMES

The present invention relates to novel DNA sequences encoding a family of Protein inase C enzymes, which are useful in a novel method of detecting the tumor promoting capacity of a chemical substance or compound.

BACKGROUND

Protein kinase C ("PKC") is a calcium and phospholipid dependent protein kinase, originally isolated from rat brain cytosol, and known to be ubiquitious in mammalian tissues and organs. PKC is an intracellular enzyme that resides in a resting cell in a "turned-off" state. PKC is directly activated by diacylglycerols (DAG) , minor components of cellular lipids, which increase the affinity of PKC for calcium ions (Ca^+j and phospholipid. When a cell is contacted by an agent, e.g. IL-2, the enzyme phospholipase C is activated, liberating DAG from phosphatidyl inositol. DAG then binds to and activates PKC which phosphorylates its target molecules, which initiate cellular division or cellular differentiation. The response initiated depends upon the particular PKC target molecule effected. For example, PKC influences platelets to release serotonin, lysosomal enzyme and arachidonate. Its effect on both basophils and mast cells mediates histamine release. Other cellular responses mediated by PKC are known to those of skill in the art [See Y. Nishizuka, Science. 233:305-312 (1986) and references cited therein].

In a normal cell, the appearance of DAG in the cell membranes is transient due to its reconversion into an inositol phospholipid or degradation. Thus PKC is active for only a short time after its stimulation by DAG. The result of PKC activation, however, may persist for a longer period depending on the biological stability of the phospate which is attached to the target molecule. Eventually, however, the cell differentiation or division directed by PKC activation ceases in a normal cell until further PKC activation.

PKC activation also plays a role in tumor formation. Tumor formation is a two-stage process characterized by initiation, which involves the transformation of a cell into a "pre-cancerous" cell and promotion, which involves the proliferation and possible evolution of the transformed cell, thereby creating a tumor. A carcinogen, e.g. virus, chemical substance or radiation, enhances initiation by increasing the rate or probability of genetic transformation of the cells or their descendants, resulting in the emergence of cells capable of forming tumors.

A tumor promoter is a substance or process which, when exposed to cells in combination with one or more carcinogens, _^significantly increases the size^* or incidence of tumors. Tumor promoting phorbol esters, such as 1,2-0-tetra- decaroyl-phorbol-1,3-acetate (TPA) have structures similar to DAG and activate PKC directly both in vivo and in vitro. Because phorbol esters are not as easily degraded in vivo as DAG, their continued presence in the cell is believed to extend a normally limited phase of cellular response, which results in the formation of tumors. The binding of and activation of PKC by tumor promoters initiates tumor promotion. The potency of tumor promoters as determined by the standard mouse skin test assay has been shown by a number of investigators to be directly correlated with their affinity for PKC. For example, a potent tumor promoter, e.g., TPA, always binds more strongly to PKC than a weaker tumor promoter, phorbol-12,13-di-butyrate (PDBu) . [See C. Ashendel, Biochim. Biophvs. Acta. 822; 219 (1985)]. Other substanceswhichhavebeen claimedby individual investigators to be weak tumor promoters at high doses, e.g. NaCl, do not bind to PKC. [See, M- Takahashi et al. , Gann, 75:491 (1984) and H. Ohgaki et al., Gann. 75,1053 (1984).] However, all potent tumor promoters have been shown to bind to and activate PKC at nanomolar concentrations.

Tumor promoting activity of various compounds, e.g., drugs, pesticides, food additives, cosmetics and numerous other chemicals which come into contact with humans, is commonly tested using an jLn vivo assay wherein the suspected tumor promoting substance or process is administered to an animal in combination with a carcinogen that has a known level of σarcinogenicity, e.g., the mouse skin test. A variety of cellular assays involving TPA are also known to those skilled in the art. [See, e.g., U.S. Pat. 4,442,203 and references cited therein. ] However, these known assays do not provide accurate quantitative results and frequently suffer from inaccuracies due to the level of technical skill of the investigator.

BRIEF SUMMARY OF THE- INVENTION

As one aspect of the present invention-, a number of related PKC polypeptides are provided which are encoded by novel DNA sequences, including those depicted in Figs. 1 and 3 and designated as DNA sequences I, II, III and IV, in a 5' to 3' direction. These PKC polypeptides are characterized by a ino acid sequences substantially as shown in Figs. 2 and 3. Variations in the DNA and amino acid sequences of Figs. 1 through 3 which are caused by allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) , point mutations or by induced modifications to enhance the activity or production of the polypeptides are also encompassed in the invention. Similarly, synthetic polypeptides which wholly or partially duplicate continuous sequences of the amino acid residues of Figs 2 and 3 are also part of this invention. These sequences, by virtue of sharing primary, secondary or tertiary structural and conformational characteristics with naturally-oσcuring PKC polypeptides of the invention may possess biological activity and/or immunological properties in common with the naturally- occurring polypeptide. Thus, they may be employed as biologically active or immunological substitutes for naturally-occurring PKC polypeptides in the assays of the present invention.

Additionally fragments of the novel polypeptides and DNA sequences provided herein, which fragments are capable of binding substances having tumor promoting activity, are also, included in the invention. Those fragments as well as the complete sequences of Figs. 1, 2 and 3 may be employed in assays for determining tumor promoting activity.

As another aspect of the present invention, there is provided a novel method for producing the PKC polypeptides of this invention. The method of the present invention involves culturing a suitable cell or cell line, which has been transformed with a vector containing a DNA sequence coding-on expression for a novel PKC pdlypeptide. Suitable, cells or cell^' lines may be mammalian cells, such as Chinese hamster ovary cells (CHO) . Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line. Also suitable for use in the present invention are bacterial cells. For example, the various strains of E. coli are well-known as host cells in the field of biotechnology. Various strains of B. subtilis may also be employed in this method. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention.

Another aspect of the present invention provides vectors for use in the method of expression of these novel PKC polypeptides. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Alternatively, vectors incorporating modified sequences as described above are also embodiments of the present invention and useful in the production of these PKC polypeptides. The vectors employed in the method also contain selected regulatory sequences known to those skilled in the art in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.

As yet a further aspect of the present invention, there is provided a simple assay for testing the tumor promoting potential of a compound. The assay employs a standard binding reaction of a test compound to a stable PKC polypeptide or fragment of the present invention. This binding assay involves a comparison between the competitive in vitro binding of PKC by the test compound or a known labelled compound having a known PKC binding capacity. If the test compound competes with the labelled compound for activation of PKC, it is thereby identified as having .tumor promoting ability. This ability is confirmed by also acting as a cofactor in a phosphotransferase assay. If the test compound functions as an inhibitor in the phosphotransferase assay, it is an antagonist of the PKC, and may be employed further in drug design. If the test compound does not interfere at any concentration with the binding of the known labelled compound, it is not a tumor promoting agent.

The advantages of this assay over presently employed tests for tumor promoting activity are both its simplicity quantitative accuracy and speed of results. The assay permits use of a stable, pure PKC polypeptide of the invention which is in unlimited supply. Additionally, the use of members of the PKC family of polypeptides allows for use of

PKC enzymes which would not be present on mouse skin, e.g.

PKC enzymes present only in bladder.

The present invention is further disclosed by the following detailed description of preferred embodiments thereof. Brief Description of the Drawings

Fig. 1 depicts the aligned nucleotide sequences of PKC-I, II, and III. The first in-frame initiator ATG is underlined for each, as is the translation terminator. An in-frame stop codon appearing in the 5^! untranslated region of PKC-I is indicated by .***. PKC-III is substantially identical to PKC-II and nucleotides in PKC-III which differ from those in PKC-II are indicated in the row below the PKC-II sequence. The sequence of the 32mer probe is also aligned as indicated, with an X representing a mismatch.

Fig. 2 depicts the aligned amino acid sequences of PKC I, II and III. T-69, T-60, T-67 and T-57 all indicate the amino acid sequence obtained from purified tryptic fragments of rat brain protein kinase C. Amino acids in PKC-III which differ from PKCf-II are indicated below the PKC-II sequence. Homology to both cAMP and. GMP dependent protein kinases begins at residue 340 and extends over the entire carboxyi terminal region.

Fig. 3 illustrates the DNA and amino acid sequences of the human PKC enzyme, designated PKC IV.

Detailed Description of the Invention

The present invention provides a family of PKC polypeptides characterized by amino acid sequences substantially homologous to those of Figs. 2 and 3. These sequences may be encoded by the specific DNA sequences I, II and III of Fig. 1, by sequence IV of Fig. 3 or by DNA sequences capable of hybridizing thereto and exhibiting biological characteristics of PKC. The sequences illustrated in the figures are exemplary PKC sequences of the present invention. The three DNA sequences, PKC-I, PKC-II, and PKC-III, were isolated from a rat brain cDNA library, and encode closely related but distinct polypeptides. PKC- IV is a human sequence also encoding a variant of the PKC family of proteins. PKC-I and PKC-II, contain complete coding regions. PKC-I, PKC-II and PKC-IV induce in cells the synthesis of high affinity phorbol ester binding activity, as well as a Ca²⁺, phosphatidylserine and DAG/PDBu, dependent protein kinase activity. These sequences or fragments thereof may be employed as probes for the recovery of other species PKC polypeptides, particularly human polypeptides. These PKC polypeptides may also be employed as probes to detect PKC isozymes characteristic of specific tissue sources, e.g. brain, lung, bladder.

The DNA sequence for PKC-I (Fig. 1, sequence I) encodes a protein of 697 residues (78,366 daltons) . The DNA sequence for PKC-II (Fig. 1, sequence II) encodes a protein of 673 residues (76,660 daltons) . The amino terminal regions of PKC-I and PKC-II both contain an internal repeat of 50 amino acids centered about a repeating unit of 6 cysteine residues. The positions of the cysteines relative to one another are precisely conserved in both clones. The DNA sequence for PKC-III (Fig. 1, sequence III) also shows the conservation of the cysteine residues in the region ^" corresponding to the second repeat. The DNA sequence for PKC-IV, the human sequence, is homologous to the rat sequences.

The phorbol ester binding sites of these proteins are within approximately the first 95 amino terminal amino acids of the protein sequences of PKC I, PKC II and PKC IV. For example, this binding site exists in PKC I and II, from approximately amino acid position number 1 to amino acid position number 95 in each sequence of Fig. 2. In the human peptide sequence, this binding region is within the region from approximately amino acid position number 1 to amino acid position number 95 in Fig. 3.

Thus where PKC fragments, rather than the complete PKC peptides, are desired for use in an assay to detect tumor promoting activity of a compound, fragments within the above-described regions containing the binding sites may be employed. Smaller fragments within these regions are also expected to be capable of binding phorbol esters and other tumor promoters.

A search of the National Biomedical Research Foundation protein database for protein sequences homologous to PKC-I, PKC-II and PKC-III revealed that PKC-I, II and III have a 35 kd carboxyl terminal domain which is homologous to the catalytic domain of most protein kinases. The greatest degree of homology is seen with the other serine/threonine kinases, such as cAMP and cGMP dependent protein kinases, which were each approximately 40% homologous to PKC-I, II and III over their catalytic region. It was also possible to align PKC-I, II and III with a number of oncogene kinases.

Mild trypsinization of purified PKC results in the generation of two fragments of approximately 32 and 51 kd [M. noue et al., J. Biol. Chem.. 252:7610 (1977)]. The 51 kd fragment contains protein kinase activity independent of calcium, phosphatidylserine and PDBu. Based on the homology of the carboxyl terminal portions of PKC-I, II and III with other known protein kinases it is^* likely that the 51 kd fragment is derived from the carboxyl terminal region, and the 32 kd fragment is derived from the amino terminus. The 32 kd fragment of the PKC sequences confers calcium, phospholipid and phorbol ester/DAG dependence upon the kinase domain.

PKC-I, PKC-II, and PKC-III were each subcloned into a COS-cell expression vector pMT-2. The novel DNA sequences of Table I included in the COS-cell expression vector have been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD and given accession numbers ATCC 40252 for PKC-I-PMT2, ATCC 40253 for PKC-II-PMT2 and ATCC 40254 for PKC-III-PMT2. These sequences and fragments thereof may also be prepared synthetically by means conventional in the art.

The following examples illustrate the novel DNA sequences, polypeptides and assays of the present invention. EXAMPLE I Purification and Analysis of PKC

PKC was purified from Charles River CD female rat brains by the method of T. Kitano et al., Meth in Enzymol. , 124: 349-352 (1986) with slight modification. The final specific activity as measured by the mixed micelle assay [Y. Hannun et al., J. Biol. Chem.. 260: 10039-10043 (1985)] was 1.2 umol/min/mg protein.

Approximately 100 ug of the PKC preparation (volume of 7 mis) was made 0.05% in sodium dodecyl sulphate (SDS) and concentrated by electrophoresis [M.W. Hunkapiller et al. , Meth. Enzynol_f £1:227-236 (1982)]. Following sample reduction, SDS-polyacrylamide gel electrophoresis and staining, the predominant protein band of apparent molecular weight of 77,000 to 80,000 daltons was titrated to neutrality in situ with 0.1 M NH₄HC0₃ and subjected to trypsinization (1% w/w) for 22 -hours at 37°C. The tryptic digest was subjected directly, to reverse phase high pressure liquid chromatography (RPHPLC-C4 Hydac column) using a linear gradient of acetonitrile in 0.1% trifluoroacetic acid at 1% acetonitrile per minute. Eluted peptides were monitored by UV absorbance at 214 and 280 nm.

Selected peptides were subjected to N-terminal amino acid sequence analysis. The amino acid sequence of one tryptic peptide was used to design the following antisense oligodeoxyribonucleotide of 32 residues for use as a probe:

5' TAA CGG GGA CTC TAG TAA CGG ATG CTC GGG AT 3 '

EXAMPLE II cDNA Library Construction and Screening

RNA was extracted from adult male Sprague Dawley rats and Poly A⁺ selected as described by N. D. Hastie et al.. Cell. 17:449 (1979). 5 micrograms of Poly (A⁺) rat brain RNA was reverse transcribed with murine reverse transcriptase (BRL) . Following first strand synthesis the method of J. J. Toole et al., Nature. 312:342 (1984) was used with modification. The following EcoRI adaptors were substituted for the EcoRI linkers: 5^! AATTCCTCGAGAGCT 3' and a 3' GGAGCTCTCGA 5' .

1 x 10⁵ recombinant GT10 phage from the rat brain cDNA library were screened with the 32-mer described above, isolating 25 positive clones. Restriction map and partial sequence analysis identfied two types of clones which were sequenced completely. The Type I sequence is illustrated in Fig. 1 from residues 462 to 3113. The Type II sequence spans residues 800 to 2105. The appropriate open reading frame from each clone extended to the 5¹ end and did not have the capacity to encode a 77-80 kd polypeptide. Neither clone contained all of the coding region. Phage with Type II inserts containing the entire coding region were obtained by screening an additional 2 x 10⁵ phage with a 27 mer specific for the Type II clones (residues 837 to 863 of Fig. 1) . Three clones .were obtained, two of approximately 2.2. kb and one of ^'approximately 2.6 kb. Both 2.2 kb clones had restriction maps expected for a Type II insert. The 2.6 kb insert had a different restriction map.

The first 400 nucleotides of one of the 2.2 kb clones, called PKC-II, was sequenced [See, e.g., M. Poncz et al., Proc. Natl. Acad. Sci. USA. 74:5463 (1977)] and found to merge with the sequence obtained from the original shorter Type II clone. Additional partial sequencing of residues 3 ' to residue 400 failed to reveal any differences between the overlapping regions of these two clones. PKC-II extends from nucleotides 1 to 2261 in Fig. 1, which shows a composite sequence. A restriction site analysis of the 2.6 kb clone revealed the absence of a 3 ' EcoRI site present in other Type II clones. Partial sequence analysis of this clone (PKC-III) revealed 8 nucleotide differences between it and PKC-II in the 5' coding region and a large number of differences in the 3' coding and non-coding regions.

Clones containing the entire coding region of the Type I clones were obtained by constructing an oligonucleotide (26 mer nucleotide number 494 to 519 in Fig. 1) specific for Type I clones. An additional 2 x 10⁵ phage were screened with this oligonucleotide and a nick translated fragment of PKC-II corresponding to nucleotides 1-379. One phage was obtained which was positive with both probes and had an insert of approximately 2.6 kb. The first 380 bp of this clone were sequenced and found to merge with the sequence obtained from the smaller Type I clone.

Further partial sequence analysis of this clone failed to reveal any differences between these two clones. The sequence of PKC I extends from residues 1 to 2575 in Fig. 1. Nucleotide sequence analysis was performed as in M. Poncz et al., Proc. Natl Acad. Sci. U.S.A.. 79: 269-276 (1982); J. Messing and J. Vieira, Gene. 12:269:276 (1982); and F. Sanger et al., Proc. Natl Acad. Sci. U.S.A.. 74: 5463-5467 (1977)].

EXAMPLE III Construction of a PKC Vector and Cos Cell Transfection

Clone PKC-I was excised from lambda GT10 by EcoRI. Clone PKC-II was excised with Xhol. Plasmid PKC-I-PMT2 is constructed by inserting the EcoRI sequence I of Fig. 1 into the EcoRI digested COS expression vector, PMT-2, a B-laσtamase derivative of p91023 [Wong et al.. Science. 228:810-815 (1985).] Similarly a PKC-II vector may be constructed by inserting sequence II of Fig. 1 into Xhol- digested PMT2. The plasmid is then transformed by conventional techniques into a selected host cell for expression of a PKC polypeptide. Exemplary host cells are mammalian cells and cell lines, particularly primate cell lines, rodent cell lines and the like.

To obtain expression of PKC-I for use in the assays of Example VII, PKC-I-PMT2 8 ug. was transfected into 2 x 10⁶ COS cells [Gluzman, Cell. 23.:175 (1981)] in a 10 cm dish by the DEAE dextran protocol [L.M. So payrac et al., Proc. Nat'l Acad. Sci. USA, 7_8:7575 (1981) ] with the addition of a chloroquine treatment [H. Luthman et al., Nucl. Acids Res. 11:1295 (1983) ] . Generally cells wereharvested 60 hours after the addition of the DNA.

One skilled in the art can also contruct other mammalian expression vectors comparable to PKC-I-PMT2 by, e.g., cutting the DNA sequence (I) or (II) of Fig. 1 sequence of from the plasmid with EcoRI or Xhol respectively and employing well-kno.wn. recombinant genetic engineering techniques and other: known, vectors, such as pCD [Okayama et al., Mol Cell Blbl.. 2.:\L61-170 (1982)] and pJL3, pJL4 [Gough et al., EMBO J. , 4.:645-653 (1985)]. The transformation of these vectors into appropriate host cells can result in expression of a PKC polypeptide.

Similarly, one skilled in the art could manipulate the sequences of Figs. 1 through 3 by eliminating or replacing the mammalian regulatory sequences flanking the coding sequence with bacterial sequences to create bacterial vectors for intraσellular o ^'.extracellular expression by bacterial cells. For example, the coding sequences of Fig. 1 and 2 could be cut from PKC-I-PMT2 or PKC-II-PMT2 with EcoRI or Xho and further manipulated (e.g., ligated to other known linkers or modified by deleting non-coding sequences therefrom or altering nucleotides therein by other known techniques) . The modified PKC coding sequence could then be inserted into a known bacterial vector using procedures such as described in . Taniguchi et al. , Proc. Natl Acad. Sci. USA. 72:5230-5233 (1980). This exemplary bacterial vector could then be transformed into bacterial host cells and PKCX expressed thereby. For a stragegy for producing extracellular expression of PKC in bacterial cells, see, e.g. European patent application EP 177,343.

Similar manipulations can be performed for the construction of an insect vector [See, e.g., procedures described in published European patent application 155,476] for expression in insect cells. A yeast vector could also be constructed employing yeast regulatory sequences for intracellular or extracellular expression of the proteins of the present inventions by yeast cells. [See, e.g., procedures described in published PCT application WO 86 00639 and European patent application EP 123,289.]

Expression of the human PKC-IV sequence may be accomplished by using the above described vectors, linkers and procedures.

EXAMPLE IV Dependencies of Protein Kinase Activity - ΓIH] PDBu Binding A. Two 10 cm dishes were transfected with pMT-2, PKC-I- PM 2, or PKC-II-PMT-2. After 60 hours the cells were washed 3 times with Ca²⁺ and Mg²⁺ free phosphate buffered saline (PBS) . Cells were then scraped in 5 ml of PBS and concentrated. Cells were re'suspended in 1 ml of 4°C homogenizastion buffer, sonicated (Heat Systems Ultrasonics) with four 15 second bursts at full power and spun at 100,000 x g for 30 minutes. The resulting pellet was resuspended in homogenization buffer plus^' 0.3% Triton-XlOO. Each of these fractions were then applied to a DEAE Sephacel column and the peak activity fractions pooled. PKC activity was measured by the mixed micelle assay [Y. Hannun et al. , supra. ] Expression in COS cells of the two clones, PKC-I-PMT2 and PKC-II-PMT-2, resulted in a several fold induction of high affinity phorbol ester binding. To date, PKC is the only high affinity phorbol ester receptor described. An examination of the PKC activity present in COS cells transfected with either PKC-1 or PKC-II showed that maximal kinase activity is dependent upon Ca²⁺ phosphatidylserine, and DAG/PDBu.

B. To estimate the amount of PKC present, the specific binding of [³H]PDBu to COS cells transfected with PMT-2, PKC-I-PMT-2, or PKC-II-PMT-2 was determined according to the procedures of Collins and Rozengurt, J. Cell Phvsiol.. 112, 42-50 (1982) and Burgess et al., J. Cell Bio. , 102: 312-319 (1986) . Cells transfected with either PKC-I-PMT-2 or PKC- II-PMT-2 bound more [³H] PDBu than cells transfected with pMT-2 alone.

C. To measure specific [³H]PDBu binding with PKC-I-PMT- 2 or PKC-II-PMT-2, individual 6 cm, or 10 cm dishes were washed 3 times with PBS and incubated for 12 hours in DMEM plus 1% BSA. Dishes were then rinsed three times with Tyrodes solution and incubated in 1.5 ml of Tyrodes solution with 0.1 uCi (5 nM) of [³H]PDBu alone, or with cold PDBu for 30 minutes at 3 ^βC. Cells were then rinsed three times with Tyrodes solution, scraped into 1 ml of PBS and counted in 10 ml Aquasol II. The specific binding of [³H] PDBu to transfected cells competed with nanomolar concentrations of cold PDBu, indicating specific high affinity binding.

EXAMPLE V Molecular Weight of PKC Polypeptides

After PKC-II-PMT-2 was transfected into COS cells as in Ex-ample III, and the polypeptides synthesized were examined by [³⁵S] methionine labelling and SDS-polyacrylamide gel electrophoresis. A predominant polypeptide of approximately 77-80 kd apparent molecular weight was observed that was absent from control cells transfected with pMT-2. This molecular weight agrees with that predicted from the open reading frame of 76,660kd and the observed molecular weight of purified rat brain PKC.

EXAMPLE VI Tissue Distribution of PKC

To investigate the tissue distribution and size of PKC related mRNAs, a single stranded probe was prepared from a region of PKC-I which was about 75% homologous with PKC-II and used in a Northern blot analysis. A major 3.5kb mRNA was present exclusively in brain mRNA. Lower levels of 9.5kb and 6kb mRNAs were present in lung RNA. A similar analysis of the tissue distribution of PKC-II related mRNAs was performed using a probe corresponding to the first 380 nucleotides of PKC-II (less than 50% homologous to PKC-I) . The probe detected, in all tissues examined, a principal mRNA of about 8kb and a minor mRNA of about 3kb. However, these mRNAs were far more abundant in brain and spleen. This corresponds to the known tissue distribution of PKC activity.

EXAMPLE VII Human PKC Polypeptides

Based on the predicted homology between the human and rat PKC DNA sequences, human PKC polypeptides can be easily obtained in the following manner. The rat PKCI or II coding sequences of Fig. 1 are used as probes to screen a human genomic library for the human genomic clone. A portion of the rat cDNA clone for PKC is labeled with ³²P by nick-translastion or through production of an M13 single stranded DNA probe. A human genomic library is screened using this probe, [See, Toole'et al., Nature, 312:342-346 (1984)], and presumptive positives isolated and DNA sequence obtained as described above.

The human gene is then cloned in a plasmid expression vector, eg. PMT2, by standard molecular biology techniques and amplified in bacteria. This construct containing the complete human genomic PKC gene can be used to construct mammalian cell lines expressing PKC, e.g. as described by PCT W085/20610 for human erythropoietin. In addition this human genomic PKC gene can be engineered with the appropriate promoter and processing signals for expression in some other heterologous system, e.g. insect promoters for constructing insect cell culture lines. Similarly, this genomic PKC gene may be epxressed in yeast or other eukaryotic systems.

Alternatively, the expression vector containing the gene is transfected into a mammalian cell, e.g., monkey COS cells. The human gene is transcribed and the RNA correctly spliced. Media from the transfected cells is assays for PKC related activities as described above as an indication that the gene is complete. mRNA is obtained from these cells and cDNA synthesized from the mRNA. The respective human cDNA for PKC can be isolated.

Alternatively, the rat PKC coding sequence can be used as a probe to identify a human cell line or tissue containing mRNA for a human PKC. Briefly described, RNA is extracted from a selected cell or tissue source and subjected to RNA blot analysis with the rat PKC cDNA sequence as probe according to T. Maniatis et al.. Molecular Cloning- A Laboratory Manual, Cold Spring Harbor Laboratory, (1983) .

Once the source of a human RNA containing a PKC mRNA is identified, poly A⁺ RNA can be prepared from this source, converted to cDNA and hence represented as either a phage or plasmid cDNA library. The particular cDNA clone can be identified by hybridization under stringent conditions with PKC coding region DNA probe.

In order to produce a human PKC polypeptide the cDNA encoding it is transferred into an appropriate expression vector and introduced into host cells by conventional genetic engineering techniques as described above. The presently preferred expression system for biologically activerecombinant human PKC polypeptide is stablytransformed CHO cells. However, active PKC polypeptide may be produced intracellularly or extracellularly from bacteria, yeast or insect cells.

EXAMPLE VIII Assay for Detecting Tumor Promoters

To assess the tumor promoting capacity of a selected substance, a competitive binding assay is performed employing a PKC polypeptide or fragment according to the present invention.

In a first reaction mixture, one or more PKC polypeptides of selected origin, is incubated along with a known tumor promoter or a known PKC binding compound labelled with a detectable marker, in a standard binding reaction. PKC polypeptides, such as PKC-I, PKC-II, PKC-III or PKC-IV or the amino terminal fragments thereof which contribute the calcium dependence thereto may be employed in this assay. Additionally PKC fragments or polypeptides may be obtained from species other than rat, as described in Example VII for recovery of human PKCs. Further PKC isozymes characteristic of a specific tissue source, e.g., brain, lung, bladder, spleen, may be obtained as in Example VII and employed to enhance the ability of the assay to detect those substances which may have tumor promoting capacity only for specific tissues. Among the known PKC binding compounds for use in the assay are the known tumor promoter PDBu or a naturally occurring DAG, dioctonoylglyσerol (diC8) or analogues of either compound. Other PKC binding compounds are known to those of skill in the art and may also be employed in this assay. ^■ Conventional detectable markers or labels known to those skilled in the art may be used including P³², H³, and other known biological markers. The labelled compound-PKC polypeptide complex binding is determined as described in B. J. Goodwin and J. B. Weinberg, J. Clin. Invest. 70:699-706 (1982).

An unlabelled control compound (which may be the known compound of the first reaction mixture) is added to a second reaction mixture containing the components of the first reaction mixture. Again the amount of bound labelled complex is determined. Each of these unlabelled control compounds will effectively compete with the labeled compounds for binding to the PKC polypeptide or fragment, and less of the labelled compounds will associate with PKC.

Finally, a test compound at various concentrations is added to a third reaction mixture containing the PKC polypeptide and labelled compounds described above. The ability of the test compound to compete for binding with the labelled compound to PKC is determined as above.

If a test compound competes with the labelled PKC binding compound for binding to PKC, then this compound is further tested in a standard phosphotransferase assay [see Y. Hannun et al., supra] for its ability to act as a cofactor or inhibitor (agonist or antagonist) of thephosphotransferase activity of PKC which is dependent upon either PDBu or diC8 and their analogues. If the test compound is a potent agonist, it is a likely tumor promoter.

If it is an antagonist, the compound may be employed to inhibit cellular proliferation or other cellular responses medicated by PKC as detailed in Nishizuka, supra. These antagonist compounds may also be useful in drug design by identifying compounds for modification to increase or direct the PKC antagonist abilities thereof.

Numerous modifications and variations in practice of this invention are expected to occur to those skilled in the art upon considertion of the foregoing descriptions of preferred embodiment hereof. Such modifications and variations are believed to be encompassed in the appended claims.

International Application No: PCT/

Claims

WEAT IS CLAIMED IS:

T. A DNA sequence coding on expression for a protein kinase C enzyme and comprising the sequence of nucleotides in a 5' to 3' direction selected from the group consisting of a) the sequence designated I in Fig. 1; b) the sequence designated II in Fig. 1; σ) the sequence designated III in Fig. 1; d) the sequence designated IV in Fig. 3.

2. A process for producing a purified PKC polypeptide comprising culturing a cell line transformed with a vector comprising a DNA sequence according to claim 1 in operative association with an expression control sequence therefor.

3. A vector for use in transforming a cell line comprising a DNA sequence^according to claim 1 in operative association with an^' expression control sequence therefor.

4. A purified PKC polypeptide characterized by an amino acid sequence substantially as shown in Fig. 2 or Fig. 3 and encoded by a DNA sequence selected from the group consisting of a) the sequence designated I in Fig. 1; b) the sequence designated II in Fig. 1; c) the sequence designated III in Fig. 1; and d) the sequence designated IV in Fig. 3.

5. A method for detecting the tumor promoting activity of a substance comprising: determining the capacity of said substance to compete for binding to a protein sequence encoded by the DNA sequence according to claim 1 or a fragment thereof containing the phorbol ester binding site with a known PKC binding compound; and determining the activity of said substance to act as an agonist in a phosphotransferase assay.