Nature 333, 470 - 473 (02 June 1988); doi:10.1038/333470a0

Molecular cloning and expression of the major protein kinase C substrate of platelets

Michael Tyers^*, Richard A. Rachubinski^*, Mary I. Stewart^†, Angela M. Varrichio^‡, Robert G. L. Shorr^‡, Richard J. Haslam^*† & Calvin B. Harley^*

^*Department of Biochemistry and ^† Department of Pathology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
^‡Smith Kline and French Laboratories, PO Box 1539, King of Prussia, Pennsylvania 19406, USA

In platelets, agonists that stimulate phosphoinositide turnover cause the rapid phosphorylation of a protein of apparent relative molecular mass (Mr) 40–47,000, called P47, by protein kinase C (PKC)^1–4. Diverse identities have been ascribed to P47 including lipocortin⁵, inositol 1,4,5-trisphosphate 5-phosphomonoesterase⁶, pyruvate dehydrogenase subunit⁷ and an actin regulatory protein⁸. We have isolated human P47 clones by immunological screening of a gtll complementary DNA library from HL-60 cells, a human promyelocytic leukaemia cell line⁹. P47 recom-binants thus identified hybridized to a 3.0 kilobase (kb) messenger RNA in mature white blood cell lines; the same mRNA was induced in HL-60 cells during differentiation. A 1,050 base pair (bp) open reading frame that could encode a protein of Mr 40,087 was confirmed by comparison with peptide sequences from platelet P47, and by expression of the putative recombinant P47 in E. coli and in vitro. The P47 sequence appears to have been conserved throughout vertebrate evolution, and is not similar to any other known sequence including human lipocortin and the subunit of pyruvate dehydrogenase. The P47 protein contains a potential Ca²⁺-binding 'EF-hand' structure and a region that strongly resembles known PKC phosphorylation sites.

References

1.	Nishizuka, Y. Nature 308, 693−698 (1984). | Article | PubMed | ISI | ChemPort |
2.	Lyons, R. M., Stanford, N. & Majerus, P. W. J. clin. Invest. 56, 924−936 (1975). | PubMed | ISI | ChemPort |
3.	Haslam, R. J. & Lynham, J. A. Biochem. biophys. Res. Commun. 77, 714−722 (1977). | Article | PubMed | ISI | ChemPort |
4.	Haslam, R. J., Lynham, J. A. & Fox, J. E. B. Biochem. J. 178, 397−406 (1979). | PubMed | ISI | ChemPort |
5.	Touqui, L. et al. Nature 321, 177−180 (1986). | Article | PubMed | ISI | ChemPort |
6.	Connolly, T. M., Lawing, W. J. Jr. & Majerus, P. W. Cell 46, 951−958 (1986). | Article | PubMed | ChemPort |
7.	Chiang, T. M., Kang, E. S. & Rang, A. H. Archs Biochem. Biophys. 252, 15−23 (1987). | ChemPort |
8.	Hashimoto, K. et al. Biochem. International 14, 759−767 (1987). | ChemPort |
9.	Collins, S. J. Blood 70, 1233−1244 (1987). | PubMed | ISI | ChemPort |
10.	Tyers, M., Rachubinski, R. A., Sartori, C. S., Harley, C. B. & Haslam, R. J. Biochem. J. 243, 249−253 (1987). | PubMed | ChemPort |
11.	Lee, W., Mitchell, P. & Tjian, R. Cell 47, 741−752 (1987).
12.	Clarke, B. J., Liao, S., Leeds, C., Soamboonsrup, P. & Neame, P. B. Cancer Res. 47, 4254−4259 (1987). | PubMed | ChemPort |
13.	Imaoka, T., Lynham, J. A. & Haslam, R. J. J. biol. Chem. 258, 11404−11414 (1983). | PubMed | ChemPort |
14.	Kozak, M. Nucleic Acids Res. 15, 8125−8149 (1987). | PubMed | ISI | ChemPort |
15.	Augen, J. & Wold, F. Trends biochem. Sci. 11, 494−497 (1986). | Article | ISI | ChemPort |
16.	Proudfoot, N. J. & Brownless, G. G. Nature 263, 211−214 (1976). | Article | PubMed | ISI | ChemPort |
17.	Kretsinger, R. H. Ann. N.Y. Acad. Sci. 356, 14−19 (1980). | PubMed | ChemPort |
18.	Bacon, D. & Anderson, W. F. J. molec. Biol. 191, 153−161 (1986). | Article | PubMed | ChemPort |
19.	Watterson, D. M., Sharief, F. & Vanaman, T. C. J. biol. Chem. 255, 962−975 (1980). | PubMed | ISI | ChemPort |
20.	Gerke, V. & Weber, K. EMBO J. 4, 2917−2920 (1985). | PubMed | ISI | ChemPort |
21.	Woodgett, J. R., Gould, K. L. & Hunter, T. Eur. J. Biochem. 161, 177−184 (1986). | Article | PubMed | ISI | ChemPort |
22.	House, C. & Kemp, B. E. Science 238, 1726−1728 (1987). | PubMed | ISI | ChemPort |
23.	Turner, R. S., Kemp, B. E., Su, H. & Kuo, J. F. J. biol. Chem. 260, 11503−11507 (1985). | PubMed | ChemPort |
24.	Ikebe, M., Hartshorne, D. J. & Elzinga, M. J. biol. Chem. 262, 9569−9573 (1987). | PubMed | ChemPort |
25.	Wallner, B. P. et al. Nature 320, 77−81 (1986). | Article | PubMed | ISI | ChemPort |
26.	Koike, K., Ohta, S., Urata, Y., Kagawa, Y. & Koike, M. Proc. natn. Acad. Sci. U.S.A. 85, 41−45 (1988). | ChemPort |
27.	Harley, C. B. Gene anal. Tech. 4, 17−22 (1987). | Article | PubMed | ChemPort |
28.	Wickens, M. P., Buell, G. N. & Schimke, R. T. J. biol. Chem. 253, 2483−2495 (1978). | PubMed | ISI | ChemPort |
29.	Henikoff, S. Gene 28, 351−359 (1984). | Article | PubMed | ISI | ChemPort |
30.	Hawke, D., Yuan, P. & Shively, J. E. Analyt. Biochem. 120, 302−311 (1982). | PubMed | ISI | ChemPort |
31.	Zoller, M. J. & Smith, M. DNA 3, 479−488 (1984). | PubMed | ISI | ChemPort |