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. 1993 Jul 1;90(13):6130–6134. doi: 10.1073/pnas.90.13.6130

Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites.

J Zhang 1, L Wang 1, J Petrin 1, W R Bishop 1, R W Bond 1
PMCID: PMC46881  PMID: 8327493

Abstract

The autophosphorylation sites of the beta 2 isozyme of protein kinase C (PKC) were recently identified as Ser-16/Thr-17 near the NH2 terminus, Thr-314/Thr-324 in the hinge region, and Thr-634/Thr-641 near the COOH terminus [Flint, A.J., Paladini, R.D. & Koshland, D.E. (1990) Science 294, 408-411]. To define the role of autophosphorylation we constructed three site-directed mutants of PKC beta 1 isozyme in which each pair of phosphorylatable residues is changed to alanine. Wild-type PKC beta 1 and the mutant proteins were transiently overexpressed in COS cells, resulting in at least a 20-fold increase in [3H]phorbol 12,13-dibutyrate binding compared with control transfectants. Enzyme assays of PKC partially purified from transfected cells indicated at least a 5-fold increase in PKC activity upon expression of the wild-type protein or the NH2-terminal and hinge mutants. In contrast, no increased activity was detected upon expression of the COOH-terminal mutant. Immunoblot analysis using a beta isoform-specific antibody showed that wild-type, NH2-terminal mutant, and hinge mutant proteins are similarly distributed between the Triton-soluble and insoluble fractions. In contrast, the COOH-terminal mutant protein is largely Triton-insoluble. Immunoblot analysis also indicated that this mutant is resistant to down-regulation upon chronic exposure of cells to phorbol ester. Moreover, RNA blot analysis showed that overexpression of wild-type PKC but not of the COOH-terminal mutant enhances phorbol ester induction of c-FOS and c-JUN mRNA. Our results indicate that (i) alteration in the NH2-terminal and hinge autophosphorylation sites has no effect on PKC function by the criteria examined and (ii) the COOH-terminal autophosphorylation sites are critical for PKC function and possibly subcellular localization in COS cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aderem A. The MARCKS brothers: a family of protein kinase C substrates. Cell. 1992 Nov 27;71(5):713–716. doi: 10.1016/0092-8674(92)90546-o. [DOI] [PubMed] [Google Scholar]
  2. Bell R. M., Burns D. J. Lipid activation of protein kinase C. J Biol Chem. 1991 Mar 15;266(8):4661–4664. [PubMed] [Google Scholar]
  3. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  4. Flint A. J., Paladini R. D., Koshland D. E., Jr Autophosphorylation of protein kinase C at three separated regions of its primary sequence. Science. 1990 Jul 27;249(4967):408–411. doi: 10.1126/science.2377895. [DOI] [PubMed] [Google Scholar]
  5. Freisewinkel I., Riethmacher D., Stabel S. Downregulation of protein kinase C-gamma is independent of a functional kinase domain. FEBS Lett. 1991 Mar 25;280(2):262–266. doi: 10.1016/0014-5793(91)80307-o. [DOI] [PubMed] [Google Scholar]
  6. House C., Robinson P. J., Kemp B. E. A synthetic peptide analog of the putative substrate-binding motif activates protein kinase C. FEBS Lett. 1989 Jun 5;249(2):243–247. doi: 10.1016/0014-5793(89)80632-5. [DOI] [PubMed] [Google Scholar]
  7. Housey G. M., Johnson M. D., Hsiao W. L., O'Brian C. A., Murphy J. P., Kirschmeier P., Weinstein I. B. Overproduction of protein kinase C causes disordered growth control in rat fibroblasts. Cell. 1988 Feb 12;52(3):343–354. doi: 10.1016/s0092-8674(88)80027-8. [DOI] [PubMed] [Google Scholar]
  8. Huang K. P., Chan K. F., Singh T. J., Nakabayashi H., Huang F. L. Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase. J Biol Chem. 1986 Sep 15;261(26):12134–12140. [PubMed] [Google Scholar]
  9. Jaken S., Leach K., Klauck T. Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J Cell Biol. 1989 Aug;109(2):697–704. doi: 10.1083/jcb.109.2.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Karin M., Smeal T. Control of transcription factors by signal transduction pathways: the beginning of the end. Trends Biochem Sci. 1992 Oct;17(10):418–422. doi: 10.1016/0968-0004(92)90012-x. [DOI] [PubMed] [Google Scholar]
  11. Kikkawa U., Kishimoto A., Nishizuka Y. The protein kinase C family: heterogeneity and its implications. Annu Rev Biochem. 1989;58:31–44. doi: 10.1146/annurev.bi.58.070189.000335. [DOI] [PubMed] [Google Scholar]
  12. Kikkawa U., Takai Y., Minakuchi R., Inohara S., Nishizuka Y. Calcium-activated, phospholipid-dependent protein kinase from rat brain. Subcellular distribution, purification, and properties. J Biol Chem. 1982 Nov 25;257(22):13341–13348. [PubMed] [Google Scholar]
  13. Mitchell F. E., Marais R. M., Parker P. J. The phosphorylation of protein kinase C as a potential measure of activation. Biochem J. 1989 Jul 1;261(1):131–136. doi: 10.1042/bj2610131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mochly-Rosen D., Koshland D. E., Jr Domain structure and phosphorylation of protein kinase C. J Biol Chem. 1987 Feb 15;262(5):2291–2297. [PubMed] [Google Scholar]
  15. Molina C. A., Ashendel C. L. Tumor promoter 12-O-tetradecanoylphorbol-13-acetate and sn-1,2-dioctanoylglycerol increase the phosphorylation of protein kinase C in cells. Cancer Res. 1991 Sep 1;51(17):4624–4630. [PubMed] [Google Scholar]
  16. Muramatsu M., Kaibuchi K., Arai K. Protein kinase C mutants in the auto-inhibitory region exhibit two distinct properties. FEBS Lett. 1992 Oct 12;311(1):75–79. doi: 10.1016/0014-5793(92)81371-r. [DOI] [PubMed] [Google Scholar]
  17. Newton A. C., Koshland D. E., Jr Protein kinase C autophosphorylates by an intrapeptide reaction. J Biol Chem. 1987 Jul 25;262(21):10185–10188. [PubMed] [Google Scholar]
  18. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  19. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  20. Ohno S., Konno Y., Akita Y., Yano A., Suzuki K. A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation. J Biol Chem. 1990 Apr 15;265(11):6296–6300. [PubMed] [Google Scholar]
  21. Ono Y., Fujii T., Ogita K., Kikkawa U., Igarashi K., Nishizuka Y. The structure, expression, and properties of additional members of the protein kinase C family. J Biol Chem. 1988 May 15;263(14):6927–6932. [PubMed] [Google Scholar]
  22. Pears C. J., Kour G., House C., Kemp B. E., Parker P. J. Mutagenesis of the pseudosubstrate site of protein kinase C leads to activation. Eur J Biochem. 1990 Nov 26;194(1):89–94. doi: 10.1111/j.1432-1033.1990.tb19431.x. [DOI] [PubMed] [Google Scholar]
  23. Pears C., Parker P. J. Down-regulation of a kinase defective PKC-alpha. FEBS Lett. 1991 Jun 17;284(1):120–122. doi: 10.1016/0014-5793(91)80776-y. [DOI] [PubMed] [Google Scholar]
  24. Spudich A., Meyer T., Stryer L. Association of the beta isoform of protein kinase C with vimentin filaments. Cell Motil Cytoskeleton. 1992;22(4):250–256. doi: 10.1002/cm.970220405. [DOI] [PubMed] [Google Scholar]
  25. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wartmann M., Jans D. A., Parker P. J., Nagamine Y., Hemmings B. A., Jaken S., Eppenberger U., Fabbro D. Overexpression of the alpha-type protein kinase (PK) C in LLC-PK1 cells does not lead to a proportional increase in the induction of two 12-O-tetradecanoylphorbol-13-acetate-inducible genes. Cell Regul. 1991 Jun;2(6):491–502. doi: 10.1091/mbc.2.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wolf M., Sahyoun N. Protein kinase C and phosphatidylserine bind to Mr 110,000/115,000 polypeptides enriched in cytoskeletal and postsynaptic density preparations. J Biol Chem. 1986 Oct 5;261(28):13327–13332. [PubMed] [Google Scholar]
  28. van Straaten F., Müller R., Curran T., Van Beveren C., Verma I. M. Complete nucleotide sequence of a human c-onc gene: deduced amino acid sequence of the human c-fos protein. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3183–3187. doi: 10.1073/pnas.80.11.3183. [DOI] [PMC free article] [PubMed] [Google Scholar]

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