Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Feb 2;116(4):863–874. doi: 10.1083/jcb.116.4.863

Deletion of the regulatory domain of protein kinase C alpha exposes regions in the hinge and catalytic domains that mediate nuclear targeting

PMCID: PMC2289337  PMID: 1734020

Abstract

Members of the protein kinase C (PKC) family are characterized by an NH2-terminal regulatory domain containing binding sites for calcium, phosphatidylserine, and diacylglycerol (or tumor-promoting phorbol esters), a small central hinge region and a COOH-terminal catalytic domain. We have constructed fusion proteins in which the regulatory domain of PKC alpha was removed and replaced by a 19-amino acid leader sequence containing a myristoylation consensus or by the same sequence in which the amino-terminal glycine was changed to alanine to prevent myristoylation. The goal was to generate constitutively active mutants of PKC that were either membrane bound, due to their myristoylation, or cytoplasmic. Western blotting of fractions from COS cells transfected with plasmids encoding wild-type and mutant proteins revealed that PKC alpha resided entirely in a Triton X-100 soluble (TS) fraction, whereas both the myristoylated and nonmyristoylated mutants were associated primarily with the nuclear envelope fraction. A similar mutant that lacked the 19 amino acid leader sequence was also found almost entirely in the nuclear envelope, as was a truncation mutant containing only the regulatory domain, hinge region, and a small portion of the catalytic domain. However, an additional truncation mutant consisting of only the regulatory domain plus the first one-third of the hinge region was almost entirely in the TS fraction. A nonmyristoylated fusion protein containing only the catalytic domain was also found in the nuclear envelope. Immunostaining of cells transfected with these constructs revealed that both the myristoylated and nonmyristoylated mutants were localized in nuclei, whereas wild-type PKC alpha was primarily cytoplasmic and perinuclear. Phorbol dibutyrate treatment of PKC alpha- transfected cells resulted in increased perinuclear and nuclear staining. The results are consistent with a model in which activation of PKC, by phorbol esters or by deletion of the regulatory domain, exposes regions in the hinge and catalytic domains that interact with a PKC "receptor" present in the nuclear envelope, and may explain the ability of wild-type PKC to be translocated to the nucleus under certain conditions.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Albert K. A., Walaas S. I., Wang J. K., Greengard P. Widespread occurrence of "87 kDa," a major specific substrate for protein kinase C. Proc Natl Acad Sci U S A. 1986 May;83(9):2822–2826. doi: 10.1073/pnas.83.9.2822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ase K., Berry N., Kikkawa U., Kishimoto A., Nishizuka Y. Differential down-regulation of protein kinase C subspecies in KM3 cells. FEBS Lett. 1988 Aug 29;236(2):396–400. doi: 10.1016/0014-5793(88)80064-4. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bell R. M. Protein kinase C activation by diacylglycerol second messengers. Cell. 1986 Jun 6;45(5):631–632. doi: 10.1016/0092-8674(86)90774-9. [DOI] [PubMed] [Google Scholar]
  5. Ben-Ze'ev A., Duerr A., Solomon F., Penman S. The outer boundary of the cytoskeleton: a lamina derived from plasma membrane proteins. Cell. 1979 Aug;17(4):859–865. doi: 10.1016/0092-8674(79)90326-x. [DOI] [PubMed] [Google Scholar]
  6. Brennan T. J., Chakraborty T., Olson E. N. Mutagenesis of the myogenin basic region identifies an ancient protein motif critical for activation of myogenesis. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5675–5679. doi: 10.1073/pnas.88.13.5675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coussens L., Parker P. J., Rhee L., Yang-Feng T. L., Chen E., Waterfield M. D., Francke U., Ullrich A. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science. 1986 Aug 22;233(4766):859–866. doi: 10.1126/science.3755548. [DOI] [PubMed] [Google Scholar]
  8. Duronio R. J., Rudnick D. A., Adams S. P., Towler D. A., Gordon J. I. Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli. J Biol Chem. 1991 Jun 5;266(16):10498–10504. [PubMed] [Google Scholar]
  9. Fields A. P., Kaufmann S. H., Shaper J. H. Analysis of the internal nuclear matrix. Oligomers of a 38 kD nucleolar polypeptide stabilized by disulfide bonds. Exp Cell Res. 1986 May;164(1):139–153. doi: 10.1016/0014-4827(86)90461-1. [DOI] [PubMed] [Google Scholar]
  10. Fields A. P., Pettit G. R., May W. S. Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C. J Biol Chem. 1988 Jun 15;263(17):8253–8260. [PubMed] [Google Scholar]
  11. Fields A. P., Pincus S. M., Kraft A. S., May W. S. Interleukin-3 and bryostatin 1 mediate rapid nuclear envelope protein phosphorylation in growth factor-dependent FDC-P1 hematopoietic cells. A possible role for nuclear protein kinase C. J Biol Chem. 1989 Dec 25;264(36):21896–21901. [PubMed] [Google Scholar]
  12. 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]
  13. Graff J. M., Gordon J. I., Blackshear P. J. Myristoylated and nonmyristoylated forms of a protein are phosphorylated by protein kinase C. Science. 1989 Oct 27;246(4929):503–506. doi: 10.1126/science.2814478. [DOI] [PubMed] [Google Scholar]
  14. Halsey D. L., Girard P. R., Kuo J. F., Blackshear P. J. Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens. J Biol Chem. 1987 Feb 15;262(5):2234–2243. [PubMed] [Google Scholar]
  15. Hocevar B. A., Fields A. P. Selective translocation of beta II-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells. J Biol Chem. 1991 Jan 5;266(1):28–33. [PubMed] [Google Scholar]
  16. House C., Kemp B. E. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science. 1987 Dec 18;238(4834):1726–1728. doi: 10.1126/science.3686012. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Huang F. L., Yoshida Y., Cunha-Melo J. R., Beaven M. A., Huang K. P. Differential down-regulation of protein kinase C isozymes. J Biol Chem. 1989 Mar 5;264(7):4238–4243. [PubMed] [Google Scholar]
  19. Isakov N., McMahon P., Altman A. Selective post-transcriptional down-regulation of protein kinase C isoenzymes in leukemic T cells chronically treated with phorbol ester. J Biol Chem. 1990 Feb 5;265(4):2091–2097. [PubMed] [Google Scholar]
  20. 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]
  21. James G., Olson E. N. Fatty acylated proteins as components of intracellular signaling pathways. Biochemistry. 1990 Mar 20;29(11):2623–2634. doi: 10.1021/bi00463a001. [DOI] [PubMed] [Google Scholar]
  22. James G., Olson E. N. Identification of a novel fatty acylated protein that partitions between the plasma membrane and cytosol and is deacylated in response to serum and growth factor stimulation. J Biol Chem. 1989 Dec 15;264(35):20998–21006. [PubMed] [Google Scholar]
  23. James G., Olson E. N. Myristoylation, phosphorylation, and subcellular distribution of the 80-kDa protein kinase C substrate in BC3H1 myocytes. J Biol Chem. 1989 Dec 15;264(35):20928–20933. [PubMed] [Google Scholar]
  24. Kaibuchi K., Fukumoto Y., Oku N., Takai Y., Arai K., Muramatsu M. Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C. J Biol Chem. 1989 Aug 15;264(23):13489–13496. [PubMed] [Google Scholar]
  25. Kariya K., Karns L. R., Simpson P. C. Expression of a constitutively activated mutant of the beta-isozyme of protein kinase C in cardiac myocytes stimulates the promoter of the beta-myosin heavy chain isogene. J Biol Chem. 1991 Jun 5;266(16):10023–10026. [PubMed] [Google Scholar]
  26. Kaufmann S. H. Additional members of the rat liver lamin polypeptide family. Structural and immunological characterization. J Biol Chem. 1989 Aug 15;264(23):13946–13955. [PubMed] [Google Scholar]
  27. Kaufmann S. H., Gibson W., Shaper J. H. Characterization of the major polypeptides of the rat liver nuclear envelope. J Biol Chem. 1983 Feb 25;258(4):2710–2719. [PubMed] [Google Scholar]
  28. 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]
  29. Kishimoto A., Mikawa K., Hashimoto K., Yasuda I., Tanaka S., Tominaga M., Kuroda T., Nishizuka Y. Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain). J Biol Chem. 1989 Mar 5;264(7):4088–4092. [PubMed] [Google Scholar]
  30. Knopf J. L., Lee M. H., Sultzman L. A., Kriz R. W., Loomis C. R., Hewick R. M., Bell R. M. Cloning and expression of multiple protein kinase C cDNAs. Cell. 1986 Aug 15;46(4):491–502. doi: 10.1016/0092-8674(86)90874-3. [DOI] [PubMed] [Google Scholar]
  31. Leach K. L., Powers E. A., Ruff V. A., Jaken S., Kaufmann S. Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells. J Cell Biol. 1989 Aug;109(2):685–695. doi: 10.1083/jcb.109.2.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lee M. H., Bell R. M. The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium- and phosphatidylserine-dependent phorbol diester binding activity. J Biol Chem. 1986 Nov 15;261(32):14867–14870. [PubMed] [Google Scholar]
  33. Melloni E., Pontremoli S., Michetti M., Sacco O., Sparatore B., Salamino F., Horecker B. L. Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring proteinase. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6435–6439. doi: 10.1073/pnas.82.19.6435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mizuta K., Hashimoto E., Yamamura H. Proteolytic activation of protein kinase C by membrane-bound protease in rat liver plasma membrane. Biochem Biophys Res Commun. 1985 Sep 30;131(3):1262–1268. doi: 10.1016/0006-291x(85)90227-x. [DOI] [PubMed] [Google Scholar]
  35. Mochly-Rosen D., Khaner H., Lopez J. Identification of intracellular receptor proteins for activated protein kinase C. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3997–4000. doi: 10.1073/pnas.88.9.3997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Parker P. J., Coussens L., Totty N., Rhee L., Young S., Chen E., Stabel S., Waterfield M. D., Ullrich A. The complete primary structure of protein kinase C--the major phorbol ester receptor. Science. 1986 Aug 22;233(4766):853–859. doi: 10.1126/science.3755547. [DOI] [PubMed] [Google Scholar]
  39. Rogue P., Labourdette G., Masmoudi A., Yoshida Y., Huang F. L., Huang K. P., Zwiller J., Vincendon G., Malviya A. N. Rat liver nuclei protein kinase C is the isozyme type II. J Biol Chem. 1990 Mar 5;265(7):4161–4165. [PubMed] [Google Scholar]
  40. Seed B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. 1987 Oct 29-Nov 4Nature. 329(6142):840–842. doi: 10.1038/329840a0. [DOI] [PubMed] [Google Scholar]
  41. Sternberg E. A., Spizz G., Perry W. M., Vizard D., Weil T., Olson E. N. Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene. Mol Cell Biol. 1988 Jul;8(7):2896–2909. doi: 10.1128/mcb.8.7.2896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stumpo D. J., Graff J. M., Albert K. A., Greengard P., Blackshear P. J. Molecular cloning, characterization, and expression of a cDNA encoding the "80- to 87-kDa" myristoylated alanine-rich C kinase substrate: a major cellular substrate for protein kinase C. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4012–4016. doi: 10.1073/pnas.86.11.4012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tapley P. M., Murray A. W. Platelet Ca2+-activated, phospholipid-dependent protein kinase: evidence for proteolytic activation of the enzyme in cells treated with phospholipase C1. Biochem Biophys Res Commun. 1984 Feb 14;118(3):835–841. doi: 10.1016/0006-291x(84)91470-0. [DOI] [PubMed] [Google Scholar]
  44. Ways K., Riddle R., Ways M., Cook P. Effect of phorbol esters on cytosolic protein kinase C content and activity in the human monoblastoid U937 cell. J Biol Chem. 1991 Jan 15;266(2):1258–1264. [PubMed] [Google Scholar]
  45. Wilcox C., Hu J. S., Olson E. N. Acylation of proteins with myristic acid occurs cotranslationally. Science. 1987 Nov 27;238(4831):1275–1278. doi: 10.1126/science.3685978. [DOI] [PubMed] [Google Scholar]
  46. Young S., Parker P. J., Ullrich A., Stabel S. Down-regulation of protein kinase C is due to an increased rate of degradation. Biochem J. 1987 Jun 15;244(3):775–779. doi: 10.1042/bj2440775. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES