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. 2000 Dec 1;352(Pt 2):475–482.

Inhibition of growth-factor-induced phosphorylation and activation of protein kinase B/Akt by atypical protein kinase C in breast cancer cells.

M Mao 1, X Fang 1, Y Lu 1, R Lapushin 1, R C Bast Jr 1, G B Mills 1
PMCID: PMC1221479  PMID: 11085941

Abstract

The protein kinase B/Akt serine/threonine kinase, located downstream of phosphoinositide 3-kinase (PI-3K), is a major regulator of cellular survival and proliferation. Atypical protein kinase C (aPKC) family members are activated by PI-3K and also contribute to cell proliferation, suggesting that Akt and aPKC might interact to activate signalling through the PI-3K cascade. Here we demonstrate that blocking PKC activity in MDA-MB-468 breast cancer cells increased the phosphorylation and activity of Akt. Functional PI-3K was required for the PKC inhibitors to increase Akt phosphorylation and activation, potentially owing to the activation of specific PKC isoforms by PI-3K. The concentration dependence of the action of the PKC inhibitors implicates aPKC in the inhibition of Akt phosphorylation and activity. In support of a role for aPKC in the regulation of Akt, Akt and PKCzeta or PKClambda/iota were readily co-precipitated from the BT-549 breast cancer cell line. Furthermore, the overexpression of PKCzeta inhibited growth-factor-induced increases in Akt phosphorylation and activity. Thus PKCzeta associates physically with Akt and decreases Akt phosphorylation and enzyme activity. The effects of PKC on Akt were transmitted through the PI-3K cascade as indicated by changes in p70 s6 kinase (p70(s6k)) phosphorylation. Thus PKCzeta, and potentially other PKC isoenzymes, regulate growth-factor-mediated Akt phosphorylation and activation, which is consistent with a generalized role for PKCzeta in limiting growth factor signalling through the PI-3K/Akt pathway.

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Selected References

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  1. Aman M. J., Lamkin T. D., Okada H., Kurosaki T., Ravichandran K. S. The inositol phosphatase SHIP inhibits Akt/PKB activation in B cells. J Biol Chem. 1998 Dec 18;273(51):33922–33928. doi: 10.1074/jbc.273.51.33922. [DOI] [PubMed] [Google Scholar]
  2. Andjelković M., Maira S. M., Cron P., Parker P. J., Hemmings B. A. Domain swapping used to investigate the mechanism of protein kinase B regulation by 3-phosphoinositide-dependent protein kinase 1 and Ser473 kinase. Mol Cell Biol. 1999 Jul;19(7):5061–5072. doi: 10.1128/mcb.19.7.5061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Balendran A., Casamayor A., Deak M., Paterson A., Gaffney P., Currie R., Downes C. P., Alessi D. R. PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr Biol. 1999 Apr 22;9(8):393–404. doi: 10.1016/s0960-9822(99)80186-9. [DOI] [PubMed] [Google Scholar]
  4. Bondeva T., Pirola L., Bulgarelli-Leva G., Rubio I., Wetzker R., Wymann M. P. Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK. Science. 1998 Oct 9;282(5387):293–296. doi: 10.1126/science.282.5387.293. [DOI] [PubMed] [Google Scholar]
  5. Borner C., Ueffing M., Jaken S., Parker P. J., Weinstein I. B. Two closely related isoforms of protein kinase C produce reciprocal effects on the growth of rat fibroblasts. Possible molecular mechanisms. J Biol Chem. 1995 Jan 6;270(1):78–86. doi: 10.1074/jbc.270.1.78. [DOI] [PubMed] [Google Scholar]
  6. Cardone M. H., Roy N., Stennicke H. R., Salvesen G. S., Franke T. F., Stanbridge E., Frisch S., Reed J. C. Regulation of cell death protease caspase-9 by phosphorylation. Science. 1998 Nov 13;282(5392):1318–1321. doi: 10.1126/science.282.5392.1318. [DOI] [PubMed] [Google Scholar]
  7. Carlin S., Yang K. X., Donnelly R., Black J. L. Protein kinase C isoforms in human airway smooth muscle cells: activation of PKC-zeta during proliferation. Am J Physiol. 1999 Mar;276(3 Pt 1):L506–L512. doi: 10.1152/ajplung.1999.276.3.L506. [DOI] [PubMed] [Google Scholar]
  8. Chou M. M., Hou W., Johnson J., Graham L. K., Lee M. H., Chen C. S., Newton A. C., Schaffhausen B. S., Toker A. Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr Biol. 1998 Sep 24;8(19):1069–1077. doi: 10.1016/s0960-9822(98)70444-0. [DOI] [PubMed] [Google Scholar]
  9. Coffer P. J., Jin J., Woodgett J. R. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J. 1998 Oct 1;335(Pt 1):1–13. doi: 10.1042/bj3350001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conway A. M., Rakhit S., Pyne S., Pyne N. J. Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase. Biochem J. 1999 Jan 15;337(Pt 2):171–177. [PMC free article] [PubMed] [Google Scholar]
  11. Dennis P. B., Pullen N., Pearson R. B., Kozma S. C., Thomas G. Phosphorylation sites in the autoinhibitory domain participate in p70(s6k) activation loop phosphorylation. J Biol Chem. 1998 Jun 12;273(24):14845–14852. doi: 10.1074/jbc.273.24.14845. [DOI] [PubMed] [Google Scholar]
  12. Doornbos R. P., Theelen M., van der Hoeven P. C., van Blitterswijk W. J., Verkleij A. J., van Bergen en Henegouwen P. M. Protein kinase Czeta is a negative regulator of protein kinase B activity. J Biol Chem. 1999 Mar 26;274(13):8589–8596. doi: 10.1074/jbc.274.13.8589. [DOI] [PubMed] [Google Scholar]
  13. Franke T. F., Kaplan D. R., Cantley L. C., Toker A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science. 1997 Jan 31;275(5300):665–668. doi: 10.1126/science.275.5300.665. [DOI] [PubMed] [Google Scholar]
  14. Goekjian P. G., Jirousek M. R. Protein kinase C in the treatment of disease: signal transduction pathways, inhibitors, and agents in development. Curr Med Chem. 1999 Sep;6(9):877–903. [PubMed] [Google Scholar]
  15. Hashimoto A., Hirose K., Okada H., Kurosaki T., Iino M. Inhibitory modulation of B cell receptor-mediated Ca2+ mobilization by Src homology 2 domain-containing inositol 5'-phosphatase (SHIP). J Biol Chem. 1999 Apr 16;274(16):11203–11208. doi: 10.1074/jbc.274.16.11203. [DOI] [PubMed] [Google Scholar]
  16. Jiang B. H., Aoki M., Zheng J. Z., Li J., Vogt P. K. Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2077–2081. doi: 10.1073/pnas.96.5.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kampfer S., Hellbert K., Villunger A., Doppler W., Baier G., Grunicke H. H., Uberall F. Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKC-lambda, epsilon and zeta. EMBO J. 1998 Jul 15;17(14):4046–4055. doi: 10.1093/emboj/17.14.4046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim S., Jung Y., Kim D., Koh H., Chung J. Extracellular zinc activates p70 S6 kinase through the phosphatidylinositol 3-kinase signaling pathway. J Biol Chem. 2000 Aug 25;275(34):25979–25984. doi: 10.1074/jbc.M001975200. [DOI] [PubMed] [Google Scholar]
  19. Klippel A., Reinhard C., Kavanaugh W. M., Apell G., Escobedo M. A., Williams L. T. Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways. Mol Cell Biol. 1996 Aug;16(8):4117–4127. doi: 10.1128/mcb.16.8.4117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Konishi H., Kuroda S., Kikkawa U. The pleckstrin homology domain of RAC protein kinase associates with the regulatory domain of protein kinase C zeta. Biochem Biophys Res Commun. 1994 Dec 30;205(3):1770–1775. doi: 10.1006/bbrc.1994.2874. [DOI] [PubMed] [Google Scholar]
  21. Konishi H., Kuroda S., Tanaka M., Matsuzaki H., Ono Y., Kameyama K., Haga T., Kikkawa U. Molecular cloning and characterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protein kinase C subspecies and beta gamma subunits of G proteins. Biochem Biophys Res Commun. 1995 Nov 13;216(2):526–534. doi: 10.1006/bbrc.1995.2654. [DOI] [PubMed] [Google Scholar]
  22. Le Good J. A., Ziegler W. H., Parekh D. B., Alessi D. R., Cohen P., Parker P. J. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science. 1998 Sep 25;281(5385):2042–2045. doi: 10.1126/science.281.5385.2042. [DOI] [PubMed] [Google Scholar]
  23. Leevers S. J., Vanhaesebroeck B., Waterfield M. D. Signalling through phosphoinositide 3-kinases: the lipids take centre stage. Curr Opin Cell Biol. 1999 Apr;11(2):219–225. doi: 10.1016/s0955-0674(99)80029-5. [DOI] [PubMed] [Google Scholar]
  24. Liu Q., Sasaki T., Kozieradzki I., Wakeham A., Itie A., Dumont D. J., Penninger J. M. SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival. Genes Dev. 1999 Apr 1;13(7):786–791. doi: 10.1101/gad.13.7.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lu Y., Cuevas B., Gibson S., Khan H., LaPushin R., Imboden J., Mills G. B. Phosphatidylinositol 3-kinase is required for CD28 but not CD3 regulation of the TEC family tyrosine kinase EMT/ITK/TSK: functional and physical interaction of EMT with phosphatidylinositol 3-kinase. J Immunol. 1998 Nov 15;161(10):5404–5412. [PubMed] [Google Scholar]
  26. Lu Y., Lin Y. Z., LaPushin R., Cuevas B., Fang X., Yu S. X., Davies M. A., Khan H., Furui T., Mao M. The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene. 1999 Nov 25;18(50):7034–7045. doi: 10.1038/sj.onc.1203183. [DOI] [PubMed] [Google Scholar]
  27. Martelli A. M., Sang N., Borgatti P., Capitani S., Neri L. M. Multiple biological responses activated by nuclear protein kinase C. J Cell Biochem. 1999 Sep 15;74(4):499–521. [PubMed] [Google Scholar]
  28. Mills G. B., Girard P., Grinstein S., Gelfand E. W. Interleukin-2 induces proliferation of T lymphocyte mutants lacking protein kinase C. Cell. 1988 Oct 7;55(1):91–100. doi: 10.1016/0092-8674(88)90012-8. [DOI] [PubMed] [Google Scholar]
  29. Mochly-Rosen D., Kauvar L. M. Modulating protein kinase C signal transduction. Adv Pharmacol. 1998;44:91–145. doi: 10.1016/s1054-3589(08)60126-x. [DOI] [PubMed] [Google Scholar]
  30. Peterson R. T., Schreiber S. L. Translation control: connecting mitogens and the ribosome. Curr Biol. 1998 Mar 26;8(7):R248–R250. doi: 10.1016/s0960-9822(98)70152-6. [DOI] [PubMed] [Google Scholar]
  31. Ramaswamy S., Nakamura N., Vazquez F., Batt D. B., Perera S., Roberts T. M., Sellers W. R. Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2110–2115. doi: 10.1073/pnas.96.5.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Romanelli A., Martin K. A., Toker A., Blenis J. p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex. Mol Cell Biol. 1999 Apr;19(4):2921–2928. doi: 10.1128/mcb.19.4.2921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sable C. L., Filippa N., Hemmings B., Van Obberghen E. cAMP stimulates protein kinase B in a Wortmannin-insensitive manner. FEBS Lett. 1997 Jun 9;409(2):253–257. doi: 10.1016/s0014-5793(97)00518-8. [DOI] [PubMed] [Google Scholar]
  34. Scaglione-Sewell B., Abraham C., Bissonnette M., Skarosi S. F., Hart J., Davidson N. O., Wali R. K., Davis B. H., Sitrin M., Brasitus T. A. Decreased PKC-alpha expression increases cellular proliferation, decreases differentiation, and enhances the transformed phenotype of CaCo-2 cells. Cancer Res. 1998 Mar 1;58(5):1074–1081. [PubMed] [Google Scholar]
  35. Schönwasser D. C., Marais R. M., Marshall C. J., Parker P. J. Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol. 1998 Feb;18(2):790–798. doi: 10.1128/mcb.18.2.790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Standaert M. L., Bandyopadhyay G., Sajan M. P., Cong L., Quon M. J., Farese R. V. Okadaic acid activates atypical protein kinase C (zeta/lambda) in rat and 3T3/L1 adipocytes. An apparent requirement for activation of Glut4 translocation and glucose transport. J Biol Chem. 1999 May 14;274(20):14074–14078. doi: 10.1074/jbc.274.20.14074. [DOI] [PubMed] [Google Scholar]
  37. Stokoe D., Stephens L. R., Copeland T., Gaffney P. R., Reese C. B., Painter G. F., Holmes A. B., McCormick F., Hawkins P. T. Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science. 1997 Jul 25;277(5325):567–570. doi: 10.1126/science.277.5325.567. [DOI] [PubMed] [Google Scholar]
  38. Summers S. A., Kao A. W., Kohn A. D., Backus G. S., Roth R. A., Pessin J. E., Birnbaum M. J. The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism. J Biol Chem. 1999 Jun 18;274(25):17934–17940. doi: 10.1074/jbc.274.25.17934. [DOI] [PubMed] [Google Scholar]
  39. Takeda H., Matozaki T., Takada T., Noguchi T., Yamao T., Tsuda M., Ochi F., Fukunaga K., Inagaki K., Kasuga M. PI 3-kinase gamma and protein kinase C-zeta mediate RAS-independent activation of MAP kinase by a Gi protein-coupled receptor. EMBO J. 1999 Jan 15;18(2):386–395. doi: 10.1093/emboj/18.2.386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tan Y., Ruan H., Demeter M. R., Comb M. J. p90(RSK) blocks bad-mediated cell death via a protein kinase C-dependent pathway. J Biol Chem. 1999 Dec 3;274(49):34859–34867. doi: 10.1074/jbc.274.49.34859. [DOI] [PubMed] [Google Scholar]
  41. Uberall F., Hellbert K., Kampfer S., Maly K., Villunger A., Spitaler M., Mwanjewe J., Baier-Bitterlich G., Baier G., Grunicke H. H. Evidence that atypical protein kinase C-lambda and atypical protein kinase C-zeta participate in Ras-mediated reorganization of the F-actin cytoskeleton. J Cell Biol. 1999 Feb 8;144(3):413–425. doi: 10.1083/jcb.144.3.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wymann M. P., Pirola L. Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta. 1998 Dec 8;1436(1-2):127–150. doi: 10.1016/s0005-2760(98)00139-8. [DOI] [PubMed] [Google Scholar]
  43. Xu Y., Fang X. J., Casey G., Mills G. B. Lysophospholipids activate ovarian and breast cancer cells. Biochem J. 1995 Aug 1;309(Pt 3):933–940. doi: 10.1042/bj3090933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yano S., Tokumitsu H., Soderling T. R. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature. 1998 Dec 10;396(6711):584–587. doi: 10.1038/25147. [DOI] [PubMed] [Google Scholar]
  45. Yoshiji H., Kuriyama S., Ways D. K., Yoshii J., Miyamoto Y., Kawata M., Ikenaka Y., Tsujinoue H., Nakatani T., Shibuya M. Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis. Cancer Res. 1999 Sep 1;59(17):4413–4418. [PubMed] [Google Scholar]

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