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. 2003 Oct 15;375(Pt 2):313–321. doi: 10.1042/BJ20030727

Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of beta2-chimaerin, a 'non-protein kinase C' phorbol ester receptor.

Maria Jose Caloca 1, HongBin Wang 1, Marcelo G Kazanietz 1
PMCID: PMC1223697  PMID: 12877655

Abstract

The regulation and function of beta2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to beta2-chimaerin with high affinity and promote its subcellular distribution. beta2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. beta2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the beta2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of beta2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor beta2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.

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Selected References

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  1. Ahmed S., Kozma R., Monfries C., Hall C., Lim H. H., Smith P., Lim L. Human brain n-chimaerin cDNA encodes a novel phorbol ester receptor. Biochem J. 1990 Dec 15;272(3):767–773. doi: 10.1042/bj2720767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahmed S., Lee J., Wen L. P., Zhao Z., Ho J., Best A., Kozma R., Lim L. Breakpoint cluster region gene product-related domain of n-chimaerin. Discrimination between Rac-binding and GTPase-activating residues by mutational analysis. J Biol Chem. 1994 Jul 1;269(26):17642–17648. [PubMed] [Google Scholar]
  3. Anand-Apte B., Zetter B. R., Viswanathan A., Qiu R. G., Chen J., Ruggieri R., Symons M. Platelet-derived growth factor and fibronectin-stimulated migration are differentially regulated by the Rac and extracellular signal-regulated kinase pathways. J Biol Chem. 1997 Dec 5;272(49):30688–30692. doi: 10.1074/jbc.272.49.30688. [DOI] [PubMed] [Google Scholar]
  4. Areces L. B., Kazanietz M. G., Blumberg P. M. Close similarity of baculovirus-expressed n-chimaerin and protein kinase C alpha as phorbol ester receptors. J Biol Chem. 1994 Jul 29;269(30):19553–19558. [PubMed] [Google Scholar]
  5. Benard V., Bohl B. P., Bokoch G. M. Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem. 1999 May 7;274(19):13198–13204. doi: 10.1074/jbc.274.19.13198. [DOI] [PubMed] [Google Scholar]
  6. Betz A., Ashery U., Rickmann M., Augustin I., Neher E., Südhof T. C., Rettig J., Brose N. Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release. Neuron. 1998 Jul;21(1):123–136. doi: 10.1016/s0896-6273(00)80520-6. [DOI] [PubMed] [Google Scholar]
  7. Betz A., Thakur P., Junge H. J., Ashery U., Rhee J. S., Scheuss V., Rosenmund C., Rettig J., Brose N. Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming. Neuron. 2001 Apr;30(1):183–196. doi: 10.1016/s0896-6273(01)00272-0. [DOI] [PubMed] [Google Scholar]
  8. Caloca M. J., Fernandez N., Lewin N. E., Ching D., Modali R., Blumberg P. M., Kazanietz M. G. Beta2-chimaerin is a high affinity receptor for the phorbol ester tumor promoters. J Biol Chem. 1997 Oct 17;272(42):26488–26496. doi: 10.1074/jbc.272.42.26488. [DOI] [PubMed] [Google Scholar]
  9. Caloca M. J., Wang H., Delemos A., Wang S., Kazanietz M. G. Phorbol esters and related analogs regulate the subcellular localization of beta 2-chimaerin, a non-protein kinase C phorbol ester receptor. J Biol Chem. 2001 Feb 14;276(21):18303–18312. doi: 10.1074/jbc.M011368200. [DOI] [PubMed] [Google Scholar]
  10. Chiariello M., Marinissen M. J., Gutkind J. S. Regulation of c-myc expression by PDGF through Rho GTPases. Nat Cell Biol. 2001 Jun;3(6):580–586. doi: 10.1038/35078555. [DOI] [PubMed] [Google Scholar]
  11. Crespo P., Schuebel K. E., Ostrom A. A., Gutkind J. S., Bustelo X. R. Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature. 1997 Jan 9;385(6612):169–172. doi: 10.1038/385169a0. [DOI] [PubMed] [Google Scholar]
  12. Diekmann D., Brill S., Garrett M. D., Totty N., Hsuan J., Monfries C., Hall C., Lim L., Hall A. Bcr encodes a GTPase-activating protein for p21rac. Nature. 1991 May 30;351(6325):400–402. doi: 10.1038/351400a0. [DOI] [PubMed] [Google Scholar]
  13. Ebinu J. O., Bottorff D. A., Chan E. Y., Stang S. L., Dunn R. J., Stone J. C. RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs. Science. 1998 May 15;280(5366):1082–1086. doi: 10.1126/science.280.5366.1082. [DOI] [PubMed] [Google Scholar]
  14. Fujii T., García-Bermejo M. L., Bernabó J. L., Caamaño J., Ohba M., Kuroki T., Li L., Yuspa S. H., Kazanietz M. G. Involvement of protein kinase C delta (PKCdelta) in phorbol ester-induced apoptosis in LNCaP prostate cancer cells. Lack of proteolytic cleavage of PKCdelta. J Biol Chem. 2000 Mar 17;275(11):7574–7582. doi: 10.1074/jbc.275.11.7574. [DOI] [PubMed] [Google Scholar]
  15. Garrett M. D., Self A. J., van Oers C., Hall A. Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins. J Biol Chem. 1989 Jan 5;264(1):10–13. [PubMed] [Google Scholar]
  16. Hall C., Monfries C., Smith P., Lim H. H., Kozma R., Ahmed S., Vanniasingham V., Leung T., Lim L. Novel human brain cDNA encoding a 34,000 Mr protein n-chimaerin, related to both the regulatory domain of protein kinase C and BCR, the product of the breakpoint cluster region gene. J Mol Biol. 1990 Jan 5;211(1):11–16. doi: 10.1016/0022-2836(90)90006-8. [DOI] [PubMed] [Google Scholar]
  17. Hall C., Sin W. C., Teo M., Michael G. J., Smith P., Dong J. M., Lim H. H., Manser E., Spurr N. K., Jones T. A. Alpha 2-chimerin, an SH2-containing GTPase-activating protein for the ras-related protein p21rac derived by alternate splicing of the human n-chimerin gene, is selectively expressed in brain regions and testes. Mol Cell Biol. 1993 Aug;13(8):4986–4998. doi: 10.1128/mcb.13.8.4986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hurley J. H., Newton A. C., Parker P. J., Blumberg P. M., Nishizuka Y. Taxonomy and function of C1 protein kinase C homology domains. Protein Sci. 1997 Feb;6(2):477–480. doi: 10.1002/pro.5560060228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jaken S. Protein kinase C isozymes and substrates. Curr Opin Cell Biol. 1996 Apr;8(2):168–173. doi: 10.1016/s0955-0674(96)80062-7. [DOI] [PubMed] [Google Scholar]
  20. Kazanietz M. G., Caloca M. J., Eroles P., Fujii T., García-Bermejo M. L., Reilly M., Wang H. Pharmacology of the receptors for the phorbol ester tumor promoters: multiple receptors with different biochemical properties. Biochem Pharmacol. 2000 Nov 15;60(10):1417–1424. doi: 10.1016/s0006-2952(00)00470-6. [DOI] [PubMed] [Google Scholar]
  21. Kazanietz M. G. Eyes wide shut: protein kinase C isozymes are not the only receptors for the phorbol ester tumor promoters. Mol Carcinog. 2000 May;28(1):5–11. doi: 10.1002/(sici)1098-2744(200005)28:1<5::aid-mc2>3.0.co;2-g. [DOI] [PubMed] [Google Scholar]
  22. Kazanietz M. G., Krausz K. W., Blumberg P. M. Differential irreversible insertion of protein kinase C into phospholipid vesicles by phorbol esters and related activators. J Biol Chem. 1992 Oct 15;267(29):20878–20886. [PubMed] [Google Scholar]
  23. Kim B. C., Yi J. Y., Yi S. J., Shin I. C., Ha K. S., Jhun B. H., Hwang S. B., Kim J. H. Rac GTPase activity is essential for EGF-induced mitogenesis. Mol Cells. 1998 Feb 28;8(1):90–95. [PubMed] [Google Scholar]
  24. Leung T., How B. E., Manser E., Lim L. Cerebellar beta 2-chimaerin, a GTPase-activating protein for p21 ras-related rac is specifically expressed in granule cells and has a unique N-terminal SH2 domain. J Biol Chem. 1994 Apr 29;269(17):12888–12892. [PubMed] [Google Scholar]
  25. Lorenzo P. S., Kung J. W., Bottorff D. A., Garfield S. H., Stone J. C., Blumberg P. M. Phorbol esters modulate the Ras exchange factor RasGRP3. Cancer Res. 2001 Feb 1;61(3):943–949. [PubMed] [Google Scholar]
  26. Mellor H., Parker P. J. The extended protein kinase C superfamily. Biochem J. 1998 Jun 1;332(Pt 2):281–292. doi: 10.1042/bj3320281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Menna Pablo Lorenzano, Skilton Guillermo, Leskow Federico Coluccio, Alonso Daniel F., Gomez Daniel E., Kazanietz Marcelo G. Inhibition of aggressiveness of metastatic mouse mammary carcinoma cells by the beta2-chimaerin GAP domain. Cancer Res. 2003 May 1;63(9):2284–2291. [PubMed] [Google Scholar]
  28. Mira J. P., Benard V., Groffen J., Sanders L. C., Knaus U. G. Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway. Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):185–189. doi: 10.1073/pnas.97.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Newton A. C. Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev. 2001 Aug;101(8):2353–2364. doi: 10.1021/cr0002801. [DOI] [PubMed] [Google Scholar]
  30. Olson M. F., Ashworth A., Hall A. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science. 1995 Sep 1;269(5228):1270–1272. doi: 10.1126/science.7652575. [DOI] [PubMed] [Google Scholar]
  31. Qiu R. G., Chen J., Kirn D., McCormick F., Symons M. An essential role for Rac in Ras transformation. Nature. 1995 Mar 30;374(6521):457–459. doi: 10.1038/374457a0. [DOI] [PubMed] [Google Scholar]
  32. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  33. Ridley A. J. Rho family proteins: coordinating cell responses. Trends Cell Biol. 2001 Dec;11(12):471–477. doi: 10.1016/s0962-8924(01)02153-5. [DOI] [PubMed] [Google Scholar]
  34. Ron D., Kazanietz M. G. New insights into the regulation of protein kinase C and novel phorbol ester receptors. FASEB J. 1999 Oct;13(13):1658–1676. [PubMed] [Google Scholar]
  35. Stam J. C., Michiels F., van der Kammen R. A., Moolenaar W. H., Collard J. G. Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling. EMBO J. 1998 Jul 15;17(14):4066–4074. doi: 10.1093/emboj/17.14.4066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tognon C. E., Kirk H. E., Passmore L. A., Whitehead I. P., Der C. J., Kay R. J. Regulation of RasGRP via a phorbol ester-responsive C1 domain. Mol Cell Biol. 1998 Dec;18(12):6995–7008. doi: 10.1128/mcb.18.12.6995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ueda Akiko, Hamadeh Hisham K., Webb Heather K., Yamamoto Yukio, Sueyoshi Tatsuya, Afshari Cynthia A., Lehmann Jürgen M., Negishi Masahiko. Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital. Mol Pharmacol. 2002 Jan;61(1):1–6. doi: 10.1124/mol.61.1.1. [DOI] [PubMed] [Google Scholar]
  38. Wang HongBin, Kazanietz Marcelo G. Chimaerins, novel non-protein kinase C phorbol ester receptors, associate with Tmp21-I (p23): evidence for a novel anchoring mechanism involving the chimaerin C1 domain. J Biol Chem. 2001 Oct 31;277(6):4541–4550. doi: 10.1074/jbc.M107150200. [DOI] [PubMed] [Google Scholar]
  39. Yuan S., Miller D. W., Barnett G. H., Hahn J. F., Williams B. R. Identification and characterization of human beta 2-chimaerin: association with malignant transformation in astrocytoma. Cancer Res. 1995 Aug 1;55(15):3456–3461. [PubMed] [Google Scholar]
  40. Zhang Q., Calafat J., Janssen H., Greenberg S. ARF6 is required for growth factor- and rac-mediated membrane ruffling in macrophages at a stage distal to rac membrane targeting. Mol Cell Biol. 1999 Dec;19(12):8158–8168. doi: 10.1128/mcb.19.12.8158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. van Leeuwen F. N., van der Kammen R. A., Habets G. G., Collard J. G. Oncogenic activity of Tiam1 and Rac1 in NIH3T3 cells. Oncogene. 1995 Dec 7;11(11):2215–2221. [PubMed] [Google Scholar]

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