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Genetic Manipulation of Protein Kinase C In Vivo

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Protein Kinase C Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 233))

Abstract

Studies since 1980 have established that protein kinase C (PKC) regulates a plethora of downstream signaling pathways leading to numerous cellular responses. Although much is known concerning PKC regulation by lipid cofactors and phosphorylation, the direct protein substrates that relay the PKC signal remain largely undescribed, although proteins such as myristoylated alanine-rich C kinase (MARCKS) and pleckstrin are well-known examples. Much of the work aimed at defining substrates of PKC has relied on the use of phorbol esters (see Chapter 34) as well as small molecule inhibitors (see Chapter 33). Because of concerns with lack of specificity and cytotoxicity associated with these approaches, new methodologies have been developed to more accurately and specifically manipulate PKC activity in cells and thus tackle the mechanisms by which PKC regulates cell function.

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References

  1. Letiges, M., Plomann, M., Standaert, M. L., Bandyopadhyay, G., Sajan, M. P., Kanoh, Y., et al. (2002) Knockout of PKCalpha Enhances Insulin Signaling Through PI3K. Mol. Endocrinol. 16, 847–858.

    Article  PubMed  CAS  Google Scholar 

  2. Leitges, M., Schmedt, C., Guinamard, R., Davoust, J., Schaal, S., Stabel, S., et al. (1996) Immunodeficiency in protein kinase cbeta-deficient mice. Science 273, 788–791.

    Article  PubMed  CAS  Google Scholar 

  3. Standaert, M. L., Bandyopadhyay, G., Galloway, L., Soto, J., Ono, Y., Kikkawa, U., et al. (1999) Effects of knockout of the protein kinase C beta gene on glucose transport and glucose homeostasis. Endocrinology 140, 4470–4477.

    Article  PubMed  CAS  Google Scholar 

  4. Weeber, E. J., Atkins, C. M., Selcher, J. C., Varga, A. W., Mirnikjoo, B., Paylor, R., et al. (2000) A role for the beta isoform of protein kinase C in fear conditioning. J. Neurosci. 20, 5906–5914.

    PubMed  CAS  Google Scholar 

  5. Suzuma, K., Takahara, N., Suzuma, I., Isshiki, K., Ueki, K., Leitges, M., et al. (2002) Characterization of protein kinase C beta isoform’s action on retinoblastoma protein phosphorylation, vascular endothelial growth factorinduced endothelial cell proliferation, and retinal neovascularization. Proc. Natl. Acad. Sci. USA 99, 721–726.

    Article  PubMed  CAS  Google Scholar 

  6. Narita, M., Aoki, T., Ozaki, S., Yajima, Y., and Suzuki, T. (2001) Involvement of protein kinase Cgamma isoform in morphine-induced reinforcing effects. Neuroscience 103, 309–314.

    Article  PubMed  CAS  Google Scholar 

  7. Miyamoto, A., Nakayama, K., Imaki, H., Hirose, S., Jiang, Y., Abe, M., et al. (2002) Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cdelta. Nature 416, 865–869.

    Article  PubMed  CAS  Google Scholar 

  8. Mecklenbrauker, I., Saijo, K., Zheng, N. Y., Leitges, M., and Tarakhovsky, A. (2002) Protein kinase Cdelta controls self-antigen-induced B-cell tolerance. Nature 416, 860–865.

    Article  PubMed  Google Scholar 

  9. Olive, M. F., Mehmert, K. K., Messing, R. O., and Hodge, C. W. (2000) Reduced operant ethanol self-administration and in vivo mesolimbic dopamine responses to ethanol in PKCepsilon-deficient mice. Eur. J. Neurosci. 12, 4131–4140.

    Article  PubMed  CAS  Google Scholar 

  10. Castrillo, A., Pennington, D. J., Otto, F., Parker, P. J., Owen, M. J., and Bosca, L. (2001) Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection. J. Exp. Med. 194, 1231–1242.

    Article  PubMed  CAS  Google Scholar 

  11. Sun, Z., Arendt, C. W., Ellmeier, W., Schaeffer, E. M., Sunshine, M. J., Gandhi, L., et al. (2000) PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes. Nature 404, 402–407.

    Article  PubMed  CAS  Google Scholar 

  12. Leitges, M., Sanz, L., Martin, P., Duran, A., Braun, U., Garcia, J. F., et al. (2001) Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway. Mol. Cell. 8, 771–780.

    Article  PubMed  CAS  Google Scholar 

  13. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.

    Article  PubMed  CAS  Google Scholar 

  14. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553.

    Article  PubMed  CAS  Google Scholar 

  15. Kisielow, M., Kleiner, S., Nagasawa, M., Faisal, A., and Nagamine, Y. (2002) Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA. Biochem. J. 363, 1–5.

    Article  PubMed  CAS  Google Scholar 

  16. Garcia-Paramio, P., Cabrerizo, Y., Bornancin, F., and Parker, P. J. (1998) The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation. Biochem. J. 333, 631–636.

    PubMed  CAS  Google Scholar 

  17. Soh, J. W., Lee, E. H., Prywes, R., and Weinstein, I. B. (1999) Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol. Cell. Biol. 19, 1313–1324.

    PubMed  CAS  Google Scholar 

  18. Cenni, V., Doppler, H., Sonnenburg, E. D., Maraldi, N., Newton, A. C., and Toker, A. (2002) Regulation of novel protein kinase C epsilon by phosphorylation. Biochem. J. 363, 537–545.

    Article  PubMed  CAS  Google Scholar 

  19. House, C. and Kemp, B. E. (1987) Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science 238, 1726–1728.

    Article  PubMed  CAS  Google Scholar 

  20. House, C. and Kemp, B. E. (1990) Protein kinase C pseudosubstrate prototope: structure-function relationships. Cell Signal. 2, 187–190.

    Article  PubMed  CAS  Google Scholar 

  21. Orr, J. W., Keranen, L. M., and Newton, A. C. (1992) Reversible exposure of the pseudosubstrate domain of protein kinase-C by phosphatidylserine and diacylglycerol. J. Biol. Chem. 267, 15,263–15,266.

    PubMed  CAS  Google Scholar 

  22. Dutil, E. M. and Newton, A. C. (2000) Dual role of pseudosubstrate in the coordinated regulation of protein kinase C by phosphorylation and diacylglycerol. J. Biol. Chem. 275, 10,697–10,701.

    Article  PubMed  CAS  Google Scholar 

  23. Pears, C. J., Kour, G., House, C., Kemp, B. E., and Parker, P. J. (1990) Mutagenesis of the pseudosubstrate site of protein kinase C leads to activation. Eur. J. Biochem. 194, 89–94.

    Article  PubMed  CAS  Google Scholar 

  24. Genot, E. M., Parker, P. J., and Cantrell, D. A. (1995) Analysis of the role of protein kinase C-alpha,-epsilon, and-zeta in T cell activation. J. Biol. Chem. 270, 9833–9839.

    Article  PubMed  CAS  Google Scholar 

  25. Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. H., Chen, C. S., et al. (1998) Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr. Biol. 8, 1069–1077.

    Article  PubMed  CAS  Google Scholar 

  26. Kampfer, S., Hellbert, K., Villunger, A., Doppler, W., Baier, G., Grunicke, H. H., et al. (1998) 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. 17, 4046–4055.

    Article  PubMed  CAS  Google Scholar 

  27. Lu, Y., Jamieson, L., Brasier, A. R., and Fields, A. P. (2001) NF-kappaB/RelA transactivation is required for atypical protein kinase C iota-mediated cell survival. Oncogene 20, 4777–4792.

    Article  PubMed  CAS  Google Scholar 

  28. Wang, A., Nomura, M., Patan, S., and Ware, J. A. (2002) Inhibition of protein kinase Calpha prevents endothelial cell migration and vascular tube formation in vitro and myocardial neovascularization in vivo. Circ. Res. 90, 609–616.

    Article  PubMed  CAS  Google Scholar 

  29. Lu, D., Yang, H., Lenox, R. H., and Raizada, M. K. (1998) Regulation of angiotensin II-induced neuromodulation by MARCKS in brain neurons. J. Cell. Biol. 142, 217–227.

    Article  PubMed  CAS  Google Scholar 

  30. Liedtke, C. M. and Cole, T. (1997) Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport. Am. J. Physiol. 273, C1632–C1640.

    PubMed  CAS  Google Scholar 

  31. Mehta, K. D., Radominska-Pandya, A., Kapoor, G. S., Dave, B., and Atkins, B. A. (2002) Critical role of diacylglycerol-and phospholipid-regulated protein kinase C epsilon in induction of low-density lipoprotein receptor transcription in response to depletion of cholesterol. Mol. Cell. Biol. 22, 3783–3793.

    Article  PubMed  CAS  Google Scholar 

  32. Hussaini, I. M., Karns, L. R., Vinton, G., Carpenter, J. E., Redpath, G. T., Sando, J. J., et al. (2000) Phorbol 12-myristate 13-acetate induces protein kinase cetaspecific proliferative response in astrocytic tumor cells. J. Biol. Chem. 275, 22,348–22,354.

    Article  PubMed  CAS  Google Scholar 

  33. Sparatore, B., Patrone, M., Passalacqua, M., Pedrazzi, M., Pontremoli, S., and Melloni, E. (2000) Human neuroblastoma cell differentiation requires protein kinase C-theta. Biochem. Biophys. Res. Commun. 279, 589–594.

    Article  PubMed  CAS  Google Scholar 

  34. Spitaler, M., Villunger, A., Grunicke, H., and Uberall, F. (2000) Unique structural and functional properties of the ATP-binding domain of atypical protein kinase C-iota. J. Biol. Chem. 275, 33,289–33,296.

    Article  PubMed  CAS  Google Scholar 

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© 2003 Humana Press Inc., Totowa, NJ

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Toker, A. (2003). Genetic Manipulation of Protein Kinase C In Vivo. In: Newton, A.C. (eds) Protein Kinase C Protocols. Methods in Molecular Biology™, vol 233. Humana Press. https://doi.org/10.1385/1-59259-397-6:475

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  • DOI: https://doi.org/10.1385/1-59259-397-6:475

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-068-7

  • Online ISBN: 978-1-59259-397-2

  • eBook Packages: Springer Protocols

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