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. 1999 Aug;77(2):973–984. doi: 10.1016/S0006-3495(99)76948-1

Distribution of active protein kinase C in smooth muscle.

G A Meininger 1, E D Moore 1, D J Schmidt 1, L M Lifshitz 1, F S Fay 1
PMCID: PMC1300388  PMID: 10423442

Abstract

To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.

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Selected References

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  1. Ali S., Becker M. W., Davis M. G., Dorn G. W., 2nd Dissociation of vasoconstrictor-stimulated basic fibroblast growth factor expression from hypertrophic growth in cultured vascular smooth muscle cells. Relevant roles of protein kinase C. Circ Res. 1994 Nov;75(5):836–843. doi: 10.1161/01.res.75.5.836. [DOI] [PubMed] [Google Scholar]
  2. Andrea J. E., Walsh M. P. Protein kinase C of smooth muscle. Hypertension. 1992 Nov;20(5):585–595. doi: 10.1161/01.hyp.20.5.585. [DOI] [PubMed] [Google Scholar]
  3. Assender J. W., Kontny E., Fredholm B. B. Expression of protein kinase C isoforms in smooth muscle cells in various states of differentiation. FEBS Lett. 1994 Mar 28;342(1):76–80. doi: 10.1016/0014-5793(94)80588-1. [DOI] [PubMed] [Google Scholar]
  4. Bradley A. B., Morgan K. G. Alterations in cytoplasmic calcium sensitivity during porcine coronary artery contractions as detected by aequorin. J Physiol. 1987 Apr;385:437–448. doi: 10.1113/jphysiol.1987.sp016500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collins E. M., Walsh M. P., Morgan K. G. Contraction of single vascular smooth muscle cells by phenylephrine at constant [Ca2+]i. Am J Physiol. 1992 Mar;262(3 Pt 2):H754–H762. doi: 10.1152/ajpheart.1992.262.3.H754. [DOI] [PubMed] [Google Scholar]
  6. Donnelly R., Yang K., Omary M. B., Azhar S., Black J. L. Expression of multiple isoenzymes of protein kinase C in airway smooth muscle. Am J Respir Cell Mol Biol. 1995 Sep;13(3):253–256. doi: 10.1165/ajrcmb.13.3.7654381. [DOI] [PubMed] [Google Scholar]
  7. Etter E. F., Kuhn M. A., Fay F. S. Detection of changes in near-membrane Ca2+ concentration using a novel membrane-associated Ca2+ indicator. J Biol Chem. 1994 Apr 1;269(13):10141–10149. [PubMed] [Google Scholar]
  8. Etter E. F., Minta A., Poenie M., Fay F. S. Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5368–5373. doi: 10.1073/pnas.93.11.5368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fay F. S., Carrington W., Fogarty K. E. Three-dimensional molecular distribution in single cells analysed using the digital imaging microscope. J Microsc. 1989 Feb;153(Pt 2):133–149. [PubMed] [Google Scholar]
  10. Fay F. S., Hoffmann R., Leclair S., Merriam P. Preparation of individual smooth muscle cells from the stomach of Bufo marinus. Methods Enzymol. 1982;85(Pt B):284–292. doi: 10.1016/0076-6879(82)85027-1. [DOI] [PubMed] [Google Scholar]
  11. Gabella G., Blundell D. Effect of stretch and contraction on caveolae of smooth muscle cells. Cell Tissue Res. 1978 Jul 5;190(2):255–271. doi: 10.1007/BF00218174. [DOI] [PubMed] [Google Scholar]
  12. Haller H., Quass P., Lindschau C., Luft F. C., Distler A. Platelet-derived growth factor and angiotensin II induce different spatial distribution of protein kinase C-alpha and -beta in vascular smooth muscle cells. Hypertension. 1994 Jun;23(6 Pt 2):848–852. doi: 10.1161/01.hyp.23.6.848. [DOI] [PubMed] [Google Scholar]
  13. Haller H., Smallwood J. I., Rasmussen H. Protein kinase C translocation in intact vascular smooth muscle strips. Biochem J. 1990 Sep 1;270(2):375–381. doi: 10.1042/bj2700375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanks S. K., Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 1995 May;9(8):576–596. [PubMed] [Google Scholar]
  15. Himpens B., Matthijs G., Somlyo A. V., Butler T. M., Somlyo A. P. Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle. J Gen Physiol. 1988 Dec;92(6):713–729. doi: 10.1085/jgp.92.6.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hug H., Sarre T. F. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J. 1993 Apr 15;291(Pt 2):329–343. doi: 10.1042/bj2910329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Khalil R. A., Lajoie C., Resnick M. S., Morgan K. G. Ca(2+)-independent isoforms of protein kinase C differentially translocate in smooth muscle. Am J Physiol. 1992 Sep;263(3 Pt 1):C714–C719. doi: 10.1152/ajpcell.1992.263.3.C714. [DOI] [PubMed] [Google Scholar]
  19. Khalil R. A., Morgan K. G. Imaging of protein kinase C distribution and translocation in living vascular smooth muscle cells. Circ Res. 1991 Dec;69(6):1626–1631. doi: 10.1161/01.res.69.6.1626. [DOI] [PubMed] [Google Scholar]
  20. Khalil R. A., Morgan K. G. Phenylephrine-induced translocation of protein kinase C and shortening of two types of vascular cells of the ferret. J Physiol. 1992 Sep;455:585–599. doi: 10.1113/jphysiol.1992.sp019317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Langlands J. M., Diamond J. Translocation of protein kinase C in bovine tracheal smooth muscle strips: the effect of methacholine and isoprenaline. Eur J Pharmacol. 1992 Oct 1;227(2):131–138. doi: 10.1016/0922-4106(92)90120-k. [DOI] [PubMed] [Google Scholar]
  22. Lifshitz L. M. Determining data independence on a digitized membrane in three dimensions. IEEE Trans Med Imaging. 1998 Apr;17(2):299–303. doi: 10.1109/42.700742. [DOI] [PubMed] [Google Scholar]
  23. Liliental J., Chang D. D. Rack1, a receptor for activated protein kinase C, interacts with integrin beta subunit. J Biol Chem. 1998 Jan 23;273(4):2379–2383. doi: 10.1074/jbc.273.4.2379. [DOI] [PubMed] [Google Scholar]
  24. Liu J. D., Wood J. G., Raynor R. L., Wang Y. C., Noland T. A., Jr, Ansari A. A., Kuo J. F. Subcellular distribution and immunocytochemical localization of protein kinase C in myocardium, and phosphorylation of troponin in isolated myocytes stimulated by isoproterenol or phorbol ester. Biochem Biophys Res Commun. 1989 Aug 15;162(3):1105–1110. doi: 10.1016/0006-291x(89)90787-0. [DOI] [PubMed] [Google Scholar]
  25. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  26. Marsault R., Murgia M., Pozzan T., Rizzuto R. Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells. EMBO J. 1997 Apr 1;16(7):1575–1581. doi: 10.1093/emboj/16.7.1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mineo C., Ying Y. S., Chapline C., Jaken S., Anderson R. G. Targeting of protein kinase Calpha to caveolae. J Cell Biol. 1998 May 4;141(3):601–610. doi: 10.1083/jcb.141.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mochly-Rosen D., Henrich C. J., Cheever L., Khaner H., Simpson P. C. A protein kinase C isozyme is translocated to cytoskeletal elements on activation. Cell Regul. 1990 Aug;1(9):693–706. doi: 10.1091/mbc.1.9.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mochly-Rosen D., Koshland D. E., Jr A general procedure for screening inhibitory antibodies: application for identifying anti-protein kinase C antibodies. Anal Biochem. 1988 Apr;170(1):31–37. doi: 10.1016/0003-2697(88)90085-1. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Mochly-Rosen D. Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science. 1995 Apr 14;268(5208):247–251. doi: 10.1126/science.7716516. [DOI] [PubMed] [Google Scholar]
  32. Moore E. D., Etter E. F., Philipson K. D., Carrington W. A., Fogarty K. E., Lifshitz L. M., Fay F. S. Coupling of the Na+/Ca2+ exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle. Nature. 1993 Oct 14;365(6447):657–660. doi: 10.1038/365657a0. [DOI] [PubMed] [Google Scholar]
  33. Morgan J. P., Morgan K. G. Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J Physiol. 1984 Jun;351:155–167. doi: 10.1113/jphysiol.1984.sp015239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Oka N., Yamamoto M., Schwencke C., Kawabe J., Ebina T., Ohno S., Couet J., Lisanti M. P., Ishikawa Y. Caveolin interaction with protein kinase C. Isoenzyme-dependent regulation of kinase activity by the caveolin scaffolding domain peptide. J Biol Chem. 1997 Dec 26;272(52):33416–33421. doi: 10.1074/jbc.272.52.33416. [DOI] [PubMed] [Google Scholar]
  37. Rasmussen H., Takuwa Y., Park S. Protein kinase C in the regulation of smooth muscle contraction. FASEB J. 1987 Sep;1(3):177–185. [PubMed] [Google Scholar]
  38. Rembold C. M., Murphy R. A. Myoplasmic [Ca2+] determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Circ Res. 1988 Sep;63(3):593–603. doi: 10.1161/01.res.63.3.593. [DOI] [PubMed] [Google Scholar]
  39. Schwartz M. A. Transmembrane signalling by integrins. Trends Cell Biol. 1992 Oct;2(10):304–308. doi: 10.1016/0962-8924(92)90120-c. [DOI] [PubMed] [Google Scholar]
  40. Singer H. A., Schworer C. M., Sweeley C., Benscoter H. Activation of protein kinase C isozymes by contractile stimuli in arterial smooth muscle. Arch Biochem Biophys. 1992 Dec;299(2):320–329. doi: 10.1016/0003-9861(92)90281-z. [DOI] [PubMed] [Google Scholar]
  41. Smart E. J., Ying Y. S., Anderson R. G. Hormonal regulation of caveolae internalization. J Cell Biol. 1995 Nov;131(4):929–938. doi: 10.1083/jcb.131.4.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stull J. T., Gallagher P. J., Herring B. P., Kamm K. E. Vascular smooth muscle contractile elements. Cellular regulation. Hypertension. 1991 Jun;17(6 Pt 1):723–732. doi: 10.1161/01.hyp.17.6.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Turner C. E., Burridge K. Transmembrane molecular assemblies in cell-extracellular matrix interactions. Curr Opin Cell Biol. 1991 Oct;3(5):849–853. doi: 10.1016/0955-0674(91)90059-8. [DOI] [PubMed] [Google Scholar]
  44. Turner C. E., Pavalko F. M., Burridge K. The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity. J Biol Chem. 1989 Jul 15;264(20):11938–11944. [PubMed] [Google Scholar]
  45. Walsh M. P., Carmichael J. D., Kargacin G. J. Characterization and confocal imaging of calponin in gastrointestinal smooth muscle. Am J Physiol. 1993 Nov;265(5 Pt 1):C1371–C1378. doi: 10.1152/ajpcell.1993.265.5.C1371. [DOI] [PubMed] [Google Scholar]

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