Abstract

Sustained smooth muscle contraction is mediated by protein kinase C (PKC) through a signal transduction cascade leading to contraction. Heat-shock protein 27 (HSP27) appears to be the link between these two major events, i.e., signal transduction and sustained smooth muscle contraction. We have investigated the involvement of HSP27 in signal transduction and HSP27 association with contractile proteins (e.g., actin, myosin, tropomyosin, and caldesmon) resulting in sustained smooth muscle contraction. We have carried out confocal microscopy to investigate the cellular reorganization and colocalization of proteins and immunoprecipitation of HSP27 with actin, myosin, tropomyosin, and caldesmon as detected by sequential immunoblotting. Our results indicate that1) translocation of Raf-1 to the membrane when stimulated with ceramide is inhibited by vasoactive intestinal peptide (VIP), a relaxant neuropeptide;2) PKC-α and mitogen-activated protein kinase translocate and colocalize on the membrane in response to ceramide, and PKC-α translocation is inhibited by VIP;3) HSP27 colocalizes with actin when contraction occurs; and 4) HSP27 immunoprecipitates with actin and with the contractile proteins myosin, tropomyosin, and caldesmon. We propose a model in which HSP27 is involved in sustained smooth muscle contraction and modulates the interaction of actin, myosin, tropomyosin, and caldesmon.

  • heat-shock protein 27
  • protein kinase C
  • colocalization
  • actin
  • immunoprecipitation
  • sustained smooth muscle contraction
  • Raf-1

Footnotes

  • Address for reprint requests and other correspondence: K. N. Bitar, Division of Pediatric Gastroenterology, Univ. of Michigan Medical School, 1150 W. Medical Center Dr., MSRB I, Rm. A520, Ann Arbor, MI 48109-0656 (E-mail: bitar{at}umich.edu).

  • This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-42876.

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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