HSP27 in signal transduction and association with contractile proteins in smooth muscle cells

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 that 1) 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.

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PKC-mediated redistribution of mitogen-activated protein kinase during smooth muscle cell activation

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.2.c406 ◽

1993 ◽

Vol 265 (2) ◽

pp. C406-C411 ◽

Cited By ~ 108

Author(s):

R. A. Khalil ◽

K. G. Morgan

Keyword(s):

Signal Transduction ◽

Smooth Muscle ◽

Protein Kinase ◽

Muscle Contraction ◽

Map Kinase ◽

Surface Membrane ◽

Smooth Muscle Contraction ◽

Cell Contraction ◽

Signal Transduction Cascade ◽

Mitogen Activated Protein

Protein kinase C (PKC) translocates from the cytosol to the surface membrane at the time it mediates agonist-induced contraction of ferret vascular smooth muscle cells (R. A. Khalil and K. G. Morgan. J. Physiol. Lond. 455: 585-599, 1992). However, no direct communication between membrane-associated PKC and the contractile filaments has been identified. Mitogen-activated protein (MAP) kinase is a substrate for PKC and is also capable of phosphorylating the actin-binding protein caldesmon at sites phosphorylated during smooth muscle contraction in vivo (L. P. Adam, C. J. Gapinski, and D. R. Hathaway. FEBS Lett. 302: 223-226, 1992). In the present study, the hypothesis that PKC and MAP kinase are involved in a signal-transduction cascade leading to smooth muscle contraction was tested. Immunofluorescence and digital-imaging microscopy were used to localize the epsilon-PKC isoform and MAP kinase during phenylephrine-induced Ca(2+)-independent activation of ferret aorta cells. We report that maintained phenylephrine-induced translocation of cytosolic PKC to the surface membrane is associated with transient redistribution of cytosolic MAP kinase to the surface membrane before cell contraction. Coincident with cell contraction, MAP kinase undergoes a second redistribution away from the plasmalemma and toward the vicinity of contractile filaments. Redistribution of MAP kinase is not stimulated by Ca2+ but is completely prevented by PKC inhibitors. The transient Ca(2+)-independent but PKC-dependent redistribution of MAP kinase points to MAP kinase as a missing link in the signal-transduction cascade between membrane-bound PKC and smooth muscle activation.

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Mitogen-activated protein kinase activation: An alternate signaling pathway for sustained vascular smooth muscle contraction

Journal of Vascular Surgery ◽

10.1016/s0741-5214(97)70196-4 ◽

1997 ◽

Vol 26 (2) ◽

pp. 327-332 ◽

Cited By ~ 43

Author(s):

Aaron M. Epstein ◽

Douglas Throckmorton ◽

Colleen M. Brophy

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Vascular Smooth Muscle ◽

Muscle Contraction ◽

Smooth Muscle Contraction ◽

Mitogen Activated Protein Kinase ◽

Kinase Activation ◽

Protein Kinase Activation ◽

Mitogen Activated Protein ◽

Vascular Smooth Muscle Contraction

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Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1998.275.6.g1454 ◽

1998 ◽

Vol 275 (6) ◽

pp. G1454-G1462 ◽

Cited By ~ 39

Author(s):

Pinglang Wang ◽

Khalil N. Bitar

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Muscle Contraction ◽

Smooth Muscle Contraction ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Actin Binding ◽

C3 Exoenzyme ◽

Mitogen Activated Protein ◽

Rho A

The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction. However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and C2 ceramide were used. Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60 src , respectively. Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10−7 M) or ceramide (10−7 M) (∼30% increase at 30 s that was sustained at 4 min). The translocation of Rho A to the membrane was confirmed by immunostaining. The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60 src inhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60 src and PKC or may belong to a different pathway than these proteins. Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways. Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.

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