Hsp27 Association with Contractile Proteins

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DESCRIPTION (Applicant's Abstract): The objective of this project is to understand the role of HSP27 in normal physiologic mechanisms that modulate smooth muscle contraction of the colon. The investigator propose the existence of an intracellular signaling cascade in colonic smooth muscle that results in the phosphorylation of HSP27 and the association of HSP27 with other contractile proteins, particularly actin, tropomyosin, myosin and caldesmon, leading to smooth muscle contraction. This cascade is initiated by PKC activation, by PKC translocation to the membrane, leading to activation of MAP kinases. The hypothesis is that HSP27 plays an important role in maintaining cytoskeletal integrity, and cytoskeletal association between the contractile proteins within smooth muscle cells. The applicant's studies will determine how phosphorylation of HSP27 confers association with the contractile proteins, leading to contraction. Emerging data suggest that phosphorylation of HSP27 plays a role in cytoskeletal reorganization by increasing stability and association of the contractile proteins in smooth muscle cells. HSP27 is phosphorylated in response to different contractile agonists like bombesin, endothelin, carbachol and ceramide. Preliminary data also suggest that phosphorylation of HSP27 is accompanied by an increase in the complexing of HSP27 with actin and with tropomyosin. Contraction is also accompanied with a strong colocalization of HSP27 with actin and with tropomyosin. The relaxant neuropeptide VIP and the PKC inhibitor calphostin C cause both an inhibition of contraction and an inhibition of HSP27 phosphorylation, suggesting that phosphorylation of HSP27 and contraction may be related. The specific aims of this proposal are to: 1) examine the effect of PKC mediated contraction on HSP27 phosphorylation and cytoskeletal reorganization: 2) examine the effect of PKC mediated contraction on the association of HSP27 with actin, and other contractile proteins that are members of the contractile machinery in smooth muscle, namely myosin, caldesmon, and tropomyosin; and 3) examine the effect of altered expression of HSP27 on the association of HSP27 with actin, myosin, caldesmon and tropomyosin in transfected cells and in transgenic mice.