Abstract
Activation of cyclic nucleotide-dependent signaling pathways leads to the relaxation of various smooth muscles. One of the major phosphorylation events associated with cyclic nucleotide-dependent vasorelaxation in bovine trachealis and carotid artery smooth muscle is the phosphorylation of two 20-kDa phosphoproteins with pI values of 5.7 and 5.9 (previously designated pp8 and pp3, respectively). The present studies sought to determine the identities of pp3 and pp8 in vascular smooth muscle. The phosphopeptide maps for the pp8 and pp3 proteins were similar. Preparative two-dimensional gel electrophoresis and amino acid sequencing of a peptide fragment of the pp3 protein revealed a sequence identical to a 20-kDa heat shock-related protein (HSP20) previously purified from skeletal muscle. Western blot and immunoprecipitation analysis with anti-HSP20 antibodies demonstrated that the pp3 and pp8 proteins are phosphorylated forms of HSP20. In addition, HSP20 could be phosphorylated in vitro by both cAMP-dependent protein kinase and cGMP-dependent protein kinase. These data suggest that the phosphorylation of the heat shock-related protein HSP20 is associated with cyclic nucleotide-dependent relaxation of vascular smooth muscle.
Highlights
Activation of cyclic nucleotide-dependent signaling pathways leads to the relaxation of vascular smooth muscle
There is no increase in the extent of the phosphorylation of these two proteins in human umbilical artery smooth muscle, a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation [15]
These data suggest that an increase in the phosphorylation of these two 20-kDa phosphoproteins plays a role in cyclic nucleotide-dependent vasorelaxation
Summary
Vol 272, No 17, Issue of April 25, pp. 11283–11287, 1997 Printed in U.S.A. Cyclic Nucleotide-dependent Vasorelaxation Is Associated with the Phosphorylation of a Small Heat Shock-related Protein*. HSP20 could be phosphorylated in vitro by both cAMP-dependent protein kinase and cGMP-dependent protein kinase These data suggest that the phosphorylation of the heat shock-related protein HSP20 is associated with cyclic nucleotide-dependent relaxation of vascular smooth muscle. There is no increase in the extent of the phosphorylation of these two proteins in human umbilical artery smooth muscle, a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation [15]. Using highly specific antibodies to HSP20, in situ and in vitro phosphorylation studies of HSP20 show that the two 20-kDa phosphoproteins are phosphorylated forms of HSP20 and that HSP20 is a substrate for both PKA and PKG These data indicate that there is a correlation between the ability of an intact vascular smooth muscle to undergo relaxation and an increase in the extent of the phosphorylation of a small heat shock-related protein, HSP20
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