Cyclic nucleotide-dependent vasorelaxation is associated with the phosphorylation of a small heat shock-related...

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.