Involvement of c‐Cbl a ring finger E3 ubiquitin ligase on cyclic GMP dependent protein kinase ubiquitination and degradation

Abstract

Cyclic GMP‐dependent protein kinase‐1 (PKG‐1) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell‐specific gene expression. PKG‐I expression in cultured smooth muscle cells (SMC) depends on culture conditions and is inhibited by inflammatory cytokines such as interleukin‐I and tumor necrosis factor‐α, which are known to stimulate Type II NO synthase (iNOS) and subsequent elevation of cGMP. Chronic elevation of cGMP, as seen in inflammatory conditions, triggers ubiquitination and proteasomal degradation of PKG‐Iα in smooth muscle. Previously we reported that the suppression of PKG‐I protein expression in SMC is triggered by the ubiquitin/26S proteasome pathway. In the current study we found that cGMP elevation down‐regulates PKG‐Iα, but not the I ?, isoform. PKG catalytic activity (autophosphorylation) was required for the suppression of protein expression. Mutation of the known autophosphorylation sites of PKG‐Iα to alanine uncovered a specific role for autophosphorylation of serine‐64 in cGMP‐dependent ubiquitination and suppression of PKG protein expression. We further explored the involvement of specific E3 ubiquitin ligases for this process. We examined the relationship between cCbl and PKG‐1α and the involvement of the ring finger E3 ligase Cbl in the process of ubiquitination. We observed that as a potential PKG‐1α ubiquitin E3 ligase, Cbl forms a complex with and ubiquitinates PKG. These results suggest that down regulation of PKG 1α is triggered by serine‐64 autophosphorylation and then subsequent binding of the ubiquitin E3 ligase, Cbl.