Molecular identification of human G-substrate, a possible downstream component of the cGMP-dependent protein kinase cascade in cerebellar Purkinje cells

G-substrate, an endogenous substrate for cGMP-dependent protein kinase, exi sts almost exclusively in cerebellar Purkinje cells, where it is possibly i nvolved in the induction of long-term depression. A G-substrate cDNA was id entified by screening expressed sequence tag databases from a human brain l ibrary. The deduced amino acid sequence of human G-substrate contained two putative phosphorylation sites (Thr-68 and Thr 119) with amino acid sequenc es [KPRRKDT(p)PALH] that were identical to those reported for rabbit G-subs trate. G-substrate mRNA was expressed almost exclusively in the cerebellum as a single transcript. The human G-substrate gene was mapped to human chro mosome 7p15 by radiation hybrid panel analysis. In vitro translation produc ts of the cDNA showed an apparent molecular mass of 24 kDa on SDS/PAGE whic h was close to that of purified rabbit G-substrate (23 kDa). Bacterially ex pressed human G-substrate is a heat-stable and acid-soluble protein that cr oss-reacts with antibodies raised against rabbit G-substrate. Recombinant h uman G-substrate was phosphorylated efficiently by cGMP-dependent protein k inase exclusively at Thr residues, and it was recognized by antibodies spec ific for rabbit phospho-G-substrate. The amino acid sequences surrounding t he sites of phosphorylation in G-substrate are related to those around Thr- 34 and Thr-35 of the dopamine- and cAMP-regulated phosphoprotein DARPP-32 a nd inhibitor-1, respectively, two potent inhibitors of protein phosphatase 1. However, purified G-substrate phosphorylated by cGMP-dependent protein k inase inhibited protein phosphatase 2A more effectively than protein phosph atase 1, suggesting a distinct role as a protein phosphatase inhibitor.