Phosphorylation-dependent inhibition of protein phosphatase-1 by G-substrate - A Purkinje cell substrate of the cyclic GMP-dependent protein kinase

G-substrate, a specific substrate of the cGMP-dependent protein kinase, has previously been localized to the Purkinje cells of the cerebellum. We repo rt here the isolation from mouse brain of a cDNA encoding G-substrate. This cDNA was used to localize G-substrate mRNA expression, as well as to produ ce recombinant protein for the characterization of G-substrate phosphatase inhibitory activity. Brain and eye were the only tissues in which a G-subst rate transcript was detected. Within the brain, G-substrate transcripts wer e restricted almost entirely to the Purkinje cells of the cerebellum, altho ugh transcripts were also detected at low levels in the paraventricular reg ion of the hypothalamus and the pons/medulla, Like the native protein, the recombinant protein was preferentially phosphorylated by cGMP-dependent pro tein kinase (K-m = 0.2 mu M) over cAMP-dependent protein kinase (K-m = 2.0 mu M). Phospho-G-substrate inhibited the catalytic subunit of native protei n phosphatase-1 with an IC50 of 131 +/- 27 nM. Dephospho-G-substrate was no t found to be inhibitory. Both dephospho and phospho-G-substrate were weak inhibitors of native protein phosphatase-2A(1), which dephosphorylated G-su bstrate 20 times faster than the catalytic subunit of protein phosphatase-1 . G-substrate potentiated the action of cAMP-dependent protein kinase on a cAMP-regulated luciferase reporter construct, consistent with an inhibition of cellular phosphatases in vivo. These results provide the first demonstr ation that G-substrate inhibits protein phosphatase-1 and suggest a novel m echanism by which cGMP-dependent protein kinase I can regulate the activity of the type 1 protein phosphatases.