Thr123 of rat G-substrate contributes to its action as a protein phosphatase inhibitor.
Rat G-substrate cDNA was isolated from a cerebellar library and characterized. The deduced amino acid sequence of rat G-substrate contained two putative phosphorylation sites for PKG at Thr72 and Thr123; the amino acid sequences (KPRRKDT(p)PA) around these sites are conserved in human, mouse and rabbit. G-substrate phosphorylated by PKG inhibited the catalytic subunits of both protein phosphatase-1 (IC(50) 14.1 nM) and -2A (IC(50) 5.9 nM). Mutation of Thr123 (site 2) to Ala significantly reduced the inhibition of both PP-1 and PP-2A, while mutation of Thr72 (site 1) to Ala had little effect on inhibitory activity. In situ hybridization analysis revealed that G-substrate mRNA was localized exclusively in cerebellar Purkinje cells. Immunoperoxidase staining showed that in Purkinje cells, G-substrate was present in somata, dendrites and axons. In rat cerebellar slices, activation of PKG with a nitric oxide (NO) donor, NOR3, or 8-Br-cGMP, increased phosphorylation of G-substrate, as demonstrated with a phosphorylation-specific antibody. These results characterize further the inhibition of PP-1 and PP-2A by phospho-G-substrate, and demonstrate its physiological phosphorylation in rat Purkinje cells.
Endo
Shogo
S
Neuronal Circuit Mechanisms Research Group, RIKEN, Brain Science Institute, Wako 351-0198, Japan. sendo@postman.riken.go.jp
Nairn
Angus C
AC
Greengard
Paul
P
Ito
Masao
M
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D003001:Cloning, Molecular; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis*; D004722:Endoribonucleases; D004789:Enzyme Activation; D015723:Gene Library; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009924:Organ Culture Techniques; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D016601:RNA-Binding Proteins; D051381:Rats; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D018076:DNA, Complementary; C029152:G-substrate; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; C021042:protein phosphatase inhibitor-1; D017869:Cyclic GMP-Dependent Protein Kinases; D004722:Endoribonucleases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C097081:PPP1R8 protein, human