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

Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2467-72. doi: 10.1073/pnas.96.5.2467.

Abstract

G-substrate, an endogenous substrate for cGMP-dependent protein kinase, exists almost exclusively in cerebellar Purkinje cells, where it is possibly involved in the induction of long-term depression. A G-substrate cDNA was identified by screening expressed sequence tag databases from a human brain library. The deduced amino acid sequence of human G-substrate contained two putative phosphorylation sites (Thr-68 and Thr-119) with amino acid sequences [KPRRKDT(p)PALH] that were identical to those reported for rabbit G-substrate. G-substrate mRNA was expressed almost exclusively in the cerebellum as a single transcript. The human G-substrate gene was mapped to human chromosome 7p15 by radiation hybrid panel analysis. In vitro translation products of the cDNA showed an apparent molecular mass of 24 kDa on SDS/PAGE which was close to that of purified rabbit G-substrate (23 kDa). Bacterially expressed human G-substrate is a heat-stable and acid-soluble protein that cross-reacts with antibodies raised against rabbit G-substrate. Recombinant human G-substrate was phosphorylated efficiently by cGMP-dependent protein kinase exclusively at Thr residues, and it was recognized by antibodies specific for rabbit phospho-G-substrate. The amino acid sequences surrounding the 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 and inhibitor-1, respectively, two potent inhibitors of protein phosphatase 1. However, purified G-substrate phosphorylated by cGMP-dependent protein kinase inhibited protein phosphatase 2A more effectively than protein phosphatase 1, suggesting a distinct role as a protein phosphatase inhibitor.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Brain / metabolism*
  • Cerebellum / physiology*
  • Cloning, Molecular
  • Cyclic GMP-Dependent Protein Kinases / metabolism*
  • Databases as Topic
  • Expressed Sequence Tags
  • Humans
  • Molecular Sequence Data
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism*
  • Purkinje Cells / physiology*
  • RNA, Messenger / analysis
  • Rabbits
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Transcription, Genetic

Substances

  • G-substrate
  • Nerve Tissue Proteins
  • RNA, Messenger
  • Recombinant Proteins
  • Cyclic GMP-Dependent Protein Kinases

Associated data

  • GENBANK/AF097730