Title | Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity. |
Publication Type | Journal Article |
Year of Publication | 1990 |
Authors | Hemmings HC, Nairn AC, Elliott JI, Greengard P |
Journal | J Biol Chem |
Volume | 265 |
Issue | 33 |
Pagination | 20369-76 |
Date Published | 1990 Nov 25 |
ISSN | 0021-9258 |
Keywords | Amino Acid Sequence, Animals, Brain, Cattle, Dopamine and cAMP-Regulated Phosphoprotein 32, Kinetics, Molecular Sequence Data, Muscles, Nerve Tissue Proteins, Peptides, Phosphopeptides, Phosphoprotein Phosphatases, Phosphoproteins, Phosphorylation, Protein Kinases, Protein Phosphatase 1, Protein Phosphatase 2, Rabbits, Sequence Homology, Nucleic Acid, Structure-Activity Relationship, Substrate Specificity |
Abstract | Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1. |
Alternate Journal | J Biol Chem |
PubMed ID | 2173704 |
Grant List | MH 40899 / MH / NIMH NIH HHS / United States NS 21550 / NS / NINDS NIH HHS / United States |