title: Regulation of neurabin I interaction with protein phosphatase 1 by phosphorylation.
abstract: Neurabin I is a brain-specific actin-binding protein. Here we show that neurabin I binds protein phosphatase 1 (PP1) and inhibits PP1 activity. Neurabin I interacted with PP1alpha in an overlay assay, in yeast two-hybrid interaction analysis, and in coprecipitation and co-immunoprecipitation experiments. Neurabin I also copurified with both the alpha and gamma isoforms of PP1. A glutathione S-transferase (GST)-neurabin I fusion protein (residues 318-661) containing the putative PP1 binding domain (residues 456-460) inhibited PP1 activity (K(i) = 2.7 +/- 1.2 nM). This fusion protein was also rapidly phosphorylated in vitro by PKA (K(m) = 6 microM) to a stoichiomtry of 1 mol/mol. The phosphorylated residue was identified as serine 461 by HPLC-MS analysis of a tryptic digest. Phosphorylation of GST-neurabin I (residues 318-661) by PKA significantly reduced its binding to PP1 by overlay and by glutathione-Sepharose coprecipitation assays. A 35-fold decrease in inhibitory potency was also observed using a S461E mutant, which mimics phosphorylation of S461. These findings identify a signaling mechanism involving the regulation of PP1 activity and localization mediated by the cAMP pathway.
authors.affiliation: Departments of Anesthesiology and Pharmacology, Weill Medical College of Cornell University, New York, New York 10021, USA.
authors.forename: T
authors.initials: T
authors.lastname: McAvoy
authors.forename: P B
authors.initials: PB
authors.lastname: Allen
authors.forename: H
authors.initials: H
authors.lastname: Obaishi
authors.forename: H
authors.initials: H
authors.lastname: Nakanishi
authors.forename: Y
authors.initials: Y
authors.lastname: Takai
authors.forename: P
authors.initials: P
authors.lastname: Greengard
authors.forename: A C
authors.initials: AC
authors.lastname: Nairn
authors.forename: H C
authors.initials: HC
authors.lastname: Hemmings
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D054645:Protein Phosphatase 1; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D000099273:Neurabins
chemical_list: D017868:Cyclic AMP-Dependent Protein Kinases; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D000099273:Neurabins