Regulation of Neurabin I Interaction with Protein Phosphatase 1 by Phosphorylation

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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 PP1α in an overlay assay, in yeast two-hybrid interaction analysis, and in coprecipitation and co-immunoprecipitation experiments. Neurabin I also copurified with both the α and γ 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 (Ki = 2.7 ± 1.2 nM). This fusion protein was also rapidly phosphorylated in vitro by PKA (Km = 6 μM) 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.