A protein phosphatase-1γ1 isoform selectivity determinant in dendritic spine-associated neurabin

Abstract

Protein phosphatase-1 (PP1) catalytic subunit isoforms interact with diverse proteins, typically containing a canonical (R/K)(V/I)XF motif. Despite sharing ∼90% amino acid sequence identity, PP1β and PP1γ1 have distinct subcellular localizations that may be determined by selective interactions with PP1-binding proteins. Immunoprecipitation studies from brain and muscle extracts demonstrated that PP1γ1 selectively interacts with spinophilin and neurabin, F-actin-targeting proteins, whereas PP1β selectively interacted with GM/ RGL, the striated-muscle glycogen-targeting subunit. Glutathione S-transferase (GST) fusion proteins containing residues 146-493 of neurabin (GST-Nb-(146-493)) or residues 1-240 of GM/RGL (GST-GM-(1-240)) recapitulated these isoform selectivities in binding and phosphatase activity inhibition assays. Site-directed mutagenesis indicated that this isoform selectivity was not due to sequence differences between the canonical PP1-binding motifs (neurabin, 457KIKF460; GM/RGL, 65RVSF68). A chimeric GST fusion protein containing residues 1-64 of GM/RGL fused to residues 457-493 of neurabin (GST-GM/Nb) selectively bound to and inhibited PP1γ1, whereas a GST-Nb/GM chimera containing Nb-(146-460) fused to G M-(69-240) selectively interacted with and weakly inhibited PP1β, implicating domain(s) C-terminal to the (R/K)(V/I)XF motif as determinants of PP1 isoform selectivity. Deletion of Pro464 and Ile465 in neurabin (API) to equally space a conserved cluster of amino acids from the (R/K)(V/I)XF motif as in GM/RGL severely compromised the ability of neurabin to bind and inhibit both isoforms but did not affect PP1γ1 selectivity. Further analysis of a series of C-terminal truncated GST-Nb-(146-493) proteins identified residues 473-479 of neurabin as containing a crucial PP1γ1-selectivity determinant. In combination, these data identify a novel PP1γ1-selective interaction domain in neurabin that may allow for selective regulation and/or subcellular targeting of PP1 isoforms.