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

Protein phosphatase- 1 ( PP1) catalytic subunit isoforms interact with diverse proteins, typically containing a canonical ( R/ K)( V/ I) XF motif. Despite sharing similar to 90% amino acid sequence identity, PP1beta and PP1gamma1 have distinct subcellular localizations that may be determined by selective interactions with PP1- binding proteins. Immunoprecipitation studies from brain and muscle extracts demonstrated that PP1gamma1 selectively interacts with spinophilin and neurabin, F- actin- targeting proteins, whereas PP1beta selectively interacted with G(M)/ R-GL, 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 G(M)/ R-GL ( GST-G(M)-( 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, (KIKF460)-K-457; G(M)/ R-GL, (RVSF68)-R-65). A chimeric GST fusion protein containing residues 1 - 64 of G(M)/ R-GL fused to residues 457 - 493 of neurabin ( GST- G(M)/ Nb) selectively bound to and inhibited PP1gamma1, whereas a GST- Nb/ G(M) chimera containing Nb-( 146 - 460) fused to G(M)-( 69 - 240) selectively interacted with and weakly inhibited PP1 beta, implicating domain( s) C- terminal to the ( R/ K)( V/ I) XF motif as determinants of PP1 isoform selectivity. Deletion of Pro(464) and Ile(465) in neurabin ( Delta PI) to equally space a conserved cluster of amino acids from the ( R/ K)( V/ I) XF motif as in G(M)/ R-GL severely compromised the ability of neurabin to bind and inhibit both isoforms but did not affect PP1gamma1 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 PP1gamma1- selectivity determinant. In combination, these data identify a novel PP1gamma1- selective interaction domain in neurabin that may allow for selective regulation and/ or subcellular targeting of PP1 isoforms.