A protein phosphatase-1gamma1 isoform selectivity determinant in dendritic spine-associated neurabin.

Carmody LC, Bauman PA, Bass MA, Mavila N, DePaoli-Roach AA, Colbran RJ
J Biol Chem. 2004 279 (21): 21714-23

PMID: 15016827 · DOI:10.1074/jbc.M402261200

Protein phosphatase-1 (PP1) catalytic subunit isoforms interact with diverse proteins, typically containing a canonical (R/K)(V/I)XF motif. Despite sharing approximately 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, (457)KIKF(460); G(M)/R(GL), (65)RVSF(68)). 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 PP1beta, 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 (deltaPI) 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.

MeSH Terms (29)

Actins Amino Acid Motifs Amino Acid Sequence Animals Blotting, Western Brain Catalytic Domain Dendrites Dose-Response Relationship, Drug Gene Deletion Genetic Vectors Glutathione Transferase Mice Microfilament Proteins Molecular Sequence Data Muscles Mutagenesis, Site-Directed Mutation Nerve Tissue Proteins Phosphoprotein Phosphatases Precipitin Tests Protein Binding Protein Isoforms Protein Phosphatase 1 Protein Structure, Tertiary Rats Recombinant Fusion Proteins Recombinant Proteins Sequence Homology, Amino Acid

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