A protein phosphatase-1gamma1 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 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:
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
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
J. Biol. Chem.
Date: May. 21, 2004
PubMed ID: 15016827
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