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.
Carmody
Leigh C
LC
Department of Molecular Physiology and Biophysics, The Center for Molecular Neuroscience, and The Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Bauman
Patricia A
PA
Bass
Martha A
MA
Mavila
Nirmala
N
DePaoli-Roach
Anna A
AA
Colbran
Roger J
RJ
D000199:Actins / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D020134:Catalytic Domain; D003712:Dendrites / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D000099273:Neurabins
D000199:Actins; D005982:Glutathione Transferase; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D010749:Phosphoprotein Phosphatases; D020033:Protein Isoforms; D054645:Protein Phosphatase 1; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000099273:Neurabins