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

Leigh C. Carmody, Patricia A. Bauman, Martha A. Bass, Nirmala Mavila, Anna De Paoli-Roach, Roger J. Colbran

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)21714-21723
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number21
DOIs
StatePublished - May 21 2004

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Dendritic Spines
Protein Phosphatase 1
Phosphoprotein Phosphatases
Protein Isoforms
Glutathione Transferase
Proteins
Muscle
Fusion reactions
neurabin
Amino Acids
Mutagenesis
Striated Muscle
Protein Transport
Site-Directed Mutagenesis
Glycogen
Application programming interfaces (API)
Phosphoric Monoester Hydrolases
Immunoprecipitation
Actins
Amino Acid Sequence

ASJC Scopus subject areas

  • Biochemistry

Cite this

A protein phosphatase-1γ1 isoform selectivity determinant in dendritic spine-associated neurabin. / Carmody, Leigh C.; Bauman, Patricia A.; Bass, Martha A.; Mavila, Nirmala; De Paoli-Roach, Anna; Colbran, Roger J.

In: Journal of Biological Chemistry, Vol. 279, No. 21, 21.05.2004, p. 21714-21723.

Research output: Contribution to journalArticle

Carmody, Leigh C. ; Bauman, Patricia A. ; Bass, Martha A. ; Mavila, Nirmala ; De Paoli-Roach, Anna ; Colbran, Roger J. / A protein phosphatase-1γ1 isoform selectivity determinant in dendritic spine-associated neurabin. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 21. pp. 21714-21723.
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