title: Brain actin-associated protein phosphatase 1 holoenzymes containing spinophilin, neurabin, and selected catalytic subunit isoforms.
abstract: We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1(A) holoenzymes selectively enriched in PP1gamma(1) over PP1beta isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956-9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470-3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H. , Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997) J. Cell Biol. 139, 951-961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427-470, or homologous neurabin residues 436-479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1gamma(1) from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1gamma(1) over PP1beta. Moreover, immunoprecipitation of PP1gamma(1) from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1beta immunoprecipitation did not. Thus, PP1(A) holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins.
authors.affiliation: Department of Molecular Physiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
authors.forename: L B
authors.initials: LB
authors.lastname: MacMillan
authors.forename: M A
authors.initials: MA
authors.lastname: Bass
authors.forename: N
authors.initials: N
authors.lastname: Cheng
authors.forename: E F
authors.initials: EF
authors.lastname: Howard
authors.forename: M
authors.initials: M
authors.lastname: Tamura
authors.forename: S
authors.initials: S
authors.lastname: Strack
authors.forename: B E
authors.initials: BE
authors.lastname: Wadzinski
authors.forename: R J
authors.initials: RJ
authors.lastname: Colbran
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016548:Prosencephalon / Q000201:enzymology*; D054645:Protein Phosphatase 1; D020836:Protein Structure, Quaternary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D000099273:Neurabins
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D011994:Recombinant Proteins; D000099273:Neurabins