Brain actin-associated protein phosphatase 1 holoenzymes containing spinophilin, neurabin, and selected catalytic subunit isoforms

We previously characterized PP1bp134 and PP1bp175, two neuronal proteins th at bind the protein phosphatase 1 catalytic subunit (PPI). Here we purify f rom rat brain actin-cytoskeletal extracts PP1(A) holoenzymes selectively en riched in PP1 gamma(1) over PP1 beta isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localize d F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Gr eengard, 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 T akai, Y. (1997) J. Cell Biol. 139, 951-961), respectively. Recombinant spin ophilin and neurabin interacted with endogenous PP1 and also with each othe r when co-expressed in HEK293 cells. Spinophilin residues 427-470, or homol ogous neurabin residues 436-479, were sufficient to bind PPI in gel overlay assays, and selectively bound PP1 gamma(1), from a mixture of brain protei n phosphatase catalytic subunits; additional N- and C-terminal sequences we re required for potent inhibition of PPI. Immunoprecipitation of spinophili n or neurabin from crude brain extracts selectively coprecipitated PP1 gamm a(1), over PP1 beta. Moreover, immunoprecipitation of PP1 gamma(1), from br ain extracts efficiently coprecipitated spinophilin and neurabin, whereas P P1 beta immunoprecipitation did not. Thus, PP1(A) holoenzymes containing sp inophilin and/or neurabin target specific neuronal PP1 isoforms, facilitati ng efficient regulation of synaptic phosphoproteins.