Abstract
The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets. PP1 associates with ≥200 regulatory proteins to form highly specific holoenzymes. These regulatory proteins target PP1 to its point of action within the cell and prime its enzymatic specificity for particular substrates. However, how they direct PP1's specificity is not understood. Here we show that spinophilin, a neuronal PP1 regulator, is entirely unstructured in its unbound form, and it binds PP1 through a folding-upon-binding mechanism in an elongated fashion, blocking one of PP1's three putative substrate binding sites without altering its active site. This mode of binding is sufficient for spinophilin to restrict PP1's activity toward a model substrate in vitro without affecting its ability to dephosphorylate its neuronal substrate, glutamate receptor 1 (GluR1). Thus, our work provides the molecular basis for the ability of spinophilin to dictate PP1 substrate specificity.
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Acknowledgements
This work was supported by the US National Institutes of Health (NS056128 to W.P.; MH074866 to A.C.N.). W.P. is the Manning Assistant Professor of Medical Science at Brown University. This material is based upon work supported under a National Science Foundation Graduate Research Fellowship to B.D. Crystallographic data was collected at the X6A beamline, funded by the National Institute of General Medical Sciences, under agreement GM-0080. The National Synchrotron Light Source, Brookhaven National Laboratory, is supported by the US Department of Energy under contract number DE-AC02-98CH10886.
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M.J.R. performed ITC, dephosphorylation assays and crystallization of the spinophilin–PP1, spinophilin–PP1–nodularin-R and neurabin–PP1 complexes; W.P. purified unbound spinophilin for NMR studies; B.D. performed and analyzed NMR studies of unbound spinophilin; M.J.R., D.A.C. and R.P. collected, processed and refined X-ray data; M.J.R., A.C.N., R.P. and W.P. wrote the paper; all authors discussed the data and manuscript.
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Ragusa, M., Dancheck, B., Critton, D. et al. Spinophilin directs protein phosphatase 1 specificity by blocking substrate binding sites. Nat Struct Mol Biol 17, 459–464 (2010). https://doi.org/10.1038/nsmb.1786
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DOI: https://doi.org/10.1038/nsmb.1786
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