Public Health Support and the National Institutes of Health [Grants GM56362 and CA40042 (to D.L.B.) and HL51824 and HL70881 (to T.K.)]. Notes This paper was submitted directly (Track II) to the PNAS office. Abbreviations: PP1, protein phosphatase-1; MC-LR, microcystin-LR; TP-CPI-17, thiophospho-CPI-17; I-1, inhibitor-1; Inh2, inhibitor-2.. same PP1 catalytic subunit. Phosphorylation of CPI-17 in rabbit arteries was enhanced by calyculin A but not okadaic acid or fostriecin, consistent with PP1-mediated dephosphorylation. We propose that CPI-17 binds at the PP1 active site where it is dephosphorylated, but association of MYPT1 with PP1C allosterically retards this hydrolysis, resulting in formation of a complex of MYPT1PP1CP-CPI-17, leading to an increase in smooth muscle mass contraction. Protein phosphatase-1 (PP1) is usually a dominant Ser/Thr phosphatase, controlling a plethora of events in cells, from yeast to mammals. Cellular PP1 holoenzymes consist of a catalytic subunit (PP1C) and various regulatory (targeting) subunits. PP1C in mammalian cells exists as four isoforms that contain a conserved catalytic domain name plus a variable region in the C-terminal tails (1). On the other hand, >50 PP1 regulatory subunits have been discovered in mammalian cells (examined in refs. 2 and 3). These subunits have a common PP1-binding sequence motif, VxF, that associates with PP1C around the backside reverse the active site, tethering PP1C to compartmentalize PP1 activity (4). Additionally, the conversation with regulatory subunits results in allosteric modulation of PP1C substrate specificity (5). Thus, PP1 subunits change properties of PP1C to generate diversity of function in cells (2, 3). In addition to regulatory subunits, several PP1-specific inhibitor proteins are present in mammalian cells. These inhibitor proteins potently suppress the activity of purified PP1C at nanomolar concentrations (6). Most PP1 inhibitor proteins are phosphoproteins, suggesting that cellular PP1 activities are modulated in response to kinase signaling via phosphorylation of PP1 inhibitor proteins. Originally, PP1 inhibitor proteins such as inhibitor-1 (I-1) and inhibitor-2 (Inh2) were believed to inhibit only the free PP1C released from regulatory subunits but not the PP1 holoenzymes ECT2 themselves. This concept was based on results showing that neither I-1 nor Inh2 blocked activity of the glycogen-bound PP1 holoenzyme (7) or myosin phosphatase holoenzyme (8). More recently, several lines of evidence show regulation of particular PP1 holoenzymes by the inhibitor proteins CPI-17 (9), I-1 (10), and Inh2 (11C13). These new data bring into question how inhibitor proteins identify different PP1 holoenzymes with common catalytic subunits. CPI-17 was purified as a myosin phosphatase inhibitor protein from pig aorta (9, 14). The inhibitory potency of CPI-17 is usually increased >1,000-fold by phosphorylation at Thr-38 (9). Several kinases purified from easy muscles, such as PKC/, ZIP-kinase, and integrin-linked kinase, activate CPI-17 by Emtricitabine phosphorylation at Thr-38 (15C17). Phosphorylation of CPI-17 at Thr-38 in easy muscle cells occurs in response to numerous agonists, such as histamine, endothelin-1, and angiotensin II, in parallel with induction of myosin phosphorylation and contraction (18, 19). On the other hand, phosphorylation of CPI-17 is usually Emtricitabine reversed during vasodilation induced by nitric oxide production (20). Thus, phosphorylation of CPI-17 suppresses myosin phosphatase activity, resulting in phosphorylation of myosin and contraction Emtricitabine of easy muscle mass. In addition, specific depletion of endogenous CPI-17 by small interfering RNA or antibody microinjection eliminated the cerebellar long-term synaptic depressive disorder of Purkinje cells mediated by PKC, demonstrating involvement of CPI-17 in neuronal signaling (21). Although phospho-CPI-17 inhibits monomeric PP1C in addition to myosin phosphatase, myosin phosphatase was proposed as a favored target of phospho-CPI-17 in easy muscle mass (22), fibroblasts (23), and cerebellar Purkinje cells (21). Here we investigated how phospho-CPI-17 discriminates myosin phosphatase from among other cellular PP1 holoenzymes, to mediate specific signaling. Experimental Procedures Materials. Recombinant His-6, S-tag (H6S)-CPI-17, and (H6S)-Inh2 were prepared as explained (6). Thiophosphorylation and phosphorylation were performed by using ATPS (Roche Applied Science, Indianapolis) and ATP (Sigma), respectively. Antibodies for pan-PP1C and MYPT1 were purchased from Transduction Laboratories (Lexington, KY) and Babco (Richmond, CA), respectively. Anti-myc epitope (9E10) antibody was obtained from the Lymphocyte Culture Center at the University or college of Virginia. Antibodies for catalytic subunit of PP2A (PP2Ac), CPI-17, and P-CPI-17(T38) were prepared as explained (18, 22, 24). S-protein agarose and glutathione-agarose were purchased from Novagen and Sigma, respectively. Microcystin-LR (MC-LR) was obtained from Calbiochem and coupled with Affigel 10 (Bio-Rad,.