Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits

Abstract

Inhibition of myosin phosphatase is critical for agonist-induced contractility of vascular smooth muscle. The protein CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase and, in response to agonists, Thr-38 is phosphorylated by protein kinase C, producing a >1,000-fold increase in inhibitory potency. Here, we addressed how CPI-17 could selectively inhibit myosin phosphatase among other protein phosphatase-1 (PP1) holoenzymes. PP1 in cell lysates was separated by sequential affinity chromatography into at least two fractions, one bound specifically to thiophospho-CPI-17, and another bound specifically to inhibitor-2. The MYPT1 regulatory subunit of myosin phosphatase was concentrated only in the fraction bound to thiophospho-CPI-17. This binding was eliminated by addition of excess microcystin-LR to the lysate, showing that binding at the active site of PP1 is required. Phospho-CPI-17 failed to inhibit glycogen-bound PP1 from skeletal muscle, composed primarily of PP1 with the striated muscle glycogentargeting subunit (G M) regulatory subunit. Phospho-CPI-17 was dephosphorylated during assay of glycogen-bound PP1, not MYPT1-associated PP1, even though these two holoenzymes have the 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 MYPT1⅐PP1C⅐P-CPI-17, leading to an increase in smooth muscle contraction.

A Novel RhoA/ROCK-CPI-17-MEF2C Signaling Pathway Regulates Vascular Smooth Muscle Cell Gene Expression

“…Consistent with previously published structural data, Fig. 4C demonstrates that CPI-17 physically interacts with PP1␣, evaluated by co-immunoprecipitation, which leads to inhibition of phosphatase activity (27). In addition to being potentiated by PKC, CPI-17 has also been shown to be activated by ROCK and PKN (28,29).…”

Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure

“…Thus, MYPT1 is not essential for phosphatase activity directed toward phosphorylated RLC. We, therefore, suggest that the remaining PP1c␦ is sufficient to dephosphorylate phosphorylated RLC, although the activity is less than in the holoenzyme (36).…”

17.BETA.-Estradiol Induces Gastrointestinal Motility Disorder by Decreasing CPI-17 Phosphorylation Via Changes in Rho-Family G-Protein Rnd Expression in Small Intestine

“…We previously found in rat ileal smooth muscle that carbachol-induced contraction is mainly associated with phosphorylation of CPI-17 at Thr38 rather than MYPT1 phosphorylation at Thr696 [20]. Phosphorylation of CPI-17 due to receptor stimulation is reportedly mediated by PKC, as well as by ROCKs and PKN via RhoA activation [7,10,15,16,20]. In the present study, we demonstrated that carbachol-induced increases in CPI-17 phosphorylation were significantly reduced in organ-cultured ileal tissue treated with 17-estradiol, but no changes in total CPI-17 protein expression were noted.…”