Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca(2+) sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K(+) depolarization. The Ca(2+)-sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In alpha-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5'-O-(gamma-thiotriphosphate), and histamine plus guanyl-5'-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca(2+) sensitization of smooth muscle contraction.

A novel phosphorylation-dependent inhibitory protein (IP) of porcine aorta myosin light chain phosphatase (PA-MLCP) was purified to homogeneity from porcine aorta media. The molecular mass of IP was 20 kDa. IP phosphorylated by endogenous potentiating kinase (IP-K) inhibited not only PA-MLCP activity, but also that of the catalytic subunit of protein phosphatase-1. The amino acid sequence of a peptide derived from IP phosphorylated with IP-K, RHARVT^*VK, shared one of the consensus sequences phosphorylatable by protein kinase C (PKC), where T^* was phosphorylated. IP was phosphorylated by PKC and the phosphorylated product inhibited PA-MLCP as strongly as IP phosphorylated with IP-K.

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca²⁺ sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr⁶⁹⁵ of the MLCP regulatory subunit (MYPT1) and at Thr³⁸ of the MLCP inhibitor protein CPI‐17 results in inhibition of MLCP activity. We have previously demonstrated that CPI‐17 Thr³⁸ phosphorylation plays an important role in G‐protein‐mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site‐ and phospho‐specific antibodies, phosphorylation of MYPT1 Thr⁶⁹⁵ and CPI‐17 Thr³⁸ was examined along with MYPT1 Thr⁸⁵⁰, which is a non‐inhibitory Rho‐kinase site. We found that both CPI‐17 Thr³⁸ and MYPT1 Thr⁸⁵⁰ were phosphorylated in response to agonists or GTPγS concurrently with contraction and myosin phosphorylation in α‐toxin‐permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr⁶⁹⁵ did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho‐kinase inhibitor Y‐27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr⁸⁵⁰ and CPI‐17 Thr³⁸, respectively, in intact VD while MYPT1 Thr⁶⁹⁵ phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr⁶⁹⁵ is independent of stimulation of G‐proteins, Rho‐kinase or PKC. In the phasic PV, phosphorylation of CPI‐17 Thr³⁸ may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI‐17 concentration, probably utilizes other Ca²⁺ sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI‐17.

Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosphorylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specific protein called CPI-17 (for protein kinase C-potentiatedinhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphorylated and converted to a potent inhibitor for myosin phosphatase. Phosphorylation of CPI-17 is diminished by pretreatment with either Y27632 or GF109203x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897–9900). Here we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extracts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) α, and the second kinase was purified to homogeneity as a 45-kDa protein, and identified by sequencing as PKCδ. Purified PKCδ was 3-fold more reactive with CPI-17 compared with myelin basic protein, whereas purified PKCα and recombinant RhoA-activated kinases (Rho-associated coiled-coil forming protein Ser/Thr kinase and protein kinase N) showed equal activity with CPI-17 and myelin basic protein. Y27632 inhibited CPI-17 phosphorylation by purified PKCδ with IC₅₀ of 0.6 μm (in the presence of 0.1 mm ATP) or 14 μm (2.0 mm ATP). Y27632 significantly suppressed CPI-17 phosphorylation in smooth muscle cells, and the contraction of permeabilized rabbit femoral artery induced by stimulation with phorbol ester. GF109203x inhibited phorbol ester-induced contraction of rabbit femoral artery by 80%, whereas a PKCα/β inhibitor, Go6976, reduced contraction by 47%. The results imply that histamine stimulation elicits contraction of vascular smooth muscle through activation of PKCα and especially PKCδ to phosphorylate CPI-17.