The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI17, thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both alpha-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203x, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction.

Abstract-Nitric oxide/cGMP pathway induces vasodilatation, yet the underlying mechanism is obscure. In the present study, we studied the mechanism of cGMP-induced relaxation of the smooth muscle contractile apparatus using permeabilized rabbit femoral arterial smooth muscle. 8-Br-cGMP-induced relaxation was accompanied with a decrease in myosin light chain (MLC) phosphorylation. MLC phosphatase (MLCP) activity, once decreased by agoniststimulation, recovered to the resting level on addition of 8-Br-cGMP. Because MLCP activity is regulated by the phosphorylation of a MLCP-specific inhibitor, CPI17 at Thr38 and MBS (myosin binding subunit of MLCP) at Thr696, we examined the effect of 8-Br-cGMP on the phosphorylation of these MLCP modulators. Whereas CPI17 phosphorylation was unchanged after addition of 8-Br-cGMP, MBS phosphorylation at Thr696 was significantly decreased by 8-Br-cGMP. We found that 8-Br-cGMP markedly increased MBS phosphorylation at Ser695 in the fiber pretreated with phenylephrine. MBS phosphorylation of Thr696 phosphorylated MBS at Ser695 partially resumed MLCP activity inhibited by Thr696 phosphorylation. Whereas Ser695 phosphorylation was markedly increased, the extent of diphosphorylated MBS at Ser695 and Thr696 in fibers was unchanged after cGMP-stimulation. We found that MBS phosphatase activity in arteries for both diphosphorylated MBS and monophosphorylated MBS at Thr696 significantly increased by 8-Br-cGMP, whereas MBS kinase activity was unchanged. These results suggest that the phosphorylation at Ser640 induced by cGMP shifted the equilibrium of the Thr641 phosphorylation toward dephosphorylation, thus increasing MLCP activity. This results in the decrease in MLC phosphorylation and smooth muscle relaxation. Key Words: cGMP Ⅲ myosin light chain phosphatase Ⅲ vasodilation Ⅲ phosphorylation Ⅲ smooth muscle I t has been known that endothelial-derived nitric oxide (NO) acts as a vasodilator, 1 and the pharmacological NO producing drugs have been used to prevent acute heart failure. NO has been defined as the activator of soluble guanylate cyclase, 2 thus increasing cGMP. A number of studies have indicated that cGMP induces relaxation of various smooth muscles contracted either by receptor-coupling agonists or depolarization 3 ; therefore, NO-induced vasodilation is thought to be attributable to the cGMP-induced relaxation of vascular smooth muscle. The key question is how cGMP triggers the vascular smooth muscle relaxation.Smooth muscle contraction is controlled by the phosphorylation of the regulatory light chain (RLC) of myosin at Ser19 4,5 by Ca 2ϩ /calmodulin-dependent protein kinase, called myosin light chain kinase (MLCK). 4 -6 On the other hand, MLC phosphatase (MLCP) activity is also regulated during the agonist-induced contraction of smooth muscle, thus contributing to the increase in RLC phosphorylation, but in contrast to the regulation of MLCK, the mechanism is Ca 2ϩ independent. 6 MLCP consists of 3 subunits, a myosin binding large subunit (MBS), 7,8 a 20-kDa small subunit (M2...

p116Rip was originally found to be a RhoA-binding protein, but its function has been unknown. Here, we clarify the function of p116Rip . Two critical findings were made. First, we found that p116Rip activated the GTPase activity of RhoA in vitro and that p116Rip overexpression in cells consistently diminished the epidermal growth factor-induced increase in GTP-bound RhoA. Second, p116Rip activated the myosin light chain phosphatase (MLCP) activity of the holoenzyme. p116Rip did not activate the catalytic subunit alone, indicating that the activation is due to the binding of p116Rip to the myosin phosphatase targeting subunit MYPT1. Interestingly, the activation of phosphatase was specific to myosin as substrate, and p116Rip directly bound to myosin, thus facilitating myosin/MLCP interaction. The gene silencing of p116Rip consistently and significantly increased myosin phosphorylation as well as stress fiber formation in cells. Based upon these findings, we propose that p116Rip is an important regulatory component that controls the RhoA signaling pathway, thus regulating MLCP activity and myosin phosphorylation in cells.

There are many downstream targets of mitogen-activated protein kinase (MAPK) signalling that are involved in neuronal development, cellular differentiation, cell migration, cancer, cardiovascular dysfunction and inflammation via their functions in promoting apoptosis and cell motility and regulating various cytokines. It has been reported that cyclic AMP response element-binding protein (CREB) is phosphorylated and activated by cyclic AMP signalling and calcium/calmodulin kinase. Recent evidence also points to CREB phosphorylation by the MAPK signalling pathway. However, the specific roles of CREB phosphorylation in MAPK signalling have not yet been reviewed in detail. Here, we describe the recent advances in the study of this MAPK-CREB signalling axis in human diseases. Overall, the crosstalk between extracellular signal-related kinase (ERK) 1/2 and p38 MAPK signalling has been shown to regulate various physiological functions, including central nervous system, cardiac fibrosis, alcoholic cardiac fibrosis, osteoclast differentiation, mucin production in the airway, vascular smooth muscle cell migration, steroidogenesis and asthmatic inflammation. In this review, we focus on ERK1/2 and/or p38 MAPK-dependent CREB activation associated with various diseases to provide insights for basic and clinical researchers.