Cyclic GMP regulation of myosin phosphatase: a new piece for the puzzle?

The contractile state of smooth muscle (SM) reflects the ratio of activities of myosin light-chain kinase (MLCK) and myosin light chain phosphatase (MLCP), which determines the extent of regulatory light chain (RLC) phosphorylation and actin-activated myosin II activity and can be changed by modulating the activities of the calcium-calmodulin–dependent MLCK or of MLCP. Agonists through G protein–coupled receptors, which activate the small G protein RhoA and its effector, Rho-kinase (ROK), results in inhibitory phosphorylation of the regulatory subunit (MYPT1) of MLCP leading to an increase in RLC phosphorylation and force independent of a change in [Ca ]i, a process termed Ca sensitization (reviewed in1). The importance of regulation of MLCP activity has focused attention on potential phosphorylation sites2 on MYPT1 especially Thr-696, Thr853, Ser-695, and Ser-852 (numbering for the human MW133 isoform) (Figure) but the physiologically relevant sites remain to be fully understood. CPI-17, found in some SMs is another potential mediator of Ca sensitization, which on phosphorylation by a variety of kinases inhibits the MLCP catalytic subunit, PP1c resulting in Ca -sensitized force independent of phosphorylation of MYPT1.3 Conversely, cyclic nucleotides can relax Ca -sensitized force and reduce RLC phosphorylation through activation or disinhibition of MLCP activity leading to Ca desensitization. Early studies showed that cyclic GMP decreased Ca sensitivity4 and reversed agonist-induced Ca -sensitized force at constant Ca .5,6 Urocortin-induced Ca desensitization through PKA activation leads to a decrease in both Thr-696 and Thr-853.7 Direct phosphorylation of MYPT1 has been shown for both PKA on Ser-6958 and by PKG on a C-terminal Ser residue.9 However, phosphorylation of these sites did not activate phosphatase activity raising the question as to the underlying mechanism. In an elegant study by Haystead and colleagues, all the major sites phosphorylated by PKA and PKG, which included Ser-692, Ser-695, and Ser-852, were identified in radioactive peptides of MYPT1 using Edman sequencing (Figure).10 Importantly, they demonstrated that phosphorylation of Ser-695, which is immediately adjacent to the inactivating Thr-696 prevented phosphorylation of Thr-696 by MYPT1 kinase. Additionally, 8-bromo-cGMP inhibited Thr-696 phosphorylation and Ca sensitization of ileum SM.10 Thus, phosphorylation of Ser695 prevented phosphorylation of Thr-696 and its inhibition of MLCP. The role of PKA/PKG induced phosphorylation of Ser692 and Ser852 remains to be determined. In the current issue of Circulation Research, Nakamura et al11 further examined the antagonism between MYPT1 Ser-695 and Thr-696 phosphorylation through the generation of a diphospho antibody, which only recognizes MYPT1 diphosphorylated at Ser-695 and Thr-696, and a phospho Ser-695 antibody, which specifically recognizes phospho Ser-695/ unphosphorylated Thr-695 MYPT1 and not the diphospho form. An additional phospho Thr-696 antibody recognizes both the diphospho and the monophosphorylated protein. Using these tools, Nakamura et al11 found that cGMP treatment of phenylephrine-stimulated -toxin–permeabilized, femoral arteries lead to a decrease in Thr-696 phosphorylation and a significant increase in Ser-695 phosphorylation, as expected from Haystead et al.10 They also detected 0.27 mol/mol. of diphospho-Ser695/Thr-696 MYPT1, which was not significantly different under the 3 conditions examined: pCa 6.5, phenylephrine stimulation, or phenylephrine plus 8-bromo-cGMP stimulation. This diphospho form represented approximately 20% of the total pool of MYPT1 in the femoral artery. This is surprising if phosphorylation of Ser-695 prevents phosphorylation at the adjacent Thr-696 site as demonstrated by Wooldridge et al,10 and this discrepancy remains to be resolved. Ultimately, direct site analysis showing the presence of phosphate at both sites is important. Both groups found that kinases that phosphorylate Thr-696 are much more effective in phosphorylating this site when Ser-695 is unphosphorylated, possibly reflecting decreased accessibility attributable to the bulky phosphate group. Curiously, no increase in phosphorylation of Thr-696 or the diphospho Ser-695/Thr-696 sites in the -toxin–permeabilized artery sensitized to Ca with phenylephrine was observed,11 in agreement with several previous reports using only the commercially available phospho-Thr-696 antibodies. It is unclear whether ROK directly phosphorylates MYPT1 at Thr-696, in contrast to phosphorylation at Thr-853, which is reduced by treatment with ROK inhibitors (reviewed in1). The Thr-853 site was not explored in the present study. They report that Thr-696 phosphorylation decreased by only 50% with 8-bromo-cGMP stimulation, whereas the tension and RLC phosphorylation fell to baseline presumably indicating that Ser-695 phosphorylation is not the sole explanation of PKG-induced Ca desensitization. It is important to emphasize that the increase in Ser-695 phosphorylation does not directly increase phosphatase activity as direct measurements The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville. Correspondence to Avril V. Somlyo, Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, 22908. E-mail [email protected] (Circ Res. 2007;101;645-647.) © 2007 American Heart Association, Inc.