Abstract
Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.
Highlights
Myosin phosphatase is regulated by multisite phosphorylation of its myosin targeting subunit of myosin light chain phosphatase (MLCP) (MYPT1) subunit
Ca2؉ sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr697 and/or Thr855 to inhibit phosphatase activity and increase contractile force
When signals from Western blots performed with the anti-[phospho-Ser696/ Ser854]MYPT1 antibody were analyzed, it was clear that the data resembled those obtained with the anti-[phosphoSer696]MYPT1 antibody, i.e. elevated signals (ϳ2-fold) were observed with forskolin treatment alone and forskolin followed by U46619 treatment (Fig. 8B)
Summary
Myosin phosphatase is regulated by multisite phosphorylation of its myosin targeting subunit of MLCP (MYPT1) subunit. Ca2؉ sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr697 and/or Thr855 (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser696 prevents phosphorylation at Thr697. Additional studies have described intracellular translocations of MYPT1 following Thr697 phosphorylation [19] Regardless of their molecular mechanisms, Thr697 and/or Thr855 phosphorylations are associated with decreased MLCP activity in smooth muscle tissue, serving to increase the ratio of MLCK:MLCP activity and resulting in increased LC20 phosphorylation and contractile force. These dual phosphorylation events were associated with disinhibition of Thr697 and Thr855 phosphorylation as well as smooth muscle relaxation
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