title: In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction.
abstract: CONCLUSIONS
  Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction.
  Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.
authors.affiliation: Model Animal Research Center and MOE Key Laboratory of Animal Models of Disease, Nanjing University, Nanjing, China.
authors.forename: Cai-Ping
authors.initials: CP
authors.lastname: Chen
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.forename: Yan-Ning
authors.initials: YN
authors.lastname: Qiao
authors.forename: Pei
authors.initials: P
authors.lastname: Wang
authors.forename: Wei-Qi
authors.initials: WQ
authors.lastname: He
authors.forename: Cheng-Hai
authors.initials: CH
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.forename: Yun-Qian
authors.initials: YQ
authors.lastname: Gao
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.forename: Tao
authors.initials: T
authors.lastname: Tao
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.forename: Ye
authors.initials: Y
authors.lastname: Wang
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.forename: Kristine E
authors.initials: KE
authors.lastname: Kamm
authors.forename: James T
authors.initials: JT
authors.lastname: Stull
authors.forename: Min-Sheng
authors.initials: MS
authors.lastname: Zhu
mesh_terms: D000818:Animals; D051379:Mice; D008810:Mice, Inbred C57BL; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000378:metabolism* / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase; D010766:Phosphorylation; D017354:Point Mutation; D001743:Urinary Bladder / Q000166:cytology / Q000378:metabolism* / Q000502:physiology
chemical_list: D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; C510645:Ppp1r12a protein, mouse