KEY POINTS: Force production and maintenance in smooth muscle is largely controlled by myosin @entity3917 ( @entity3917 ) phosphorylation, which relies on a balance between @entity141 /calmodulin-dependent @entity23452 ( @entity23452 ) and myosin light chain phosphatase (MLCP) activities. @entity63671 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 @entity19 lines, @entity68459 and @entity68460 , and found that phosphorylation of @entity63671 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of @entity3917 phosphorylation in vivo. Our findings reveal the role of @entity63671 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. ABSTRACT: Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin @entity3917 ( @entity3917 ) phosphorylation, which relies on a balance between @entity141 /calmodulin-dependent @entity23452 ( @entity23452 ) 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 @entity63671 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. @entity63671 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 @entity19 lines with @entity493 substitution of T694 or T852. @entity1494 smooth muscle from @entity68460 @entity19 displayed no significant changes in @entity3917 phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the @entity68459 mutation showed a significant reduction of force along with reduced @entity3917 phosphorylation. The contractile responses of @entity68459 mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of @entity63671 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of @entity3917 phosphorylation in vivo. The constitutive phosphorylation of @entity63671 T694 may provide a mechanism for regulating force maintenance of smooth muscle.

In vivo roles for XXXX phosphorylation sites T694 and T852 in bladder smooth muscle contraction.

@entity1494 :: ('MESH:D001745', 'Disease') :: ['Isolated bladder'] @entity19 :: ('10090', 'Species') :: ['mice', 'mouse'] @entity68460 :: ('c|SUB|T|852|A', 'DNAMutation') :: ['T852A'] @entity68459 :: ('c|SUB|T|694|A', 'DNAMutation') :: ['T694A'] @entity493 :: ('CHEBI:16449', 'Chemical') :: ['alanine'] @entity23452 :: ('213435(Tax:10090)', 'Gene') :: ['MLCK', 'myosin light chain kinase'] @entity141 :: ('CHEBI:29108', 'Chemical') :: ['Ca(2+)'] @entity3917 :: ('MESH:D020795', 'Disease') :: ['RLC', 'regulatory light chain'] @entity63671 :: ('17931(Tax:10090)', 'Gene') :: ['MYPT1']
@entity63671 :: (17931(Tax:10090),Gene) :: ['myosin phosphatase targeting subunit-1']
