In vivo roles for myosin phosphatase targeting subunit‐1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction

Abstract

Key pointsr 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+ /calmodulindependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities.r MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro.r 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.r Our findings reveal the role of MYPT1 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 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.

Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

“…68 The latter hypothesis is supported by the recent study using a genetic approach to knock-in nonphosphorylatable Ala for T852 or T694. 69 The MYPT1 T694A mutation was found to significantly inhibit sustained force as well as MLC phosphorylation, while the T852A mutation had no significant effect on maximal force development and little effect on force maintenance in neonatal bladder smooth muscle. These findings indicate that phosphorylated T694, but not T852, inhibits MLCP activity.…”

“…However, ROCK inhibitors still inhibited force development and force maintenance in bladder smooth muscles with the T852A mutation to similar extents as wild-type muscles, without affecting MLC phosphorylation. 69 Why did the T852 mutation have no significant effect on maximal force development and little effect on force maintenance in bladder smooth muscle, and yet ROCK inhibitors were still able to inhibit contractile responses? Much of the evidence supporting a role of MYPT1 T852 phosphorylation in contributing to enhanced MLC phosphorylation in intact tissues has been obtained using pharmacological inhibitors of ROCK.…”

“…The study of embryonic bladder smooth muscle by Chen et al showed that the constitutive MYPT1 T694 phosphorylation was not further increased by phosphatase inhibition. 69 In addition, the MYPT1 T694A mutation resulted in significantly decreased force generation and reduced MLC20 phosphorylation. 69 These findings indicate that MYPT1 T694 may be 100% phosphorylated, and is responsible for suppressing MLCP activity under resting conditions.…”

“…69 In addition, the MYPT1 T694A mutation resulted in significantly decreased force generation and reduced MLC20 phosphorylation. 69 These findings indicate that MYPT1 T694 may be 100% phosphorylated, and is responsible for suppressing MLCP activity under resting conditions. 69 Interestingly, these findings may explain why MYPT1 T694 phosphorylation is sometimes not further increased by contractile agonists.…”

Rho Kinases in Health and Disease: From Basic Science to Translational Research

“…Nevertheless, in the C57BL/6 mice background, open eyelids at birth and presence of the omphalocele phenotype observed in Rock1 2/2 and Rock2 2/2 mice indicate that both Rock1 and Rock2 are necessary for eyelids and ventricular body wall closure, and each isoform does not compensate for the absence of the other. Interestingly, mice expressing mutated MYPT1 that cannot be phosphorylated by ROCK exhibit a similar omphalocele phenotype, thus supporting a role of ROCK in signaling cascades that regulate closure of the ventral body wall at the umbilical ring (Chen et al, 2015). This particular example suggests that the correlation between the phenotype of mice carrying genetic manipulation of known ROCK targets, as well as upstream ROCK regulators, and ROCK knockout phenotypes can provide useful data to identify ROCK signaling pathway components and to understand their biologic functions.…”