A tale of two threonines: myosin phosphatase inhibition and calcium sensitization of smooth muscle

Smooth muscle (SM) contraction is a dynamic and highly regulated process. The contractile properties of SM are primarily governed by the phosphorylation of the regulatory light chain (LC20) of myosin II. To initiate contraction, an increase in intracellular [Ca2+] activates myosin light chain kinase (MLCK), a Ca2+/calmodulin-dependent enzyme. MLCK phosphorylates LC20 on Ser19, resulting in contraction of SM through increases in myosin ATPase activity and cross-bridge cycling. Myosin light chain phosphatase (MLCP) is responsible for the dephosphorylation of LC20 (Shirazi et al. 1994). Although intracellular [Ca2+] is the primary determinant of SM contraction, it is the balance between MLCK and MLCP activities that predicates the precise contractile activity. Indeed, MLCP functions independently of Ca2+ and can be regulated by a variety of signalling pathways. Inhibition of MLCP leads to an increase in both LC20 phosphorylation and contractile force development without any changes in [Ca2+], a phenomenon commonly referred to as ‘calcium sensitization’. Once thought to possess ‘housekeeping activity’, MLCP is now known to be under exquisite regulatory control (Grassie et al. 2011). A number of studies demonstrate that MLCP activity (and hence Ca2+ sensitization) is regulated by phosphorylation of the myosin-targeting subunit (MYPT1). Although several phosphorylation sites on MYPT1 have been identified since its discovery a decade ago, two threonine residues (i.e. T694 and T852, numbering of the mouse isoform) are the most extensively studied in the context of Ca2+ sensitization. Experimental evidence suggests that stimulation of SM by agonists elicits variable degrees of Ca2+ sensitization depending on the agonist used and the SM employed. These contractile responses have been associated with a degree of variability regarding the nature and stoichiometry of MYPT1 phosphorylation. Much debate has been directed toward the physiological importance of MYPT1 phosphorylation at the two inhibitory threonine residues. Indeed, the physiological relevance of T694 and T852 phosphorylation has been approached by a number of laboratories in the recent past. Currently, phosphorylation of T694 is thought to directly inhibit MLCP activity towards LC20 whereas T852 phosphorylation is believed to cause dissociation of MLCP from myosin and/or inhibit MLCP activity directly. In this issue of The Journal of Physiology, Chen and colleagues (2015) examine the importance of T694 and T852 phosphorylations of MYPT1 from bladder detrusor SM. In completing this assessment, the authors generate novel knockin mice (namely heterozygous T694A/– and T694A/+ along with homozygous T852A null animals). Although not the specific focus of the study, an omphalocele phenotype was coincidently found with the knockin of either T694A or T852A that suggests a Rho-associated kinase (ROCK) and MYPT1 dependency. Moreover, the authors’ examinations confirm individual and unique roles for the threonine phosphorylation sites of MYPT1 during Ca2+ sensitization of bladder SM. In this regard, the MYPT1 T694A mutation was found to significantly inhibit sustained force as well as LC20 phosphorylation while the T852 mutation had no significant effect on maximal force development and little effect on force maintenance. The study also supports the presence of a constitutive Ca2+ sensitization mechanism which contributes to force maintenance via the phosphorylation of MYPT1 at T694. These findings offer confirmation of recent in vitro data that suggested T694 phosphorylation inhibited MLCP activity whereas T852 phosphorylation did not (Khasnis et al. 2014). An additional intriguing development reported for the T852A mice is the absence of changes in LC20 phosphorylation status and force responses while an attenuation of force was observed upon exposure to ROCK inhibitors. An emergent precept regarding Ca2+ sensitization and SM tone now embraces dynamic cytoskeletal reorganization as a contributing process that operates along with MLCP regulation (Walsh & Cole, 2013). Indeed, the observations provided for bladder SM of the T852A knockin mouse are congruent with ROCK acting to enhance connections between the actin cytoskeleton, plasma membrane and extracellular matrix to augment force transmission. In this SM tissue, agonist-induced T852 phosphorylation appears to have no apparent role in regulating contractile responses. One regulatory mechanism for the control of MYPT1 phosphorylation and MLCP activity was not addressed by the study, namely the dual phosphorylation of the adjacent S693/T694 and S851/T852 residues. MLCP activity appears to be dependent on the relative phosphorylation of inhibitory (T) and adjacent disinhibitory (S) sites of MYPT1 (Grassie et al. 2012). Future investigations with the knockin mice may provide illumination on how cross-talk between cyclic nucleotide and ROCK signalling pathways dictates SM contractile force. Although the study does have its limitations (e.g. a lack of adult tissues from the knockin animals), these interesting findings shed new light on the specific function of the threonine phosphorylation sites of MYPT1. In all likelihood, the mice (especially the viable homozygous T852A) will have appreciable importance for investigators’ examination of various SM beds and processes. SM tissues display diverse contractile phenotypes and possess a variety of functional signalling mechanisms to regulate force development. However, our understanding of the unique regulatory pathways operating to modulate MLCP activity and Ca2+ sensitization is still incomplete. Obviously, Ca2+ sensitization is a complex process involving multiple pathways that allow for subtle functional differentiations to be achieved. None declared. J.A.M.'s research is funded by the Canadian Institutes of Health Research (CIHR).

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