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This review has presented some of the recent data on myosin phosphatase from smooth muscle. Although it is not conclusive, it is likely that most of the myosin phosphatase activity is represented by a holoenzyme composed of three subunits. These are: a catalytic subunit of 38 kDa of the type 1 phosphatase, probably the delta isoform (i.e. PP1c delta); a subunit of about 20 kDa whose function is not established; and a larger subunit that is thought to act as a target subunit. This is termed the myosin phosphatase target subunit, MYPT. Various isoforms of MYPT exist and the relatively minor distinctions are in the C-terminal leucine zipper motifs and/or with inserts in the central region. Many regions of the molecule are highly conserved, including the ankyrin repeats in the N-terminal part of the molecule and the sequence around the phosphorylation site. In addition, these isoforms all contain the four residue PP1c-binding motif (Arg/Lys-Val/Ile-Xaa-Phe). MYPT has been detected in a variety of cells and thus is not unique to smooth muscle. With phosphorylated myosin as substrate, the phosphatase activity of PP1c is low and is enhanced on addition of MYPT. It is assumed that MYPT functions as a target subunit and binds to both PP1c and substrate. The N-terminal fragment of MYPT is responsible for the activation of PP1c activity, but how much of the N-terminal sequence is required is not established. An important point is that activation is not a general effect and is specific for myosin. It is not known if other substrates may be targeted to MYPT. There are two binding sites for PP1c on MYPT: a strong site in the N-terminal segment (containing the 4-residue motif) and a weaker site in the ankyrin repeats, possibly in repeats 5, 6 and 7. The location(s) of the myosin-binding sites on MYPT is controversial, and binding of myosin, or light chain, to both N- and C-terminal fragments has been reported. Regulation of myosin phosphatase activity involves changes in subunit interactions, although molecular mechanisms are not defined. There are basically two theories proposed for phosphatase inhibition (i.e. as seen in the agonist-induced increase in Ca2+ sensitivity). One hypothesis is that phosphorylation of Myosin light chain phosphatase MYPT (at residue 654 or 695 of the gizzard MYPT isoforms or an equivalent residue) inhibits the activity of the MP holoenzyme. The kinase involved is not established, but may be an unidentified endogenous kinase or a RhoA-activated kinase. The latter is an attractive possibility because there is convincing evidence that RhoA plays a crucial role in the Ca(2+)-sensitizing process in smooth muscle. A second idea involves arachidonic acid. This is released via phospholipase A2 and could either interact directly with MYPT and cause dissociation of the holoenzyme (thus effectively reducing the phosphatase activity to that of the isolated catalytic subunit), or it could activate a kinase that would phosphorylate MYPT and inhibit the phosphatase. It is possible that MP activity may also be activated, for example, following increases in cAMP and/or cGMP. Evidence in support of this is very limited and under in vivo conditions the phosphorylation of MYPT by the respective kinases has not been demonstrated. There is, however, a tentative hypothesis based on in vitro data that phosphorylation of MYPT by PKA alters its cellular localization. This involves a shuttle between the dephosphorylated membrane-bound and inhibited state (at least towards P-myosin) to a phosphorylated cytosolic or cytoskeletal, and active state. The pathway(s) discussed above originates at the cell membrane and is carried via one or more messengers to the level of the contractile apparatus where it is manifested by regulation of phosphatase activity. Various components of the route have been identified, including RhoA and the atypical PKC isoforms, but more remain to be discovered. It is possible that more than one pathway, or cascade, is |
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