Myosin Light Chain Phosphatase
It Gets Around
The importance of Ca2+calmodulin-dependent myosin light chain kinase (MLCK) for smooth muscle contraction is well documented.1 Until recently, myosin light chain (MLC) phosphatase was thought to be unregulated and constitutively active. However, studies have demonstrated that smooth muscle contraction is dependent on regulation of MLC phosphatase activity.2,3⇓ The inhibition of phosphatase activity increases force at a constant [Ca2+], whereas a stimulation of MLC phosphatase activity reduces force at a constant [Ca2+].
MLC phosphatase isolated from smooth muscle is a holoenzyme consisting of 3 subunits2: a small ≈20-kDa subunit, an ≈38-kDa catalytic subunit, and a myosin-targeting subunit (MYPT) of 110 to 133 kDa. The small subunit has no established function and is not required for either catalytic activity or activation of PP1c.2 The catalytic subunit is a PP1c phosphatase, and the δ isoform is associated with the holoenzyme.4 The large subunit is the MYPT. Isoform diversity of MYPT was first shown in chicken where 2 isoforms (M130 and M133)2 were shown to differ by the presence of a central insert between aa residues 512 to 552.5 Similar isoform diversity has been demonstrated for MYPT isolated from other species.2,6⇓ In addition to the isoform diversity produced by the central insert, alternative splicing of a COOH-terminal exon leads to MYPT isoforms, which differ by the presence or absence of a leucine zipper.7
Agonist-Induced Force Enhancement
At any intracellular [Ca+2], force for an agonist stimulated contraction is higher than for depolarization.8,9⇓ This phenomenon has been termed agonist-induced force enhancement, or Ca+2 sensitization, and several mechanisms have been proposed.2,3⇓ Although the signaling pathway(s) for Ca+2 sensitization have not been elucidated, most proposed mechanisms converge on an inhibition of MLC phosphatase activity.2
In smooth muscle, G protein–coupled receptor activation increases MLC20 phosphorylation and force.10–12⇓⇓ Trinkle-Mulcahy et al13 demonstrated that MYPT was thiophosphorylated by ATPγS, resulting in a decrease of MLC phosphatase activity and force enhancement. These data are consistent with a signaling pathway involving heterotrimeric G protein–induced activation of Rho A and Rho kinase. Rho kinase has been shown to phosphorylate MYPT at Thr695.5 Phosphorylation at this site decreases MLC phosphatase activity5 to increase both MLC20 phosphorylation and force,14 although MYPT phosphorylation and force enhancement do not always correlate.15 Another mechanism for Ca2+ sensitization involves an inhibitor protein for MLC phosphatase, CPI-17.16 CPI-17 can be phosphorylated by protein kinase C17,18⇓ and Rho kinase,19 and phosphorylated CPI-17 inhibits MLC phosphatase activity.17,18⇓ An additional mechanism is dissociation of the subunits of MLC phosphatase by arachidonic acid.20 Activation of the G proteins increases the activity of phospholipase A2 and production of arachidonic acid, which binds to and dissociates MLC phosphatase to reduce phosphatase activity. The Rho kinase signaling pathway may also lead to a direct phosphorylation of MLC20 by Zip-like kinase21 and/or integrin-linked kinase22 to produce a Ca2+-independent increase in force.
In this issue of Circulation Research, the study from Morgan’s laboratory23 has evidence for another mechanism for Ca2+ sensitization. Their data shows that there is an agonist-specific translocation of MLC phosphatase to the smooth muscle cell membrane. PGF2α stimulation of freshly dispersed smooth muscle cells results in phosphorylation of MYPT at Thr697 (Rho kinase site of mammalian sequence) and a translocation of MLC phosphatase from the cytosol to the membrane. The Rho kinase inhibitor, Y27632, inhibited both MYPT phosphorylation and MLC phosphatase translocation, suggesting that the initial event in the signaling cascade is an activation of Rho kinase. After MLC phosphatase translocates to the membrane, there is a dissociation of MYPT and PP1c; PP1c returns to the cytosol. Phenylephrine stimulation produced a distinctly different pattern of MLC phosphatase movement. MYPT did not translocate but remained homogenously distributed throughout the cell, whereas PP1c initially demonstrated a homogenous pattern that became transiently nonhomogenous after stimulation.
Agonist-induced protein translocation is not unprecedented. Translocation of PKC,24 calponin,25 and Rho A26,27⇓ have all been reported. Translocation of Rho A from the cytosol to the cell membrane has been suggested to lead to spatial activation of Rho kinase, which would phosphorylate MYPT to inhibit MLC phosphatase activity at the cell membrane and produce a sustained increase in MLC20 phosphorylation at the cortical region of the smooth muscle cell.26 The data of Shin et al23 show a translocation of MLC phosphatase from the cytosol to the cell membrane. The first event in this process is a Rho kinase–induced phosphorylation of MYPT, which would inhibit MLC phosphatase activity.5 Thus, their data23 are consistent with and could produce the sustained increase in MLC20 phosphorylation in the cortical region of the smooth muscle cell observed by others.26
Isolated PP1c is known to have a reduced phosphatase activity toward phosphorylated MLC20.28 Thus, the results of Morgan’s group23 make it intriguing to speculate that dissociation of PP1c from MYPT decreases MLC phosphatase activity to produce the sustained increase in MLC20 phosphorylation observed during PGF2α stimulation. Phenylephrine stimulation does not lead to dissociation of MLC phosphatase, and MLC phosphatase activity would remain elevated to decrease the level of MLC20 phosphorylation. For phenylephrine, force would have to be maintained by a mechanism outside of actomyosin.29 However, one would expect both PGF2α and phenylephrine to activate Rho A and Rho kinase to lead to a phosphorylation of MYPT and force enhancement. Consistent with this hypothesis is the data demonstrating that the Rho kinase inhibitor Y27632 almost completely inhibited force for both agonists.23 This could suggest the effects of Y27632 are nonspecific,30 Rho kinase has more than one substrate,31 or the differential translocation of MYPT and PP1c is unrelated to inhibition of MLC phosphatase activity. There could be a difference in the PGF2α and phenylephrine signaling pathways, which led to this differential translocation of MLC phosphatase; ie, there could be an agonist-dependent activation of phospholipase A2 and production of arachidonic acid. This would result in an agonist-specific dissociation of the MLC holoenzyme and be consistent with the results of Morgan’s group, but again, it is difficult to envision how Rho kinase inhibition produces a similar decrease in force for both agonists.
The study by Morgan’s group23 provides a provocative molecular mechanism for Ca2+ sensitization. Others have shown that both the dose response relationship and the magnitude of Ca2+ sensitization is tissue-specific.11,18,27,32⇓⇓⇓ In addition, it has been shown that the distribution of the M130/M133 isoforms of the MYPT6,33⇓ as well as CPI-1734 are tissue-dependent and correlate with the magnitude of Ca2+ sensitization.15,34⇓ Therefore, the role and physiological importance of differential translocation of MLC phosphatase, MYPT isoforms, CPI-17, and arachidonic acid for the molecular mechanism of force enhancement remains to be determined.
The molecular mechanism for Ca2+ sensitization has broad clinical implications. Ca2+ sensitization and desensitization have been implicated in the pathogenesis of hypertension,35 erectile dysfunction,36 and could lead to asthma, vasospasm, and both the resting vasoconstriction and resistance to nitric oxide–associated vasodilatation associated with congestive heart failure. Many cellular signaling pathways involve protein phosphorylation, and modulation of MLC phosphatase activity could also play a role in the metastasis and invasion of malignant cells,37 as well as the growth and development of neurons.38 Therefore, the molecular mechanism for the regulation of phosphatase activity is important for both normal physiology and pathophysiology of human disease, and agents designed to modulate MLC phosphatase activity may have broad therapeutic potential.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Hartshorne DJ. Biochemistry of the contractile process in smooth muscle.In: Johnson LR, ed. Physiology of the Gastrointestinal Tract. New York, NY: Raven Press; 1987: 432–482.
- ↵Alessi D, MacDougall LK, Sola MM, Ikebe M, Cohen P. The control of protein phosphatase-1 by targeting subunits. Eur J Biochem. 1999; 210; 1023–1035.
- ↵Ichikawa K, Ito M, Hartshorne DJ. Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. J Biol Chem. 1996; 271: 4733–4740.
- ↵Dirksen WP, Vladic F, Fisher SA. A myosin phosphatase targeting subunit isoform transition defines a smooth muscle developmental phenotypic switch. Am J Physiol. 2000; 278: C589–C600.
- ↵Khatri JJ, Joyce KM, Brozovich FV, Fisher SA. Role of myosin phosphatase isoforms in cGMP-mediated smooth muscle relaxation. J Biol Chem. 2001; 276: 37250–37257.
- ↵Morgan JP, Morgan KG. Stimulus specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J Physiol. 1984; 184;351: 155–167.
- ↵Kitazawa T, Kobayashi S, Horiuti K, Somlyo AV, Somlyo AP. Receptor-coupled, permeabilized smooth muscle: Role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca2+. J Biol Chem. 1989; 264: 5339–5342.
- ↵Kitazawa T, Gaylinn BD, Denney GH, Somlyo AP. G-protein–mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem. 1991; 266: 1708–1715.
- ↵Trinkle-Mulcahy L, Ichikawa K, Hartshorne DJ, Siegman MJ, Butler TM. Thiophosphorylation of the 130-kDa subunit is associated with a decreased activity of myosin light chain phosphatase in alpha-toxin-permeabilized smooth muscle. J Biol Chem. 1995; 270: 18191–18194.
- ↵Richards CT, Ogut O, Brozovich FV. Agonist-induced force enhancement: the role of isoforms and phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase. J Biol Chem. 2002; 277: 4422–4427.
- ↵Eto M, Ohmori T, Suzuki M, Furuya K, Morita F. A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C. Isolation from porcine aorta media and characterization. J Biochem. 1995; 118: 1104–1107.
- ↵Masuo M, Reardon S, Ikebe M, Kitazawa T. A novel mechanism for the Ca2+-sensitizing effect of protein kinase C on vascular smooth muscle: Inhibition of myosin light chain phosphatase. J Gen Physiol. 1994; 104: 265–286.
- ↵Gong MC, Fuglsang A, Alessi D, Kobayashi S, Cohen P, Somlyo AV, Somlyo AP. Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium. J Biol Chem. 1992; 267: 21492–21498.
- ↵Niiro N, Ikebe M. Zipper-interacting protein kinase induces Ca2+-free smooth muscle contraction via myosin light chain phosphorylation. J Biol Chem. 2001; 276: 29567–29574.
- ↵Deng JT, Van Lierop JE, Sutherland C, Walsh MP. Ca2+-independent smooth muscle contraction: a novel function for integrin-linked kinase. J Biol Chem. 2001; 276: 16365–16373.
- ↵Shin HM, Je HD, Gallant C, Tao TC, Hartshorne DJ, Ito M, Morgan KG. Differential association and localization of myosin phosphatase subunits during agonist-induced signal transduction in smooth muscle. Circ Res. 2002; 90: 546–553.
- ↵Khalil RA, Morgan KG. Imaging of protein kinase C distribution and translocation in living vascular smooth muscle cells. Circ Res. 1991; 69: 1626–1631.
- ↵Miyazaki K, Yano T, Schmidt DJ, Tokui T, Shibata M, Lifshitz L.M, Kimura S, Tuft RA, Ikebe M. Rho-dependent agonist-induced spatio-temporal change in myosin phosphorylation in smooth muscle cells. J Biol Chem. 2002; 277: 725–734.
- ↵Gong MC, Fujihara H, Somlyo AV, Somlyo AP. Translocation of rhoA associated with Ca2+ sensitization of smooth muscle. J Biol Chem. 1997; 272: 10704–10709.
- ↵Menice CB, Hulvershorn J, Adam LP, Wang CL, Morgan KG. Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle. J Biol Chem. 1997; 272: 25157–25161.
- ↵Eto M, Kitazawa T, Yazawa A, Mukai H, Ono Y, Brautigan DL. Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C α and δ isoforms. J Biol Chem. 2001; 276: 29072–29078.
- ↵Solaro RJ. Myosin light chain phosphatase: a Cinderella of cellular signaling. Circ Res. 2000; 87: 173–175.
- ↵Gong MC, Cohen P, Kitazawa T, Ikebe M, Masuo M, Somlyo AP, Somlyo AV. Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle. J Biol Chem. 1992; 267: 14662–14668.
- ↵Ogut O, Brozovich FV. Determinants of the contractile properties in the embryonic chicken gizzard and aorta. Am J Physiol. 2000; 279: C1722–C1732.
- ↵Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem J. 2000; 348: 241–255.