Characterization of protein kinase pathways responsible for Ca2+ sensitization in rat ileal longitudinal smooth muscle

We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca2+ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 microM microcystin induced LC20 diphosphorylation and contraction of beta-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF-109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 muM, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca2+ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle.     

The regulation of smooth muscle contractility by zipper-interacting protein kinase

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.     

Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2

Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.     

Staurosporine inhibition of zipper-interacting protein kinase contractile effects in gastrointestinal smooth muscle.

Zipper-interacting protein kinase (ZIPK) is a serine-threonine kinase that has been implicated in Ca2+-independent myosin II phosphorylation and contractile force generation in vascular smooth muscle. However, relatively little is known about the contribution of this kinase to gastrointestinal smooth muscle contraction. The addition of a recombinant version of ZIPK that lacked the leucine zipper domain to permeabilized ileal strips evoked a Ca2+-independent contraction and resulted in myosin regulatory light chain diphosphorylation at Ser19 and Thr18. Neither Ca2+-independent force development nor myosin regulatory light chain phosphorylation was elicited by the addition of kinase-dead ZIPK to the ileal strips. The sensitivity of ZIPK-induced contraction to various kinase inhibitors was similar to the in vitro sensitivity of purified ZIPK to these inhibitors. Staurosporine was the most effective ZIPK inhibitor, with a Ki value calculated to be 2.6 +/- 0.3 micromol/L. Through the use of specific kinase inhibitors, we determined that Rho-associated protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C) do not mitigate ZIPK-induced contraction in ileum. Our findings support a role for ZIPK in Ca2+-independent contractile force generation in gastrointestinal smooth muscle.     

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.     

Vascular smooth muscle myosin light chain diphosphorylation: mechanism, function, and pathological implications

Smooth muscle contraction is activated primarily by phosphorylation at S19 of the 20-kDa regulatory light chain subunits of myosin II (LC(20) ) catalyzed by Ca(2+) /calmodulin-dependent myosin light chain kinase. Other kinases, for example, integrin-linked kinase (ILK), Rho-associated kinase (ROCK), and zipper-interacting protein kinase (ZIPK), can phosphorylate T18 in addition to S19, which increases the actin-activated myosin MgATPase activity at subsaturating actin concentrations ～3-fold. These phosphorylatable residues and the amino acid sequence surrounding them are highly conserved throughout the animal kingdom; they are also found in an LC(20) homolog within the genome of Monosiga brevicollis, the closest living relative of metazoans. LC(20) diphosphorylation has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli and in pathophysiological situations associated with hypercontractility. LC(20) diphosphorylation has also been observed frequently in cultured cells where it activates force generation. Kinases such as ILK, ROCK, and ZIPK, therefore, are potential therapeutic targets in the treatment of, for example, cerebral vasospasm following subarachnoid hemorrhage and atherosclerosis.     

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts

Thus far, determining the relative contribution of Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) and Ca²⁺-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca²⁺, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO₄/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO₄/mol RLC, respectively, within 2.5 min. Ca²⁺ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells. myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation     

Signaling through Myosin Light Chain Kinase in Smooth Muscles

Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca²⁺/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca²⁺ sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.     

Diphosphorylation of platelet myosin by myosin light chain kinase.

Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration (greater than 20 micrograms/ml) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca2+, KCl, MgCl2-dependence. Maximal reaction was seen at [Ca2+] greater than 10(-6) M, 60 mM KCl and 2 mM MgCl2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca2+, KCl-dependence of ATPase activity but distinct MgCl2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCl2 and monophosphorylated myosin showed a maximum at 10 mM MgCl2. (4) The addition of tropomyosin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human platelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.     

ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells.

A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper-interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998) Mol. Cell. Biol. 18, 1642-1651]. Here we found that HeLa ZIPK phosphorylated the regulatory light chain of myosin II (MRLC) at both serine 19 and threonine 18 in a Ca2+/calmodulin independent manner. Phosphorylation of myosin II by HeLa ZIPK resulted in activation of actin-activated MgATPase activity of myosin II. HeLa ZIPK is the first non-muscle MLCK that phosphorylates MRLC at two sites.     

Mode of inhibition of smooth muscle myosin light chain kinase by synthetic peptide analogs of the regulatory site

The functions associated with the inhibitory region and calmodulin binding region of smooth muscle myosin light chain kinase (MLCK) were studied using various synthetic peptide analogs. Peptides 480-501 and 483-498 strongly inhibited 61 kDa Ca2+/calmodulin-independent MLCK activity with Ki of 25 nM. Peptides 493-512 and 493-504 were considerably less effective as inhibitor of the Ca2+/calmodulin-independent MLCK and Kiapp. were 2 and 3 microM, respectively. Inhibition of Ca2+/calmodulin-independent MLCK by the peptides 480-501 and 483-498 were competitive with ATP and 20,000 dalton smooth muscle myosin light chain. The inhibition of native MLCK by peptide 493-512 was explained by the calmodulin depletion model in which the peptide binds to free calmodulin and prevents it from activating MLCK. On the other hand, the inhibition of native MLCK by the peptides 480-501 and 483-498 was explained by the binding of these peptides to the MLCK-calmodulin complex. The present study suggests that the inhibitory region of MLCK directly binds to MLCK active site and competes with both ATP and 20,000 dalton light chain so as to inhibit the enzyme.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation

Fibroblasts form fibers when grown inculture medium containing native type 1 collagen. The contractileforces generated can be precisely quantified and used to analyze thesignal transduction pathways regulating fibroblast contraction. Calfserum (30%) induces a sustained contraction that is accompanied by atransient increase in intracellular calcium([Ca²⁺]_i). W-7, a calmodulin inhibitor,KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase, andML-7, a myosin light-chain kinase inhibitor, had no effects on eitherthe contraction or the [Ca²⁺]_i responses.Neither genistein, a tyrosine kinase inhibitor, nor calphostin C, aprotein kinase C inhibitor, had major effects on force or[Ca²⁺]_i. In contrast, the Rho kinaseinhibitors(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and HA1077 depressed the contraction in a dose-dependent manner without affecting the [Ca²⁺]_iresponse. Stress fiber formation was also suppressed by Y-27632. Surprisingly, calf serum, Y-27632, and calf serum plus Y-27632 did notalter mono- or diphosphorylation of the myosin regulatory light chain(MRLC) compared with control untreated fibers. These results suggestthat the sustained contraction of NIH 3T3 fibroblast fibers induced bycalf serum is mediated by Rho kinase but is independent of a sustainedincrease in [Ca²⁺]_i, calcium/calmodulin- orprotein kinase C-dependent pathways, or increases in MRLC phosphorylation.

     

Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles

Contraction of smooth muscle depends on the balance of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. Because MLCK activation depends on the activation of calmodulin, which requires a high Ca²⁺ concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic Ca²⁺ levels. The link between activation of the Ca²⁺-independent protein kinase C (PKC) and MLC phosphorylation observed in the esophagus (ESO) (Sohn UD, Cao W, Tang DC, Stull JT, Haeberle JR, Wang CLA, Harnett KM, Behar J, and Biancani P. Am J Physiol Gastrointest Liver Physiol 281: G467–G478, 2001), however, has not been elucidated. We used phosphatase and kinase inhibitors and antibodies to signaling enzymes in combination with intact and saponin-permeabilized isolated smooth muscle cells from ESO and lower esophageal sphincter (LES) to examine PKC-dependent, Ca²⁺-independent signaling in ESO. The phosphatase inhibitors okadaic acid and microcystin-LR, as well as an antibody to the catalytic subunit of type 1 protein serine/threonine phosphatase, elicited similar contractions in ESO and LES. MLCK inhibitors (ML-7, ML-9, and SM-1) and antibodies to MLCK inhibited contraction induced by phosphatase inhibition in LES but not in ESO. The PKC inhibitor chelerythrine and antibodies to PKC, but not antibodies to PKCII, inhibited contraction of ESO but not of LES. In ESO, okadaic acid triggered translocation of PKC from cytosolic to particulate fraction and increased activity of integrin-linked kinase (ILK). Antibodies to the mitogen-activated protein (MAP) kinases ERK1/ERK2 and to ILK, and the MAP kinase kinase (MEK) inhibitor PD-98059, inhibited okadaic acid-induced ILK activity and contraction of ESO. We conclude that phosphatase inhibition potentiates the effects of MLCK in LES but not in ESO. Contraction of ESO is mediated by activation of PKC, MEK, ERK1/2, and ILK. protein kinase C; myosin light chain kinase; phosphatase; integrin-linked kinase     

AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser(815). Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-beta, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-beta inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which alpha1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.     

Potent peptide inhibitors of smooth muscle myosin light chain kinase: mapping of the pseudosubstrate and calmodulin binding domains

Smooth muscle myosin light chain kinase (MLCK) is activated by calcium-calmodulin and, in turn, phosphorylates and activates the smooth muscle actomyosin ATPase, resulting in muscle contraction. The amino acid sequence of the regulatory domain of MLCK is known, and it contains a region that binds calmodulin and also bears a strong homology to the phosphorylation site in the substrate. Thus, it has been called the "pseudosubstrate". It has been proposed that calmodulin activates MLCK by binding to and reversing the autoinhibitory function of the pseudosubstrate. Synthetic peptides based on this sequence inhibit MLCK both by binding to calmodulin and by competing with the substrate at the active site. In the work reported here, we have synthesized a large number of peptides from the regulatory region of MLCK (MLCK 480-516). The region was systematically analyzed by dividing it into fragments of two to six amino acids, each containing one or more basic residues, in order to map in detail the calmodulin binding site and the autoinhibitory region. It was observed that both calmodulin binding and autoinhibition are mediated by several different fragments of the regulatory sequence. Two nonoverlapping peptides, MLCK 480-493 and MLCK 494-504, are similar in potency in inhibiting the enzyme (IC50's of 2 and 6 microM, respectively). Larger fragments, combining multiple inhibitory regions, are more potent inhibitors. For example, MLCK 480-504 is extremely potent, with an IC50 of 13 nM. The calmodulin binding site and active site directed inhibitory regions overlap, but are not identical. Residues 505-512 are important only for calmodulin binding.     

Catalytic fragment of protein kinase C exhibits altered substrate specificity toward smooth muscle myosin light chain.

Smooth muscle myosin light chain (LC) can be phosphorylated by myosin light chain kinase (MLCK) at Ser19 and Thr18 and by protein kinase C (PKC) at Thr9 and Ser1 or Ser2 under the in vitro assay conditions. Conversion of PKC to the spontaneously active protein kinase M (PKM) by proteolysis resulted in a change in the substrate specificity of the kinase. PKM phosphorylated both sets of sites in LC recognized by MLCK and PKC as analyzed by peptide mapping analysis. The PKM-catalyzed phosphorylation of these sites was not greatly affected by a MLCK inhibitor, ML-9, nor by the activators of MLCK, Ca2+ and calmodulin.     

Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.     

Cardiac Myosin Is a Substrate for Zipper-interacting Protein Kinase (ZIPK)

Zipper-interacting protein kinase (ZIPK) is a member of the death-associated protein kinase family associated with apoptosis in nonmuscle cells where it phosphorylates myosin regulatory light chain (RLC) to promote membrane blebbing. ZIPK mRNA and protein are abundant in heart tissue and isolated ventricular neonatal rat cardiac myocytes. An unbiased substrate search performed with purified ZIPK on heart homogenates led to the discovery of a prominent 20-kDa protein substrate identified as RLC of ventricular myosin. Biochemical analyses showed ZIPK phosphorylated cardiac RLC at Ser-15 with a V_max value 2-fold greater than the value for smooth/nonmuscle RLC; cardiac RLC is a favorable biochemical substrate. Knockdown of ZIPK in cardiac myocytes by small interfering RNA significantly decreased the extent of RLC Ser-15 phosphorylation. Thus, ZIPK may act as a cardiac RLC kinase and thereby affect contractility.     

Depolarization induces Rho-Rho kinase-mediated myosin light chain phosphorylation in kidney tubular cells

Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been elucidated. Herein we show that plasma membrane depolarization provoked by distinct means, including high extracellular K⁺, the lipophilic cation tetraphenylphosphonium, or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a Ca²⁺ signal, and removal of external Ca²⁺ promoted rather than inhibited MLC phosphorylation. Moreover, elevation of intracellular Ca²⁺ did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator because 1) depolarization stimulated Rho and induced its peripheral translocation, 2) inhibition of Rho by Clostridium difficile toxin B or C3 transferase abolished MLC phosphorylation, and 3) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiological depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic Na⁺-alanine cotransporter, stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a Na⁺-dependent manner. Together, our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization triggered and Rho-ROK-mediated but Ca²⁺ signal independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport. membrane potential; Na⁺-alanine cotransport; epithelium; phosphatidylinositol 3-kinase; LLC-PK₁ cells