Calmodulin-dependent Myosin Light Chain Kinase Research Articles

Abstract 15590: Smooth Muscle Specific Deletion of P90 Ribosomal S6 Kinase Alters the Vasomotor Response of Resistance Arteries

Background: Smooth muscle (SM) contraction is triggered by Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK), which phosphorylates the regulatory light chain of myosin (RLC 20 ). However, additional regulatory mechanisms exist. We found that global Rsk2 KO mice had more dilated mesenteric arteries (MAs) with reduced myogenic tone and RLC 20 phosphorylation, and significantly lower blood pressure. Furthermore, RSK2-dependent activation of the Na + /H + exchanger resulted in an increase in pH i and increased Ca 2+ i transients. However, it is unclear whether our observations were due to developmental abnormalities observed in Rsk2 global KO mice. Hypothesis: RSK2 signaling in SM is a significant regulator of myogenic tone, arterial stiffness, and Ca 2+ signaling. Methods: To study the role of RSK2 signaling in vascular tone, we generated inducible SM-specific RSK2 KO mice. Detection of total and phospho proteins were determined by western blot. Pressure myography studies assessed vascular reactivity in 4 th - and 5 th -order MAs isolated from MYH11-Cre + Rsk2 fl/fl (RSK2 SM -/- ) and MYH11-Cre - Rsk2 fl/fl (RSK2 SM +/+ ) mice. We examined SM-specific Ca 2+ events in pressurized MAs by confocal fluorescent microscopy. Results: Western blot confirms knock down of RSK2 expression (~70%) in MAs and aorta. Intraluminal pressure-induced constriction of MAs is attenuated in RSK2 SM -/- . RSK2 SM -/- MAs display a delay in development of myogenic tone at 40 mmHg and decreased vascular stiffness, compared to RSK2 SM +/+ . Vasodilatory responses to Spermine NONOate (1-100 μM) in preconstricted (L-NNA; 10 μM) RSK2 SM -/- vessels were enhanced. RSK2 SM -/- MAs display fewer Ca 2+ events (97±19.6; n = 4) than RSK2 SM +/+ (400±24.5; n = 4) at 80 mmHg ( p = 0.0001). Additionally, Ca 2+ events are slower and longer-lasting in the RSK2 SM -/- . Decreased levels of p-RLC 20 are observed at 40 mmHg, but not 80 mmHg in RSK2 SM -/- vs. RSK2 SM +/+ mice, consistent with impeding tone development. Conclusion: RSK2 signaling contributes to myogenic tone and vessel stiffness by regulating sensitivity to NO, increasing Ca 2+ events, and RLC 20 activity. RSK2 signaling pathway is a potential therapeutic target for hypertension.

Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30–120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

Regulation of Smooth Muscle Myosin Light Chain Phosphatase by Multisite Phosphorylation of the Myosin Targeting Subunit, MYPT1.

Smooth muscle contraction is triggered primarily by activation of Ca2+/calmodulin-dependent myosin light chain kinase leading to phosphorylation of the regulatory light chains of myosin II. Numerous contractile stimuli also induce inhibition of myosin light chain phosphatase thereby prolonging the contractile response. The phosphatase is a trimeric enzyme containing a catalytic subunit, a regulatory, myosin-binding subunit (MYPT1) and a third subunit of uncertain function. MYPT1 is phosphorylated at multiple sites by several kinases, which regulate phosphatase activity, protein-protein interactions and subcellular localization. The best-characterized phosphorylation events involve phosphorylation by Rho-associated coiled-coil kinase (ROCK) at T697 and T855, which inhibits phosphatase activity, and phosphorylation by cAMP- or cGMPdependent protein kinases (PKA and PKG, respectively) at S696, T697, S854 and T855, which has no effect on phosphatase activity. Furthermore, phosphorylation by ROCK at T697 and T855 prevents phosphorylation by PKA or PKG at the neighboring serine residues. Some 30 phosphorylation sites have been identified in MYPT1 with many more suggested by large-scale phosphoproteomic studies. It is important to gain as complete understanding as possible of the complex phosphorylation-mediated mechanisms of regulation of MYPT1 functions in part because of their involvement in pathological processes. For example, dysfunctional MYPT1 phosphorylation has been implicated in the pathogenesis of several vascular disorders, including type 2 diabetes. Much effort is now being devoted to the development of novel therapeutics targeting MYPT1 and specific kinases involved in the phosphorylation of MYPT1.

Gender differences in the regulation of MLC20 phosphorylation and smooth muscle contraction in rat stomach.

Evidence of sex-related differences in gastrointestinal (GI) functions has been reported in the literature. In addition, various GI disorders have disproportionate prevalence between the sexes. An essential step in the initiation of smooth muscle contraction is the phosphorylation of the 20-kDa regulatory myosin light chain (MLC20) by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK). However, whether male stomach smooth muscle inherits different contractile signaling mechanisms for the regulation of MLC20 phosphorylation from that in females has not been established. The present study was designed to investigate sex-associated differences in the regulation of MLC20 phosphorylation and thus muscle contraction in gastric smooth muscle cells (GSMCs). Experiments were performed on GSMCs freshly isolated from male and female rats. Contraction of the GSMCs in response to acetylcholine (ACh), a muscarinic agonist, was measured via scanning micrometry in the presence or absence of the MLCK inhibitor, ML-7. Additionally, the protein levels of MLC20, MLCK and phosphorylated MLC20 were measured by ELISA. The protein levels of MLC20 and MLCK were indifferent between the sexes. ACh induced greater contraction (P<0.05) as well as greater MLC20 phosphorylation (P<0.05) in male GSMCs compared with female. Pretreatment of GSMCs with ML-7 significantly reduced the ACh-induced contraction (P<0.05) and MLC20 phosphorylation (P<0.05) in the male and female cells, and notably, abolished the contractile differences between the sexes. In conclusion, MLC20 phosphorylation and thus muscle contraction may be activated to a greater extent in male rat stomach compared with that in females.

Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle.

Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+ /calmodulin-dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock-in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction. Ca2+ /calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI-17 (PKC-potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1SM+/+ , MYPT1SM-/- and the knock-in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.

Physiological signalling to myosin phosphatase targeting subunit‐1 phosphorylation in ileal smooth muscle

The extent of myosin regulatory light chain phosphorylation (RLC) necessary for smooth muscle contraction depends on the respective activities of Ca(2+) /calmodulin-dependent myosin light chain kinase and myosin light chain phosphatase (MLCP), which contains a regulatory subunit MYPT1 bound to the phosphatase catalytic subunit and myosin. MYPT1 showed significant constitutive T696 and T853 phosphorylation, which is predicted to inhibit MLCP activity in isolated ileal smooth muscle tissues, with additional phosphorylation upon pharmacological treatment with the muscarinic agonist carbachol. Electrical field stimulation (EFS), which releases ACh from nerves, increased force and RLC phosphorylation but not MYPT1 T696 or T853 phosphorylation. The conditional knockout of MYPT1 or the knockin mutation T853A in mice had no effect on the frequency-maximal force responses to EFS in isolated ileal tissues. Physiological RLC phosphorylation and force development in ileal smooth muscle depend on myosin light chain kinase and MLCP activities without changes in constitutive MYPT1 phosphorylation. Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+) /calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in ileal smooth muscle in adult mice with (1) smooth muscle-specific deletion of MYPT1; (2) non-phosphorylatable MYPT1 containing a T853A knockin mutation; and (3) measurements of force and protein phosphorylation responses to cholinergic neurostimulation initiated by electric field stimulation. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted and relaxed rapidly with moderate differences in sustained responses to KCl and carbachol treatments and washouts, respectively. Similarly, measurements of regulatory proteins responsible for RLC phosphorylation during contractions also revealed moderate changes. There were no differences in contractile or RLC phosphorylation responses to carbachol between tissues from normal mice vs. MYPT1 T853A knockin mice. Quantitatively, there was substantial MYPT1 T696 and T853 phosphorylation in wild-type tissues under resting conditions, predicting a high extent of MLCP phosphatase inhibition. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Electric field stimulation increased RLC phosphorylation and force development in tissues from wild-type mice without an increase in MYPT1 phosphorylation. Thus, physiological RLC phosphorylation and force development in ileal smooth muscle appear to be dependent on MLCK and MLCP activities without changes in constitutive MYPT1 phosphorylation.

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

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+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. 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. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. 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.

Constitutive phosphorylation of myosin phosphatase targeting subunit-1 in smooth muscle.

Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in bladder smooth muscle containing a smooth muscle-specific deletion of MYPT1 in adult mice. Deep-sequencing analyses of mRNA and immunoblotting revealed that MYPT1 depletion reduced the amount of PP1cδ with no compensatory changes in expression of other MYPT1 family members. Phosphatase activity towards phosphorylated smooth muscle heavy meromyosin was proportional to the amount of PP1cδ in total homogenates from wild-type or MYPT1-deficient tissues. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted with moderate differences in response to KCl and carbachol treatments, and relaxed rapidly with comparable rates after carbachol removal and only 1.5-fold slower after KCl removal. Measurements of phosphorylated proteins in the RLC signalling and actin polymerization modules during contractions revealed moderate changes. Using a novel procedure to quantify total phosphorylation of MYPT1 at Thr696 and Thr853, we found substantial phosphorylation in wild-type tissues under resting conditions, predicting attenuation of MLCP activity. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to the attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Constitutive phosphorylation of MYPT1 Thr696 and Thr853 may thus represent a physiological mechanism acting in concert with agonist-induced MYPT1 phosphorylation to inhibit MLCP activity. In summary, MYPT1 deficiency may not cause significant derangement of smooth muscle contractility because the effective MLCP activity is not changed.

Involvement of myosin regulatory light chain diphosphorylation in sustained vasoconstriction under pathophysiological conditions.

Smooth muscle contraction is activated primarily by phosphorylation at Ser19 of the regulatory light chain subunits (LC20) of myosin II, catalysed by Ca2+/calmodulin-dependent myosin light chain kinase. Ca2+-independent contraction can be induced by inhibition of myosin light chain phosphatase, which correlates with diphosphorylation of LC20 at Ser19 and Thr18, catalysed by integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK). LC20 diphosphorylation at Ser19 and Thr18 has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli (e.g. endothelin-1 stimulation of rat renal afferent arterioles) and in pathophysiological situations associated with hypercontractility (e.g. cerebral vasospasm following subarachnoid hemorrhage). Comparison of the effects of LC20 monophosphorylation at Ser19 and diphosphorylation at Ser19 and Thr18 on contraction and relaxation of Triton-skinned rat caudal arterial smooth muscle revealed that phosphorylation at Thr18 has no effect on steady-state force induced by Ser19 phosphorylation. On the other hand, the rates of dephosphorylation and relaxation are significantly slower following diphosphorylation at Thr18 and Ser19 compared to monophosphorylation at Ser19. We propose that this diphosphorylation mechanism underlies the prolonged contractile response of particular vascular smooth muscle tissues to specific stimuli, e.g. endothelin-1 stimulation of renal afferent arterioles, and the vasospastic behavior observed in pathological conditions such as cerebral vasospasm following subarachnoid hemorrhage and coronary arterial vasospasm. ILK and ZIPK may, therefore, be useful therapeutic targets for the treatment of such conditions.

Notch Transcriptional Control of Vascular Smooth Muscle Regulatory Gene Expression and Function

Notch receptors and ligands mediate heterotypic cell signaling that is required for normal vascular development. Dysregulation of select Notch receptors in mouse vascular smooth muscle (VSM) and in genetic human syndromes causes functional impairment in some regional circulations, the mechanistic basis of which is undefined. In this study, we used a dominant-negative Mastermind-like (DNMAML1) to block signaling through all Notch receptors specifically in VSM to more broadly test a functional role for this pathway in vivo. Mutant DNMAML1-expressing mice exhibited blunted blood pressure responses to vasoconstrictors, and their aortic, femoral, and mesenteric arteries had reduced contractile responses to agonists and depolarization in vitro. The mutant arteries had significant and specific reduction in the expression and activity of myosin light chain kinase (MLCK), a primary regulator of VSM force production. Conversely, activated Notch signaling in VSM cells induced endogenous MLCK transcript levels. We identified MLCK as a direct target of activated Notch receptor as demonstrated by an evolutionarily conserved Notch-responsive element within the MLCK promoter that binds the Notch receptor complex and is required for transcriptional activity. We conclude that Notch signaling through the transcriptional control of key regulatory proteins is required for contractile responses of mature VSM. Genetic or pharmacological manipulation of Notch signaling is a potential strategy for modulating arterial function in human disease.

Signaling through Myosin Light Chain Kinase in Smooth Muscles

Ca(2+)/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 Ca(2+)/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 Ca(2+) sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.

Involvement of calmodulin in realization of vasoconstrictive effects of serotonin and norepinephrine

Possible involvement ofcalmodulin in adrenergic and serotoninergic regulation of vascular contractility has been studied. Calmodulin inhibitors trifluoperazine and W-13 suppress vasoconstriction of the rat aorta in response to norepinephrine, serotonin, and serotonin 5HT1A- and 5HT2A-receptor agonists (8-OH-DPAT and DOI, respectively) and do not affect the vasodilatory effect of 5HT1B-, 5HT2B-, and 5HT4-receptors. The force of aorta contraction in response to 8-OH-DPAT increases after the activation of calcium entry through voltage-gated Ca2+-channels. This effect is not related to non-specific activation of alpha1-adrenoceptors, since it is realized in the presence of prazosin. The inhibitor of calmodulin-dependent myosin light chain kinase KN93 decreases the vasoconstrictive response in response to norepinephrine and serotonin by only 20%. Calmodulin inhibitors slightly decrease aortic constriction in response to endothelin-1, vasopressin, angiotensin II, and KCl. Trifluoperazine does not suppress vasoconstriction induced by the G-protein activator AlF4(-). It is assumed that the target of trifluoperazine and W-13 is calmodulin interacting directly with alpha1-adrenoceptors and serotonin 5HT1A- and 5HT2A-receptors.

Myosin Regulatory Light Chain Diphosphorylation Slows Relaxation of Arterial Smooth Muscle

The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC(20)) at Ser(19) by Ca(2+)/calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca(2+)-independent phosphorylation at both Ser(19) and Thr(18) by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr(18) on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser(19) and Thr(18) was achieved by treatment with adenosine 5'-O-(3-thiotriphosphate) in the presence of Ca(2+), which induced stoichiometric thiophosphorylation at Ser(19), followed by microcystin (phosphatase inhibitor) in the absence of Ca(2+), which induced phosphorylation at Thr(18). Phosphorylation at Thr(18) had no effect on steady-state force induced by Ser(19) thiophosphorylation. However, phosphorylation of Ser(19) or both Ser(19) and Thr(18) to comparable stoichiometries (0.5 mol of P(i)/mol of LC(20)) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: t(&frac12;) values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser(19)) and diphosphorylated LC(20), respectively. We conclude that phosphorylation at Thr(18) decreases the rates of LC(20) dephosphorylation and smooth muscle relaxation compared with LC(20) phosphorylated exclusively at Ser(19). These effects of LC(20) diphosphorylation, combined with increased Ser(19) phosphorylation (Ca(2+)-independent), may underlie the hypercontractility that is observed in response to certain physiological contractile stimuli, and under pathological conditions such as cerebral and coronary arterial vasospasm, intimal hyperplasia, and hypertension.

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.

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.

Fractional activation of myosin light chain kinase is sufficient for robust smooth muscle contraction

Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) is necessary and sufficient for phosphorylation of myosin regulatory light chain (RLC) for smooth muscle contraction (He et al., 2008; Zhang et al., 2010). However, it was noted that a biosensor MLCK was activated only 25% for maximal agonist-induced contraction (Isotani et al., 2004). We therefore used a tamoxifen-activated and smooth muscle-specific inactivation of MLCK expression in adult mice to determine if a small fraction of MLCK was sufficient for RLC phosphorylation and contraction. An 80% decrease in MLCK content in bladder smooth muscle strips resulted in a 60% and 50% decrease in maximal RLC phosphorylation and contractile responses to KCl, respectively, but only a 20% decrease in maximal responses to 10 uM carbachol (CCh). Phosphorylation of the regulatory subunit (MYPT1) of myosin light chain phosphatase (MLCP) inhibits its activity and thus, enhances RLC phosphorylation. MLCK knockdown did not change MYPT1 Thr695 and Thr850 phosphorylation responses to CCh, but did enhance MLCP inhibitor protein CPI-17 phosphorylation. Thus, activation of a small fraction of MLCK along with MLCP inhibition may be sufficient for robust RLC phosphorylation and contractile responses in smooth muscles. Supported by HL26043.

Rho GTPases and their role in organizing the actin cytoskeleton

Cells receive extracellular stimuli in various ways: in the form of soluble molecules (growth factors, cytokines and hormones) that interact with cell-surface receptors; from adhesive interactions with the extracellular matrix; and from cell–cell adhesions. These stimuli act to generate changes in

Syntheses of Dehydroaltenusin, a Selective Inhibitor of Mammalian DNA Polymerase &amp;#945;

Dehydroaltenusin has been isolated from Alternaria tenuis and other fungal species. It exhibits various biological activities such as growth inhibition of wood-damaging fungi, inhibition of calmodulin-dependent myosin light chain kinase, anti-HIV activity, and inhibition of mammalian DNA polymerase α (pol α). These significant biological activities, as well as the unique structural properties of dehydroaltenusin, have stimulated considerable interest in its synthesis. In this review, we summarize the synthesis and structural analysis of dehydroaltenusin and its derivatives. We also focus on the structure-activity relationships of dehydroaltenusin as a mammalian pol α inhibitor. Dehydroaltenusin specifically inhibits mammalian pol α, an enzyme that plays an important role in DNA replication and cell division. The human genome encodes 14 pols that control cellular DNA synthesis, and synthetic dehydroaltenusin derivatives, which are specific inhibitors of mammalian pol α, are useful tools and molecular probes for distinguishing pols and for clarifying their biological and in vivo functions. Moreover, these compounds might be potentially useful as anti-cancer chemotherapeutic drugs for in vivo studies.

MLCK‐independent phosphorylation of MLC20 and its regulation by MAP kinase pathway in human bladder smooth muscle cells

Myosins are a superfamily of actin-based molecular motor proteins, which hydrolyze ATP and generate various forms of eukaryotic motility and muscle contraction. Myosin light chain 20 (MLC20) is small ring around the neck region of heavy chain of myosins. Phosphorylation of MLC20 is thought to play a key role in regulation of smooth muscle contraction. Calcium- and calmodulin-dependent myosin light chain kinase (MLCK) is considered the primary regulator of MLC20 phosphorylation. However, several observations in smooth muscle contraction cannot be explained by the mode of phosphorylation. By performing a series of experiments in vitro and in vivo, we report here MLCK-independent MLC20 phosphorylation. Gene expression study reveals that expression of MLCK in smooth muscles is inconsistent with MLC20 phosphorylation at Ser19. None of inactivating calmodulin/MLCK, depriving of calcium and silencing MLCK expression by siRNA blocks effectively the phosphorylation of MLC20 at Ser19. In addition, by overexpressing active human MAP (mitogen-activated protein)-ERK kinase kinase-1 (MEKK1) and blocking its downstream messengers, we have demonstrated a new regulatory system of MLC phosphorylation via MEKK1, which downregulates Ser19 phosphorylation of MLC20 through its downstream molecules, p38, JNK, and ERK in human bladder smooth muscle cells.

Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction

Different interacting signaling modules involving Ca(2+)/calmodulin-dependent myosin light chain kinase, Ca(2+)-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K(+)-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.