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1.
Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca2+-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.  相似文献   

2.
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.  相似文献   

3.
CPI-17 is a unique phosphoprotein that specifically inhibits myosin light chain phosphatase in smooth muscle and plays an essential role in agonist-induced contraction. To elucidate the in situ mechanism for G protein-mediated Ca2+-sensitization of CPI-17 phosphorylation, α-toxin-permeabilized arterial smooth muscle strips were used to monitor both force development and CPI-17 phosphorylation in response to GTPγS with varying Ca2+ concentrations. CPI-17 phosphorylation increased at unphysiologically high Ca2+ levels of pCa ? 6. GTPγS markedly enhanced the Ca2+ sensitivity of CPI-17 steady-state phosphorylation but had no enhancing effect under Ca2+-free conditions, while the potent PKC activator PDBu increased CPI-17 phosphorylation regardless of Ca2+ concentration. CPI-17 phosphorylation induced by pCa 4.5 alone was markedly inhibited by the presence of PKC inhibitor but not ROCK inhibitor. In the presence of calyculin A, a potent PP1/PP2A phosphatase inhibitor, CPI-17 phosphorylation increased with time even under Ca2+-free conditions. Furthermore, as Ca2+ concentration increased, so did CPI-17 phosphorylation rate. GTPγS markedly enhanced the rate of phosphorylation of CPI-17 at a given Ca2+. In the absence of calyculin A, either steady-state phosphorylation of CPI-17 under Ca2+-free conditions in the presence of GTPγS or at pCa 6.7 in the absence of GTPγS was negligible, suggesting a high intrinsic CPI-17 phosphatase activity. In conclusion, cooperative increases in Ca2+ and G protein activation are required for a significant activation of total kinases that phosphorylate CPI-17, which together overcome CPI-17 phosphatase activity and effectively increase the Ca2+ sensitivity of CPI-17 phosphorylation and smooth muscle contraction.  相似文献   

4.
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.  相似文献   

5.
In the canonical model of smooth muscle (SM) contraction, the contractile force is generated by phosphorylation of the myosin regulatory light chain (RLC20) by the myosin light chain kinase (MLCK). Moreover, phosphorylation of the myosin targeting subunit (MYPT1) of the RLC20 phosphatase (MLCP) by the RhoA-dependent ROCK kinase, inhibits the phosphatase activity and consequently inhibits dephosphorylation of RLC20 with concomitant increase in contractile force, at constant intracellular [Ca2+]. This pathway is referred to as Ca2+-sensitization. There is, however, emerging evidence suggesting that additional Ser/Thr kinases may contribute to the regulatory pathways in SM. Here, we report data implicating the p90 ribosomal S6 kinase (RSK) in SM contractility. During both Ca2+- and agonist (U46619) induced SM contraction, RSK inhibition by the highly selective compound BI-D1870 (which has no effect on MLCK or ROCK) resulted in significant suppression of contractile force. Furthermore, phosphorylation levels of RLC20 and MYPT1 were both significantly decreased. Experiments involving the irreversible MLCP inhibitor microcystin-LR, in the absence of Ca2+, revealed that the decrease in phosphorylation levels of RLC20 upon RSK inhibition are not due solely to the increase in the phosphatase activity, but reflect direct or indirect phosphorylation of RLC20 by RSK. Finally, we show that agonist (U46619) stimulation of SM leads to activation of extracellular signal-regulated kinases ERK1/2 and PDK1, consistent with a canonical activation cascade for RSK. Thus, we demonstrate a novel and important physiological function of the p90 ribosomal S6 kinase, which to date has been typically associated with the regulation of gene expression.  相似文献   

6.
Previous studies have shown that cGMP-dependent protein kinase (PKG) act on several targets in the contractile pathway to reduce intracellular Ca2+ and/or augment RhoA-regulated myosin light chain phosphatase (MLCP) activity and cause muscle relaxation. Recent studies have identified a novel protein M-RIP that associates with MYPT1, the regulatory subunit of MLCP. Herein, we examine whether PKG enhance MLCP activity downstream of Ca2+ and RhoA via phosphorylation of M-RIP in gastric smooth muscle cells. Treatment of permeabilized muscle cells with 10 μM Ca2+ caused an increase in MLC20 phosphorylation and muscle contraction, but had no effect on Rho kinase activity. Activators of PKG (GSNO or cGMP) decreased MLC20 phosphorylation and contraction in response to 10 μM Ca2+, implying existence of inhibitory mechanism independent of Ca2+ and RhoA. The effect of PKG on Ca2+-induced MLC20 phosphorylation was attenuated by M-RIP siRNA. Both GSNO and 8-pCPT-cGMP induced phosphorylation of M-RIP; phosphorylation was accompanied by an increase in the association of M-RIP with MYPT1 and MLCP activity. Taken together, these results provide evidence that PKG induces phosphorylation of M-RIP and enhances its association with MYPT1 to augment MLCP activity and MLC20 dephosphorylation and inhibits muscle contraction, downstream of Ca2+- or RhoA-dependent pathways.  相似文献   

7.
Ureteric peristalsis, which occurs via alternating contraction and relaxation of ureteric smooth muscle, ensures the unidirectional flow of urine from the kidney to the bladder. Understanding of the molecular mechanisms underlying ureteric excitation–contraction coupling, however, is limited. To address these knowledge deficits, and in particular to test the hypothesis that Ca2+ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway plays an important role in ureteric smooth muscle contraction, we carried out a thorough characterization of the electrical activity, Ca2+ signaling, MYPT1 (myosin targeting subunit of myosin light chain phosphatase, MLCP) and myosin regulatory light chain (LC20) phosphorylation, and force responses to membrane depolarization induced by KCl (electromechanical coupling) and carbachol (CCh) (pharmacomechanical coupling). The effects of ROK inhibition on these parameters were investigated. We conclude that the tonic, but not the phasic component of KCl- or CCh-induced ureteric smooth muscle contraction is highly dependent on ROK-catalyzed phosphorylation of MYPT1 at T855, leading to inhibition of MLCP and increased LC20 phosphorylation.  相似文献   

8.
Phosphorylation of myosin II plays an important role in many cell functions, including smooth muscle contraction. The level of myosin II phosphorylation is determined by activities of myosin light chain kinase and myosin phosphatase (MP). MP is composed of 3 subunits: a catalytic subunit of type 1 phosphatase, PPlc; a targeting subunit, termed myosin phosphatase target subunit, MYPT; and a smaller subunit, M20, of unknown function. Most of the properties of MP are due to MYPT and include binding of PP1c and substrate. Other interactions are discussed. A recent discovery is the existence of an MYPT family and members include, MYPT1, MYPT2, MBS85, MYPT3 and TIMAP. Characteristics of each are outlined. An important discovery was that the activity of MP could be regulated and both activation and inhibition were reported. Activation occurs in response to elevated cyclic nucleotide levels and various mechanisms are presented. Inhibition of MP is a major component of Ca2+-sensitization in smooth muscle and various molecular mechanisms are discussed. Two mechanisms are cited frequently: (1) Phosphorylation of an inhibitory site on MYPT1, Thr696 (human isoform) and resulting inhibition of PP1c activity. Several kinases can phosphorylate Thr696, including Rho-kinase that serves an important role in smooth muscle function; and (2) Inhibition of MP by the protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17). Examples where these mechanisms are implicated in smooth muscle function are presented. The critical role of RhoA/Rho-kinase signaling in various systems is discussed, in particular those vascular smooth muscle disorders involving hypercontractility.  相似文献   

9.
Phosphorylation of the regulatory light chain of myosin by the Ca2+/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca2+/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca2+/calmodulin required for activation and hence increases the Ca2+ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca2+ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca2+ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.  相似文献   

10.
The mammalian MYPT family consists of the products of five genes, denoted MYPT1, MYPT2, MBS85, MYPT3 and TIMAP, which function as targeting and regulatory subunits to confer substrate specificity and subcellular localization on the catalytic subunit of type 1δ protein serine/threonine phosphatase (PP1cδ). Family members share several conserved domains, including an RVxF motif for PP1c binding and several ankyrin repeats that mediate protein–protein interactions. MYPT1, MYPT2 and MBS85 contain C-terminal leucine zipper domains involved in dimerization and protein–protein interaction, whereas MYPT3 and TIMAP are targeted to membranes via a C-terminal prenylation site. All family members are regulated by phosphorylation at multiple sites by various protein kinases; for example, Rho-associated kinase phosphorylates MYPT1, MYPT2 and MBS85, resulting in inhibition of phosphatase activity and Ca2+ sensitization of smooth muscle contraction. A great deal is known about MYPT1, the myosin targeting subunit of myosin light chain phosphatase, in terms of its role in the regulation of smooth muscle contraction and, to a lesser extent, non-muscle motile processes. MYPT2 appears to be the key myosin targeting subunit of myosin light chain phosphatase in cardiac and skeletal muscles. MBS85 most closely resembles MYPT2, but little is known about its physiological function. Little is also known about the physiological role of MYPT3, although it is likely to target myosin light chain phosphatase to membranes and thereby achieve specificity for substrates involved in regulation of the actin cytoskeleton. MYPT3 is regulated by phosphorylation by cAMP-dependent protein kinase. TIMAP appears to target PP1cδ to the plasma membrane of endothelial cells where it serves to dephosphorylate proteins involved in regulation of the actin cytoskeleton and thereby control endothelial barrier function. With such a wide range of regulatory targets, MYPT family members have been implicated in diverse pathological events, including hypertension, Parkinson’s disease and cancer.  相似文献   

11.
Reduced colonic motility has been observed in aged rats with a parallel reduction in acetylcholine (ACh)-induced myosin light chain (MLC(20)) phosphorylation. MLC(20) phosphorylation during smooth muscle contraction is maintained by a coordinated signal transduction cascade requiring both PKC-alpha and RhoA. Caveolae are membrane microdomains that permit rapid and efficient coordination of different signal transduction cascades leading to sustained smooth muscle contraction of the colon. Here, we show that normal physiological contraction can be reinstated in aged colonic smooth muscle cells (CSMCs) upon transfection with wild-type caveolin-1 through the activation of both the RhoA/Rho kinase and PKC pathways. Our data demonstrate that impaired contraction in aging is an outcome of altered membrane translocation of PKC-alpha and RhoA with a concomitant reduction in the association of these molecules with the caveolae-specific protein caveolin-1, resulting in a parallel decrease in the myosin phosphatase-targeting subunit (MYPT) and CPI-17 phosphorylation. Decreased MYPT and CPI-17 phosphorylation activates MLC phosphatase activity, resulting in MLC(20) dephosphorylation, which may be responsible for decreased colonic motility in aged rats. Importantly, transfection of CSMCs from aged rats with wild-type yellow fluorescent protein-caveolin-1 cDNA restored translocation of RhoA and PKC-alpha and phosphorylation of MYPT, CPI-17, and MLC(20), thereby restoring the contractile response to levels comparable with young adult rats. Thus, we propose that caveolin-1 gene transfer may represent a promising therapeutic treatment to correct the age-related decline in colonic smooth muscle motility.  相似文献   

12.
We analyzed the signaling pathways initiated by endothelin receptors ETA and ETB in intestinal circular and longitudinal smooth muscle cells. The response to endothelin-1 (ET-1) consisted of two phases in both cell types. The initial, transient phase of contraction and phosphorylation of 20-kDa myosin light chain (MLC20) was mediated additively by ETA and ETB receptors and initiated by Gq-, Ca2+/calmodulin-dependent activation of MLC kinase. In contrast, the sustained phase was mediated selectively by ETA receptors via a pathway involving sequential activation of G13, RhoA, and Rho kinase, resulting in phosphorylation of MYPT1 at Thr696 and phosphorylation of MLC20. Although PKC was activated, CPI-17 was not phosphorylated and hence did not contribute to inhibition of MLC phosphatase. The absence of CPI-17 phosphorylation by PKC reflected active dephosphorylation of CPI-17 by protein phosphatase 2A (PP2A). PP2A was activated via a pathway involving ETB-dependent stimulation of p38 MAPK activity. CPI-17 phosphorylation was unmasked in the presence of the ETB antagonist BQ-788, but not the ETA antagonist BQ-123, and in the presence of a low concentration of okadaic acid, which selectively inactivates PP2A. The resultant phosphorylation of CPI-17 was blocked by bisindolylmaleimide, providing direct confirmation that it was PKC dependent. We conclude that the two phases of the intestinal smooth muscle response to ET-1 involve distinct receptors, G proteins, and signaling pathways. The sustained response is mediated via selective ETA-dependent phosphorylation of MYPT1. In contrast, ETB initiates an inhibitory pathway involving p38 MAPK-dependent activation of PP2A that causes dephosphorylation of CPI-17. endothelin receptor type A; endothelin receptor type B; myosin phosphatase targeting subunit  相似文献   

13.
Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosphorylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specific protein called CPI-17 (for protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphorylated and converted to a potent inhibitor for myosin phosphatase. Phosphorylation of CPI-17 is diminished by pretreatment with either or GF109203x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897--9900). Here we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extracts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) alpha, and the second kinase was purified to homogeneity as a 45-kDa protein, and identified by sequencing as PKC delta. Purified PKC delta was 3-fold more reactive with CPI-17 compared with myelin basic protein, whereas purified PKC alpha and recombinant RhoA-activated kinases (Rho-associated coiled-coil forming protein Ser/Thr kinase and protein kinase N) showed equal activity with CPI-17 and myelin basic protein. inhibited CPI-17 phosphorylation by purified PKC delta with IC(50) of 0.6 microm (in the presence of 0.1 mm ATP) or 14 microm (2.0 mm ATP). significantly suppressed CPI-17 phosphorylation in smooth muscle cells, and the contraction of permeabilized rabbit femoral artery induced by stimulation with phorbol ester. GF109203x inhibited phorbol ester-induced contraction of rabbit femoral artery by 80%, whereas a PKC alpha/beta inhibitor, Go6976, reduced contraction by 47%. The results imply that histamine stimulation elicits contraction of vascular smooth muscle through activation of PKC alpha and especially PKC delta to phosphorylate CPI-17.  相似文献   

14.
Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC20). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca2+-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K+-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC20 phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K+, Pyk2 autophosphorylation, and LC20 phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca2+, elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca2+ channel blocker nifedipine inhibited peak and sustained K+-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K+-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca2+ activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC20 phosphorylation then accounts for the tonic contractile response to membrane depolarization.  相似文献   

15.
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.  相似文献   

16.
Different interacting signaling modules involving Ca2+/calmodulin-dependent myosin light chain kinase, Ca2+-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.  相似文献   

17.
Lukas TJ 《Biophysical journal》2004,87(3):1417-1425
An agonist-initiated Ca(2+) signaling model for calmodulin (CaM) coupled to the phosphorylation of myosin light chains was created using a computer-assisted simulation environment. Calmodulin buffering was introduced as a module for directing sequestered CaM to myosin light chain kinase (MLCK) through Ca(2+)-dependent release from a buffering protein. Using differing simulation conditions, it was discovered that CaM buffering allowed transient production of more Ca(2+)-CaM-MLCK complex, resulting in elevated myosin light chain phosphorylation compared to nonbuffered control. Second messenger signaling also impacts myosin light chain phosphorylation through the regulation of myosin light chain phosphatase (MLCP). A model for MLCP regulation via its regulatory MYPT1 subunit and interaction of the CPI-17 inhibitor protein was assembled that incorporated several protein kinase subsystems including Rho-kinase, protein kinase C (PKC), and constitutive MYPT1 phosphorylation activities. The effects of the different routes of MLCP regulation depend upon the relative concentrations of MLCP compared to CPI-17, and the specific activities of protein kinases such as Rho and PKC. Phosphorylated CPI-17 (CPI-17P) was found to dynamically control activity during agonist stimulation, with the assumption that inhibition by CPI-17P (resulting from PKC activation) is faster than agonist-induced phosphorylation of MYPT1. Simulation results are in accord with literature measurements of MLCP and CPI-17 phosphorylation states during agonist stimulation, validating the predictive capabilities of the system.  相似文献   

18.
Previous work has shown that Mg2+ levels modulate the net level of myosin light chain phosphorylation in bovine aortic smooth muscle actomyosin preparations. The goal of this study was to determine the precise step, i.e. phosphorylation or dephosphorylation, where Mg2+ modulates the net phosphorylation reaction. The technique using [γ35S]ATPγS to monitor the phosphorylating step yielded no effect of either Mg2+ or Ca2+. Unfortunately the lack of Ca2+-dependence did not allow conclusions about the influence of Mg2+ on myosin light chain kinase activity. The study of the effect of Mg2+ on dephosphorylation showed that phosphatase activity in the actomyosin preparation exhibited a Mg2+ modulation only when the actomyosin was previously exposed to activating levels (3×10?5M) of Ca2+, suggesting the presence of a Ca2+ -regulation system for myosin light chain phosphatase.  相似文献   

19.
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.  相似文献   

20.
Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca(2+) sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K(+) depolarization. The Ca(2+)-sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In alpha-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5'-O-(gamma-thiotriphosphate), and histamine plus guanyl-5'-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca(2+) sensitization of smooth muscle contraction.  相似文献   

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