Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation

The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.     

Myosin light chain phosphorylation-dependent modulation of volume-regulated anion channels in macrovascular endothelium

The Rho/Rho-associated kinase (ROK) pathway has been shown to modulate volume-regulated anion channels (VRAC) in cultured calf pulmonary artery endothelial (CPAE) cells. Since Rho/ROK can increase myosin light chain phosphorylation, we have now studied the effects of inhibitors of myosin light chain kinase (MLCK) or myosin light chain phosphatase (MLCP) on VRAC in CPAE. Application of ML-9, an MLCK inhibitor, inhibited VRAC, both when applied extracellularly or when dialyzed into the cell. A similar inhibitory effect was obtained by dialyzing the cells with AV25, a specific MLCK inhibitory peptide. Conversely, NIPP1(191-210), an MLCP inhibitory peptide, potentiated the activation of VRAC by a 25% hypotonic stimulus. These data indicate that activation of VRAC is modulated by MLC phosphorylation.     

An increase or a decrease in myosin II phosphorylation inhibits macrophage motility

Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin-independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells.     

Myosin ii light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor-1

Activation of myosin II by myosin light chain kinase (MLCK) produces the force for many cellular processes including muscle contraction, mitosis, migration, and other cellular shape changes. The results of this study show that inhibition or potentiation of myosin II activation via over-expression of a dominant negative or wild type MLCK can delay or accelerate tumor necrosis factor-alpha (TNF)-induced apoptotic cell death in cells. Changes in the activation of caspase-8 that parallel changes in regulatory light chain phosphorylation levels reveal that myosin II motor activities regulate TNF receptor-1 (TNFR-1) signaling at an early step in the TNF death signaling pathway. Treatment of cells with either ionomycin or endotoxin (lipopolysaccharide) leads to activation of myosin II and increased translocation of TNFR-1 to the plasma membrane independent of TNF signaling. The results of these studies establish a new role for myosin II motor activity in regulating TNFR-1-mediated apoptosis through the translocation of TNFR-1 to or within the plasma membrane.     

Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK‐mediated MLC phosphorylation

The retinal pigment epithelium (RPE) forms the outer blood–retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial–mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR‐1 induces the time‐ and dose‐dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho‐MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y‐27632, whereas MLCK inhibitor ML‐7 and PLC‐β inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC‐β‐mediated calcium increase. Additionally, thrombin‐induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP₃/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin‐induced RPE cell transformation subsequent to BRB dysfunction. J. Cell. Physiol. 226: 414–423, 2011. © 2010 Wiley‐Liss, Inc.     

Regulation of Cell Motility by Mitogen-activated Protein Kinase

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/ mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.     

Non-muscle myosin II and myosin light chain kinase are downstream targets for vasopressin signaling in the renal collecting duct

We have previously demonstrated that vasopressin increases the water permeability of the inner medullary collecting duct (IMCD) by inducing trafficking of aquaporin-2 to the apical plasma membrane and that this response is dependent on intracellular calcium mobilization and calmodulin activation. Here, we address the hypothesis that this water permeability response is mediated in part through activation of the calcium/calmodulin-dependent myosin light chain kinase (MLCK) and regulation of non-muscle myosin II. Immunoblotting and immunocytochemistry demonstrated the presence of MLCK, the myosin regulatory light chain (MLC), and the IIA and IIB isoforms of the non-muscle myosin heavy chain in rat IMCD cells. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified two isoforms of MLC, both of which also exist in phosphorylated and non-phosphorylated forms. 32P incubation of the inner medulla followed by autoradiography of two-dimensional gels demonstrated increased 32P labeling of both isoforms in response to the V2 receptor agonist [deamino-Cys1,D-Arg8]vasopressin (DDAVP). Time course studies of MLC phosphorylation in IMCD suspensions (using immunoblotting with anti-phospho-MLC antibodies) showed that the increase in phosphorylation could be detected as early as 30 s after exposure to vasopressin. The MLCK inhibitor ML-7 blocked the DDAVP-induced MLC phosphorylation and substantially reduced [Arg8]vasopressin (AVP)-stimulated water permeability. AVP-induced MLC phosphorylation was associated with a rearrangement of actin filaments (Alexa Fluor 568-phalloidin) in primary cultures of IMCD cells. These results demonstrate that MLC phosphorylation by MLCK represents a downstream effect of AVP-activated calcium/calmodulin signaling in IMCD cells and point to a role for non-muscle myosin II in regulation of water permeability by vasopressin.     

Gi-coupled receptors mediate phosphorylation of CPI-17 and MLC20 via preferential activation of the PI3K/ILK pathway

Sustained smooth-muscle contraction or its experimental counterpart, Ca2+ sensitization, by G(q/13)-coupled receptor agonists is mediated via RhoA-dependent inhibition of MLC (myosin light chain) phosphatase and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation by a Ca2+-independent MLCK (MLC kinase). The present study identified the corresponding pathways initiated by G(i)-coupled receptors. Somatostatin acting via G(i)1-coupled sstr3 receptor, DPDPE ([D-Pen2,D-Pen5]enkephalin; where Pen is penicillamine) acting via G(i)2-coupled delta-opioid receptors, and cyclopentyl adenosine acting via G(i)3-coupled adenosine A1 receptors preferentially activated PI3K (phosphoinositide 3-kinase) and ILK (integrin-linked kinase), whereas ACh (acetylcholine) acting via G(i)3-coupled M2 receptors preferentially activated PI3K, Cdc42 (cell division cycle 42)/Rac1, PAK1 (p21-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase). Only agonists that activated ILK induced sustained CPI-17 (protein kinase C potentiated inhibitor 17 kDa protein) phosphorylation at Thr38, MLC20 phosphorylation at Ser19, and contraction, consistent with recent evidence that ILK can act as a Ca2+-independent MLCK capable of phosphorylating the MLC phosphatase inhibitor, CPI-17, at Thr38. ILK activity, and CPI-17 and MLC20 phosphorylation were inhibited by LY294002 and in muscle cells expressing ILK(R211A) or treated with siRNA (small interfering RNA) for ILK. ACh acting via M2 receptors activated ILK, and induced CPI-17 and MLC20 phosphorylation and muscle contraction, but only after inhibition of p38 MAPK; all these responses were inhibited in cells expressing ILK(R211A). Conversely, ACh activated PAK1, a step upstream of p38 MAPK, whereas the three other agonists did so only in cells transfected with ILK(R211A) or siRNA for ILK. The results demonstrate reciprocal inhibition between two pathways downstream of PI3K, with ILK inhibiting PAK1, and p38 MAPK inhibiting ILK. Sustained contraction via G(i)-coupled receptors is dependent on CPI-17 and MLC20 phosphorylation by ILK.     

Central involvement of Rho family GTPases in TNF-alpha-mediated bovine pulmonary endothelial cell apoptosis

In our recent studies, we defined a critical role for increased levels of myosin light chain (MLC) phosphorylation, a regulatory event in the interaction between actin and myosin in TNF-alpha-induced pulmonary endothelial cell actomyosin rearrangement and apoptosis. The Rho GTPase effector, Rho kinase is an important signaling effector governing levels of MLC phosphorylation which contributes to plasma membrane blebbing in several models of apoptosis. In this study, we directly assessed the role of Rho kinase in TNF-alpha-induced endothelial cell microfilament rearrangement and apoptosis. Inhibition of RhoA GTPase activity by the overexpression of dominant negative RhoA attenuates TNF-alpha-triggered stress fiber formation, consistent with Rho activation as a key event in TNF-alpha-induced cytoskeletal rearrangement. Furthermore, pharmacologic inhibition of Rho kinase as well as dominant negative RhoA overexpression dramatically reduced TNF-alpha-induced bovine endothelial apoptosis reflected by nucleosomal fragmentation as well as caspase 7, 3, and 8 activation. These results indicate that Rho kinase-dependent cytoskeletal rearrangement is critical for early apoptotic events, possibly in the assembly of the death-inducing signaling complex leading to initiator and effector caspase activation, and suggest a novel role for Rho GTPases in endothelial cell apoptosis.     

Phosphorylation is required for myosin regulatory light chain (PmMRLC) to control yellow head virus infection in shrimp hemocytes

The cellular signal-transduction process is largely controlled by protein phosphorylation. Shrimp infected with yellow head virus show dramatic changes in their hemocyte phosphoproteomic patterns, and aberrant activation of phosphorylation-based signaling networks has been implicated in a number of diseases. In this study, we focused on phosphorylation of Penaeus monodon myosin regulatory light chain (PmMRLC) that is induced at an early hour post YHV infection and is concomitant with cellular actin remodeling. In shrimp cell cultures, this phosphorylation was inhibited by the myosin light chain kinase (MLCK) inhibitors ML-7 and ML-9, suggesting that PmMLC phosphorylation is MLCK pathway-dependent. Blocking PmMRLC phosphorylation resulted in increased replication of YHV and reduction of phagocytic activities of shrimp hemocytes called semigranular cells (SGC) and granular cells (GC). Injection of MLCK inhibitors prior to YHV challenge resulted in dose-dependent elevation in quantity of YHV-positive GC and cytoplasmic YHV protein, coincident with high shrimp mortality. Altogether, we demonstrated that PmMRLC phosphorylation increases after YHV infection in shrimp and that inhibition of the phosphorylation leads to increased YHV replication, reduced hemocyte phagocytic activity (probably through actin remodeling) and subsequent shrimp death. Thus, further studies on the MLCK activation pathway may lead to new strategies in development and implementation of therapy for YHV infections in shrimp.     

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl- cotransporter?

Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na⁺-K⁺-2Cl^– cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl^– depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl^– depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC. proline-alanine-rich STE20-related kinase     

Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors.     

Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts

We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.     

The catalytic domain of xPAK1 is sufficient to induce myosin II dependent in vivo cell fragmentation independently of other apoptotic events

During apoptosis, cells are fragmented into sealed packages for safe disposal by phagocytosis, a process requiring major reorganisation of the cytoskeleton. The small p21 GTPase-activated kinases (PAKs) have been implicated in regulating cytoskeletal dynamics and a subset are activated by caspase 3/7 cleavage. However, the functional importance of this activation in apoptosis remains unknown. Using early Xenopus embryos, we have dissected xPAK1 activation from other causative events in apoptosis. An apoptotic-like cell fragmentation was observed 30 min after expression of the xPAK1 catalytic domain and occurred in the absence of other markers of apoptosis. In vitro, activated xPAK1 phosphorylated the regulatory light chain (xMLC) of myosin II at threonine 18 and serine 19, events known to activate the actin-dependent ATPase of cytoskeletal myosin. In vivo, activated xPAK1 induced hyperphosphorylation of xMLC. BDM, a myosin inhibitor, and ML-7, a MLCK inhibitor, both abrogated cell fragmentation induced by activated xPAK1, and ML-7 also inhibited xPAK1 activity. Endogenous xPAK1 was cleaved during normal apoptosis and this was associated with xPAK1 activation and increased serine 19 phosphorylation of xMLC. The data show that PAK activation is sufficient for apoptotic body formation in vivo and strongly suggest that activation of myosin II is essential for this process.     

Targeting by myosin phosphatase-RhoA interacting protein mediates RhoA/ROCK regulation of myosin phosphatase

Vascular smooth muscle cell contractile state is the primary determinant of blood vessel tone. Vascular smooth muscle cell contractility is directly related to the phosphorylation of myosin light chains (MLCs), which in turn is tightly regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. Myosin phosphatase is the principal enzyme that dephosphorylates MLCs leading to relaxation. Myosin phosphatase is regulated by both vasoconstrictors that inhibit its activity to cause MLC phosphorylation and contraction, and vasodilators that activate its activity to cause MLC dephosphorylation and relaxation. The RhoA/ROCK pathway is activated by vasoconstrictors to inhibit myosin phosphatase activity. The mechanism by which RhoA and ROCK are localized to and interact with myosin light chain phosphatase (MLCP) is not well understood. We recently found a new member of the myosin phosphatase complex, myosin phosphatase-rho interacting protein, that directly binds to both RhoA and the myosin-binding subunit of myosin phosphatase in vitro, and targets myosin phosphatase to the actinomyosin contractile filament in smooth muscle cells. Because myosin phosphatase-rho interacting protein binds both RhoA and MLCP, we investigated whether myosin phosphatase-rho interacting protein was required for RhoA/ROCK-mediated myosin phosphatase regulation. Myosin phosphatase-rho interacting protein silencing prevented LPA-mediated myosin-binding subunit phosphorylation, and inhibition of myosin phosphatase activity. Myosin phosphatase-rho interacting protein did not regulate the activation of RhoA or ROCK in vascular smooth muscle cells. Silencing of M-RIP lead to loss of stress fiber-associated RhoA, suggesting that myosin phosphatase-rho interacting protein is a scaffold linking RhoA to regulate myosin phosphatase at the stress fiber.     

Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.     

A role for Rho kinase in vascular contraction evoked by sodium fluoride

Agonist and depolarization-induced vascular smooth muscle contractions involve the activation of Rho-kinase pathway. However, there are no reports addressing the question whether this pathway is involved in NaF-induced vascular contractions. We hypothesized that Rho-kinase plays a role in vascular contraction evoked by sodium fluoride in rat aortae. In both physiological salt solution and calcium-free solution with 2 mM EGTA, cumulative addition of NaF increased vascular tension in concentration-dependent manners. Effects of Rho-kinase inhibitor (Y27632) on phosphorylation of myosin light chain (MLC20) and myosin targeting subunit (MYPT1(Thr696)) of myosin light chain phosphatase as well as NaF-induced contractions were determined using isolated tissue and the Western blot experiments. Y27632 inhibited NaF-induced contractions in a concentration-dependent manner. NaF increased phosphorylation of MLC20 and MYPT1(Thr696), which were also inhibited by Y27632. However, MLCK inhibitor (ML-7) or PKC inhibitor (Ro31-8220) did not inhibit the NaF-induced contraction. These results indicate that activation of Rho-kinase and the subsequent phosphorylation of MYPT1(Thr696) play important roles in NaF-induced contraction of rat aortae.     

G(q)-dependent signalling by the lysophosphatidic acid receptor LPA(3) in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA(3) receptors, but not LPA(1) and LPA(2), receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the G(alphaq), but not the G(alphai), minigene. Initial contraction and MLC(20) (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-beta (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC(20) and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IkappaBalpha were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr(696) was masked by phosphorylation of MYPT1 at Ser(695) by PKA derived from IkappaB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-beta and MLCK and phosphorylation of MLC(20), and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC(20). Although Rho kinase was activated, phosphorylation of MYPT1 at Thr(696) by Rho kinase was masked by phosphorylation of MYPT1 at Ser(695) via cAMP-independent PKA derived from the NF-kappaB pathway.     

Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain

Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction [IUBMB Life 51 (2001) 337, for review]. The well-established mode for its regulation is to phosphorylate the 20 kDa myosin light chain (MLC 20) to activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also stimulates myosin ATPase activity without phosphorylating MLC 20 [Proc. Natl. Acad. Sci. USA 96 (1999) 6666]. We engineered an MLCK fragment containing the myosin-binding domain but devoid of a catalytic domain to explore how myosin is stimulated by this non-kinase pathway. The recombinant fragment thus obtained stimulated myosin ATPase activity by V(max)=5.53+/-0.63-fold with K(m)=4.22+/-0.58 microM (n=4). Similar stimulation figures were obtained by measuring the ATPase activity of HMM and S1. Binding of the fragment to both HMM and S1 was also verified, indicating that the fragment exerts stimulation through the myosin heads. Since S1 is in an active form regardless of the phosphorylated state of MLC 20, we conclude that the non-kinase stimulation is independent of the phosphorylating mode for activation of myosin.