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.     

Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions.We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.     

Theoretical and experimental investigation of calcium-contraction coupling in airway smooth muscle

We investigated theoretically and experimentally the Ca²⁺-contraction coupling in rat tracheal smooth muscle. [Ca²⁺]_i, isometric contraction and myosin light chain (MLC) phosphorylation were measured in response to 1 mM carbachol. Theoretical modeling consisted in coupling a model of Ca²⁺-dependent MLC kinase (MLCK) activation with a four-state model of smooth muscle contractile apparatus. Stimulation resulted in a short-time contraction obtained within 1 min, followed by a long-time contraction up to the maximal force obtained in 30 min. ML-7 and Wortmannin (MLCK inhibitors) abolished the contraction. Chelerythrine (PKC inhibitor) did not change the short-time, but reduced the long-time contraction. [Ca²⁺ _i responses of isolated myocytes recorded during the first 90 s consisted in a fast peak, followed by a plateau phase and, in 28% of the cells, superimposed Ca²⁺ oscillations. MLC phosphorylation was maximal at 5 s and then decreased whereas isometric contraction followed a Hill-shaped curve. The model properly predicts the time course of MLC phosphorylation and force of the short-time response. With oscillating Ca²⁺ signal, the predicted force does not oscillate. According to the model, the amplitude of the plateau and the frequency of oscillations encode for the amplitude of force, whereas the peak encodes for force velocity. The long-time phase of the contraction, associated with a second increase in MLC phosphorylation, may be explained, at least partially, by MLC phosphatase (MLCP) inhibition, possibly via PKC inhibition.     

The defective protein level of myosin light chain phosphatase (MLCP) in the isolated saphenous vein, as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM)

We examined the contractile reactivity to 5-hydroxytryptamine (5-HT) in isolated human saphenous vein (SV), as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM) and non-DM (NDM). Vascular rings of endothelium-denuded SV were used for functional and biochemical experiments. The vasoconstrictions caused by 5-HT were significantly greater (hyperreactivity) in the DM group than in the NDM group. RhoA/ROCK pathway is activated by various G-protein-coupled receptor agonists and consequently induces phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin light chain phosphatase (MLCP), which inhibits MLCP activity. In the resting state of the vessels, total tissue protein levels of 5-HT_2A receptor, 5-HT_1B receptor, RhoA, ROCK1, and ROCK2 did not differ between NDM and DM groups. However, the total protein level of MYPT1 was significantly lower in the DM group than in the NDM group. Furthermore, the ratio of P(Thr⁶⁹⁶)-MYPT1 to total MYPT1 was significantly higher in the DM group than in the NDM group. These results suggest that the hyperreactivity to 5-HT in the SV smooth muscle of patients with DM is due to not only enhanced phosphorylation of MLCP but also defective protein level of MLCP. Thus, we reveal for the first time that the defective protein level of MLCP in the DM group can partially explain the poor patency of SV graft harvested from patients with DM.     

Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells

Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mDia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mDia1 by smart-pool double-stranded short-interfering RNAs (siRNA) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mDia1 siRNAs. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mDia1, and FAK reduction by siRNA blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mDia1 reduction by siRNA also blocked strain-induced FAK-Tyr(925), p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mDia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation.     

Heat shock augments myosin phosphatase target-subunit phosphorylation

Our previous study demonstrated that heat shock augmented vascular contraction. In the present study, we hypothesized that heat shock augments myosin phosphatase target-subunit (MYPT1) phosphorylation resulting in augmented vascular contraction. Endothelium-denuded rat aortic rings were mounted in organ baths, exposed to heat shock (42 degrees C for 45 min), and subjected to contraction 4 h after the heat shock followed by Western blot analysis for MLC(20) (the 20 kDa light chains of myosin II) or MYPT1. The contractile responses in both control and heat shock-treated aorta were inhibited by Y27632, an inhibitor of Rho-kinase. The level of the MLC(20) and MYPT1(Thr855) phosphorylation in response to KCl was higher in heat shock-treated aorta than that in timed-control. The increased MYPT1(Thr855) phosphorylation was inhibited by Y27632 (1.0 microM) in parallel with inhibition of MLC(20) phosphorylation and vascular contraction. These results indicate that heat shock augments MYPT1 phosphorylation resulting in augmented vascular contraction.     

Heart-specific Small Subunit of Myosin Light Chain Phosphatase Activates Rho-associated Kinase and Regulates Phosphorylation of Myosin Phosphatase Target Subunit 1

Phosphorylation of myosin regulatory light chain (MLC) plays a regulatory role in muscle contraction, and the level of MLC phosphorylation is balanced by MLC kinase and MLC phosphatase (MLCP). MLCP consists of a catalytic subunit, a large subunit (MYPT1 or MYPT2), and a small subunit. MLCP activity is regulated by phosphorylation of MYPTs, whereas the role of small subunit in the regulation remains unknown. We previously characterized a human heart-specific small subunit (hHS-M₂₁) that increased the sensitivity to Ca²⁺ in muscle contraction. In this study, we investigated the role of hHS-M₂₁ in the regulation of MLCP phosphorylation. Two isoforms of hHS-M₂₁, hHS-M₂₁A and hHS-M₂₁B, preferentially bound the C-terminal one-third region of MYPT1 and MYPT2, respectively. Amino acid substitutions at a phosphorylation site of MYPT1, Ser-852, impaired the binding of MYPT1 and hHS-M₂₁. The hHS-M₂₁ increased the phosphorylation level of MYPT1 at Thr-696, which was attenuated by Rho-associated kinase (ROCK) inhibitors and small interfering RNAs for ROCK. In addition, hHS-M₂₁ bound ROCK and enhanced the ROCK activity. These findings suggest that hHS-M₂₁ is a heart-specific effector of ROCK and plays a regulatory role in the MYPT1 phosphorylation at Thr-696 by ROCK.     

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.     

Direct Binding and Regulation of RhoA Protein by Cyclic GMP-dependent Protein Kinase Iα

Vascular smooth muscle cell (VSMC) tone is regulated by the state of myosin light chain (MLC) phosphorylation, which is in turn regulated by the balance between MLC kinase and MLC phosphatase (MLCP) activities. RhoA activates Rho kinase, which phosphorylates the regulatory subunit of MLC phosphatase, thereby inhibiting MLC phosphatase activity and increasing contraction and vascular tone. Nitric oxide is an important mediator of VSMC relaxation and vasodilation, which acts by increasing cyclic GMP (cGMP) levels in VSMC, thereby activating cGMP-dependent protein kinase Iα (PKGIα). PKGI is known to phosphorylate Rho kinase, preventing Rho-mediated inhibition of MLC phosphatase, promoting vasorelaxation, although the molecular mechanisms that mediate this are unclear. Here we identify RhoA as a target of activated PKGIα and show further that PKGIα binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGIα was demonstrated via a direct interaction between the amino terminus of RhoA (residues 1–44), containing the switch I domain of RhoA, and the amino terminus of PKGIα (residues 1–59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGIα binds RhoA, and a leucine zipper mutant PKGIα was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGIα bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGIα target and that direct binding of activated PKGIα to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway.     

Rho kinase signaling pathways during stretch in primary alveolar epithelia

Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.     

RhoA- and PKC-alpha-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain (MLC20) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-alpha. Presently, we examined the cross talk between RhoA and PKC-alpha in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-alpha, CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21+/-3.52 and 42.19+/-3.85% nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12+/-46.60% and 290.59+/-22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-alpha by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-alpha pathways, suggesting cross talk between RhoA and PKC-alpha resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of MLC20.     

Myosin‐II negatively regulates minor process extension and the temporal development of neuronal polarity

The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated “minor processes” (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live‐cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA‐kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell‐permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells

Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA-ROCK-myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.     

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

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

Cytokinesis and cancer： Polo loves ROCK＇n＇ Rho（A）

Cytokinesis is the last step of the M (mitosis) phase, yet it is crucial for the faithful division of one cell into two. Cytokinesis failure is often associated with cancer. Cytokinesis can be morphologically divided into four steps: cleavage furrow initiation, cleavage furrow ingression, midbody formation and abscission. Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis. At the same time, Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis, including cytokinesis. Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks. More specifically, Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1, thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains. Ect2 itself can be phosphorylated by Plk1 in vitro. Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity. Once activated, RhoA-GTP will activate downstream effectors, including ROCK1 and ROCK2. ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen, and Plk1 can phosphorylate ROCK2 in vitro. We review current understandings of the interplay between Plk1, RhoA proteins and other proteins (e.g., NudC, MKLP2, PRC1, CEP55) involved in cytokinesis, with particular emphasis of its clinical implications in cancer.     

Organized migration of epithelial cells requires control of adhesion and protrusion through Rho kinase effectors

Migration of epithelial cell sheets, a process involving F-actin restructuring through Rho family GTPases, is both physiologically and pathophysiologically important. Our objective was to clarify the mechanisms whereby the downstream RhoA effector Rho-associated coil-coil-forming kinase (ROCK) influences coordinated epithelial cell motility. Although cells exposed to a pharmacological ROCK inhibitor (Y-27632) exhibited increased spreading in wound closure assays, they failed to migrate in a cohesive manner. Two main phenomena were implicated: the formation of aberrant protrusions at the migrating front and the basal accumulation of F-actin aggregates. Aggregates reflected increased membrane affiliation and detergent insolubility of the actin-binding protein ezrin and enhanced coassociation of ezrin with the membrane protein CD44. While F-actin aggregation following ROCK inhibition was recapitulated by inhibiting myosin light chain (MLC) phosphorylation with the MLC kinase inhibitor ML-7, the latter did not influence protrusiveness and, in fact, significantly decreased cell migration. Our results suggest that excessive protrusiveness downstream of ROCK inhibition reflects an influence of ROCK on F-actin stability via LIM kinase 1 (LIMK-1), which phosphorylates and inactivates cofilin. Y-27632 reduced the levels of both active LIMK-1 and inactive cofilin (phospho forms), and expression of a dominant negative LIMK-1 mutant stimulated leading edge protrusiveness. Furthermore, Y-27632-induced protrusions were partially reversed by overexpression of LIMK-1 to restore cofilin phosphorylation. In summary, our results provide new evidence suggesting that adhesive and protrusive events involved in organized epithelial motility downstream of ROCK are separately coordinated through the phosphorylation of (respectively) MLC and cofilin.     

Is MLC phosphorylation essential for the recovery from ROCK inhibition in glioma C6 cells?

Inhibition of Rho-associated protein kinase (ROCK) activity in glioma C6 cells induces changes in actin cytoskeleton organization and cell morphology similar to those observed in other types of cells with inhibited RhoA/ROCK signaling pathway. We show that phosphorylation of myosin light chains (MLC) induced by P2Y? receptor stimulation in cells with blocked ROCK correlates in time with actin cytoskeleton reorganization, F-actin redistribution and stress fibers assembly followed by recovery of normal cell morphology. Presented results indicate that myosin light-chain kinase (MLCK) is responsible for the observed phosphorylation of MLC. We also found that the changes induced by P2Y? stimulation in actin cytoskeleton dynamics and morphology of cells with inhibited ROCK, but not in the level of phosphorylated MLC, depend on the presence of calcium in the cell environment.     

Neuroendocrine Signaling Via the Serotonin Transporter Regulates Clearance of Apoptotic Cells

Serotonin (5-hydroxytryptamine; 5-HT) is a CNS neurotransmitter increasingly recognized to exert immunomodulatory effects outside the CNS that contribute to the pathogenesis of autoimmune and chronic inflammatory diseases. 5-HT signals to activate the RhoA/Rho kinase (ROCK) pathway, a pathway known for its ability to regulate phagocytosis. The clearance of apoptotic cells (i.e. efferocytosis) is a key modulator of the immune response that is inhibited by the RhoA/ROCK pathway. Because efferocytosis is defective in many of the same illnesses where 5-HT has been implicated in disease pathogenesis, we hypothesized that 5-HT would suppress efferocytosis via activation of RhoA/ROCK. The effect of 5-HT on efferocytosis was examined in murine peritoneal and human alveolar macrophages, and its mechanisms were investigated using pharmacologic blockade and genetic deletion. 5-HT impaired efferocytosis by murine peritoneal macrophages and human alveolar macrophages. 5-HT increased phosphorylation of myosin phosphatase subunit 1 (Mypt-1), a known ROCK target, and inhibitors of RhoA and ROCK reversed the suppressive effect of 5-HT on efferocytosis. Peritoneal macrophages expressed the 5-HT transporter and 5-HT receptors (R) 2a, 2b, but not 2c. Inhibition of 5-HTR2a and 5-HTR2b had no effect on efferocytosis, but blockade of the 5-HT transporter prevented 5-HT-impaired efferocytosis. Genetic deletion of the 5-HT transporter inhibited 5-HT uptake into peritoneal macrophages, prevented 5-HT-induced phosphorylation of Mypt-1, reversed the inhibitory effect of 5-HT on efferocytosis, and decreased cellular peritoneal inflammation. These results suggest a novel mechanism by which 5-HT might disrupt efferocytosis and contribute to the pathogenesis of autoimmune and chronic inflammatory diseases.     

Activation of mTOR and RhoA is a major mechanism by which ceramide 1-phosphate stimulates macrophage proliferation

This study tested the hypothesis that Ceramide 1-phosphate (C1P) stimulates macrophage proliferation through activation of the mammalian target of rapamycin (mTOR). We first reported that C1P is mitogenic for fibroblasts and macrophages, but the mechanisms whereby it stimulates cell proliferation are incompletely understood. Here we demonstrate that C1P causes phosphorylation of mTOR in primary (bone marrow-derived) macrophages. Activation of this kinase was tested my measuring the phosphorylation state of its downstream target p70S6K after treatment with C1P. These actions were dependent upon prior activation of phosphoinositide 3 kinase (PI3-K), as selective inhibition of this kinase blocked mTOR phosphorylation and activation. In addition, C1P caused phosphorylation of PRAS40, a component of the mTOR complex 1 (mTORC1) that is absent in mTORC2. Furthermore, inhibition of the small G protein Ras homolog enriched in brain (Rheb), which is also a specific component of mTORC1, with FTI277, completely blocked C1P-stimulated mTOR phosphorylation, DNA synthesis and macrophage growth. In addition, C1P caused phosphorylation of another Ras homolog gene family member, RhoA, which is also involved in cell proliferation. Interestingly, inhibition of the RhoA downstream effector RhoA-associated kinase (ROCK) also blocked C1P-stimulated mTOR and cell proliferation. It can be concluded that mTORC1, and RhoA/ROCK are essential components of the mechanism whereby C1P stimulates macrophage proliferation.     

Cytokinesis and cancer:Polo loves ROCK'n' Rho(A)

Cytokinesis is the last step of the M (mitosis) phase,yet it is crucial for the faithful division of one cell into two.Cytokinesis failure is often associated with cancer.Cytokinesis can be morphologically divided into four steps:cleavage furrow initiation,cleavage furrow ingression,midbody formation and abscission.Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis.At the same time,Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis,including cytokinesis.Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks.More specifically,Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1,thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains.Ect2 itself can be phosphorylated by Plk1 in vitro.Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity.Once activated,RhoA-GTP will activate downstream effectors,including ROCK1 and ROCK2.ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen,and Plk1 can phosphorylate ROCK2 in vitro.We review current understandings of the interplay between Plk1,RhoA proteins and other proteins (e.g.,NudC,MKLP2,PRC1,CEP55) involved in cytokinesis,with partitular emphasis of its clinical implications in cancer.