Dictyostelium myosin light chain kinase. Purification and characterization

A Dictyostelium myosin light chain kinase has been purified approximately 15,000-fold to near homogeneity. The purified kinase is a single polypeptide of approximately 34 kDa that phosphorylates only the 18-kDa Dictyostelium myosin regulatory light chain and itself among substrates tested. The enzyme was purified largely by ammonium sulfate fractionation and hydrophobic (butyl) interaction chromatography. Analysis using polyclonal antibodies raised against the purified 34-kDa protein confirms that this protein is responsible for myosin light chain kinase activity. Protein microsequence of the 34-kDa protein reveals conserved protein kinase sequences. The purified Dictyostelium myosin light chain kinase exhibits a Km for Dictyostelium myosin of 4 microM and a Vmax of 8 nmol/min/mg. Unlike other characterized myosin light chain kinases, this enzyme is not regulated by calcium/calmodulin. Western blot analysis demonstrates that the purified kinase is not a proteolytic fragment that has lost calcium/calmodulin regulation. The Dictyostelium myosin light chain kinase activity is not directly regulated by cyclic nucleotides. However, this kinase undergoes an intramolecular autophosphorylation that activates the enzyme.     

Role of myosin light chain kinase and myosin light chain phosphatase in the resistance arterial myogenic response to intravascular pressure

The intrinsic ability of vascular smooth muscle cells (VSMCs) within arterial resistance vessels to respectively contract and relax in response to elevation and reduction of intravascular pressure is essential for appropriate blood flow autoregulation. This fundamental mechanism, referred to as the myogenic response, is dependent on apposite control of myosin regulatory light chain (LC₂₀) phosphorylation, a prerequisite for force generation, through the coordinated activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Here, we highlight the molecular basis of the smooth muscle contractile mechanism and review the regulatory pathways demonstrated to participate in the control of LC₂₀ phosphorylation in the myogenic response, with a focus on the Ca²⁺-dependent and Rho-associated kinase (ROK)-mediated regulation of MLCK and MLCP, respectively.     

Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum

A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.     

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

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

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

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

Isolation and properties of platelet myosin light chain kinase.

A protein kinase which phosphorylates the 20 000-dalton light chain of platelet myosin has been isolated from human blood platelets and purified approximately 600-fold. Elution of a 7.5% polyacrylamide gel following electrophoresis of the partially purified enzyme yielded a single peak of kinase activity which could be aligned with a protein band on a stained gel. Assuming a globular shape, a native molecular weight of 83 000 (+/- 10%) was determined by gel filtration on Bio-Gel P-200. The kinase requires Mg2+ for activity and is not sensitive to the removal of trace Ca2+. The enzyme purified from human platelets phosphorylates the 20 000-dalton light chain of mouse fibroblast and chicken gizzard myosin, but does not phosphorylate human skeletal and cardiac myosin.     

Purification and characterization of a phosphoprotein phosphatase that dephosphorylates myosin and the isolated light chain from chicken gizzard smooth muscle

A phosphatase that dephosphorylates myosin and the isolated light chain has been purified to near homogeneity from chicken gizzard smooth muscle. The molecular weight of the enzyme was estimated to be 100,000 and 35,000 under native and denatured conditions, respectively. It requires Mg2+ or Mn2+. The activity was measured quantitatively with a coupled enzyme system with the aid of myosin light chain kinase. The Vm and Km were determined to be 23.4 mumol/mg/min and 4.2 microM, respectively, with the isolated light chain as substrate under the optimal conditions (5 mM Mg2+ at pH 8.45). The specific activity with myosin as substrate at a concentration of 0.9 microM was found to be 1.25 mumol/mg/min, which was about one-fifth of the activity for the isolated light chain under the same conditions. The phosphatase seems to be specific to gizzard myosin. It may play an important role in the regulation of the myosin-actin interaction in smooth muscle.     

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.     

Macrophage myosin. Regulation of actin-activated ATPase, activity by phosphorylation of the 20,000-dalton light chain.

Myosin was purified from rabbit alveolar macrophages in a form that could not be activated by actin. This myosin could be phosphorylated by an endogenous myosin light chain kinase, up to 2 mol of phosphate being incorporated/mol of myosin. The site phosphorylated was located on the 20,000-dalton myosin light chain. Phosphorylation of macrophage myosin was found to be necessary for actin activation of myosin ATPase activity. Moreover, the actin-activated ATPase activity was found to vary directly with the extent of myosin phosphorylation, maximal phosphorylation (2 mol of Pi/mol of myosin) resulting in an actin-activated MgATPase activity of approximately 200 nmol of Pi/mg of myosin/min at 37 degrees C. These results establish that phosphyoyration of the 20,000-dalton light chain of myosin is sufficient to regulate the actin-activated ATPase activity of macrophage myosin.     

Modulation of myosin filament activation by telokin in smooth muscle liberation of myosin kinase and phosphatase from supramolecular complexes

The mechanism of telokin action on reversible phosphorylation of turkey gizzard myosin was investigated using a native-like filamentous myosin. This myosin contained endogenous calmodulin (CaM) and myosin light chain kinase (MLCK) at a molar ratio to myosin of about 1 to 40 or less depending on the initial extractions conditions. These levels were sufficient to fully phosphorylate myosin within 20-40 s or less after addition of [gamma-32P]ATP, but when the ATP was depleted, they became dephosphorylated indicating the presence of myosin light chain phosphatase (MLCP). Addition of telokin at the 1 to 1 or higher molar ratio to myosin caused a three- to five-fold inhibition of the initial phosphorylation rates (without reduction of the overall extent of phosphorylation) and produced a similar increase in the rate of dephosphorylation. The inhibition was also observed for myosin filaments free of MLCK and CaM together with constitutively active MLCKs produced by digestion, or by expression of a truncated mammalian kinase as well as for the wild-type enzyme. Thus, neither N- nor C-terminal of MLCK was necessary for interaction of myosin with telokin and the inhibition resulted from telokin-induced change of myosin head configuration within the filament that prevented their ordered, paracrystaline-like, aggregation. Sedimentation of the filamentous myosin in glycerol gradients showed that this change made the filaments less compact and facilitated release of the endogenous MLCK/CaM complex. For a mixture of the filaments with or without the complex, the configuration change resulted in an increase of the phosphorylation rate but not in its inhibition. The increase of the rate resulting from the liberation of the complex was also observed in mixtures of the filamentous myosin with added isolated regulatory light chain (ReLC) or soluble myosin head subfragment. This observation reinforces the above conclusions. The acceleration of the MLCP activity by telokin was shown to result from dissociation of its catalytic subunit from a MLCK/MLCP complex bound to the filamentous myosin. Analogous desensitizing effects of telokin were also demonstrated for the contraction and relaxation cycle of Triton-skinned fibers from guinea pig Teania coli. Taken together, our results indicate that telokin acted as an effective modulator or chaperone of the myosin filament and a scheme for its action in smooth muscle was proposed.     

Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age

We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cdelta) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cdelta contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.     

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.     

Phosphorylation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase regulates myosin light chain kinase

Smooth muscle myosin light chain kinase, purified to homogeneity, has a molecular weight of 130,000 +/- 5,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme has a specific activity under maximal conditions of 30 mumol Pi transferred to myosin light chain/mg kinase/min at 24 C and is totally dependent on calmodulin and calcium for activity. Incubation of myosin kinase with the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase results in the covalent incorporation of up to one mol of phosphate per mol of myosin kinase in the absence of bound calmodulin. Limited tryptic digestion of the radioactively labeled kinase indicates that all of the label has been incorporated into a single tryptic peptide (mol wt approximately 22,000), suggesting that a single site is being phosphorylated. Phosphorylation of myosin kinase lowers the rate at which the kinase phosphorylates myosin light chain. The lower rate of light chain phosphorylation is due to a weaker binding of calmodulin to the phosphorylated kinase than to the unphosphorylated kinase. Cyclic adenosine 3':5'-monophosphate-dependent phosphorylation of the kinase actin-myosin interaction represents a possible link between hormonal binding to smooth muscle receptors and muscle relaxation. A scheme for this sequence of events is presented.     

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.     

Purification of a novel Ca-binding protein that inhibits myosin light chain kinase activity in lower eukaryote Physarum polycephalum

Myosin light chain kinase (MLCK) was partially purified from the lower eukaryote Physarum polycephalum. The activity to phosphorylate Physarum myosin was maximal in the absence of Ca2+ and decreased with an increase in Ca2+ concentration with a microM-level Kd. The Ca-binding protein contained in the MLCK preparation was purified to homogeneity. The native protein had a molecular mass of 75 kDa, while under denaturing conditions, it was 38 kDa. Ca-dependent changes in the intensities of intrinsic fluorescence showed that the Kd of the protein for Ca2+ was also in the microM-range. Our results suggest that the Ca-binding protein would play a key role in the effects of Ca2+ in the MLCK preparation.     

Role of the N-terminal region of the regulatory light chain in the dephosphorylation of myosin by myosin light chain phosphatase.

Myosin regulatory light chain (RLC) is phosphorylated at various sites at its N-terminal region, and heterotrimeric myosin light chain phosphatase (MLCP) has been assigned as a physiological phosphatase that dephosphorylates myosin in vivo. Specificity of MLCP toward the various phosphorylation sites of RLC was studied, as well as the role of the N-terminal region of RLC in the dephosphorylation of myosin by MLCP. MLCP dephosphorylated phosphoserine 19, phosphothreonine 18, and phosphothreonine 9 efficiently with almost identical rates, whereas it failed to dephosphorylate phosphorylated serine 1/serine 2. Deletion of the N-terminal seven amino acid residues of RLC markedly decreased the dephosphorylation rate of phosphoserine 19 of RLC incorporated in the myosin molecule, whereas this deletion did not significantly affect the dephosphorylation rate of isolated RLC. On the other hand, deletion of only four N-terminal amino acid residues showed no effect on dephosphorylation of phosphoserine 19 of incorporated RLC. The inhibition of dephosphorylation by deletion of the seven N-terminal residues was also found with the catalytic subunit of MLCP. Phosphorylation at serine 1/serine 2 and threonine 9 did not influence the dephosphorylation rate of serine 19 and threonine 18 by MLCP. These results suggest that the N-terminal region of RLC plays an important role in substrate recognition of MLCP.     

A signal transduction pathway model prototype II: Application to Ca2+-calmodulin signaling and myosin light chain phosphorylation

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.     

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.     

Heterogeneity of smooth muscle myosin light chain kinase is revealed by anion-exchange high-performance liquid chromatography

When smooth muscle myosin light chain kinase, purified by standard procedures from chicken gizzard smooth muscle, was applied to an anion-exchange high-performance liquid chromatographic column, three well resolved peaks were obtained. Each peak contained a single protein whose electrophoretic mobility corresponded to that of MLCK. However each enzyme was characterized by a different specific activity. Peptide mapping experiments were unable to demonstrate different proteolytic patterns for the three proteins. Treatment of myosin light chain kinase with alkaline phosphatase, prior to ion chromatography, resulted in a change of elution profile. These experiments suggest that myosin light chain kinase could exist in three forms characterized by a different degree of phosphorylation.     

p116Rip decreases myosin II phosphorylation by activating myosin light chain phosphatase and by inactivating RhoA

p116Rip was originally found to be a RhoA-binding protein, but its function has been unknown. Here, we clarify the function of p116Rip. Two critical findings were made. First, we found that p116Rip activated the GTPase activity of RhoA in vitro and that p116Rip overexpression in cells consistently diminished the epidermal growth factor-induced increase in GTP-bound RhoA. Second, p116Rip activated the myosin light chain phosphatase (MLCP) activity of the holoenzyme. p116Rip did not activate the catalytic subunit alone, indicating that the activation is due to the binding of p116Rip to the myosin phosphatase targeting subunit MYPT1. Interestingly, the activation of phosphatase was specific to myosin as substrate, and p116Rip directly bound to myosin, thus facilitating myosin/MLCP interaction. The gene silencing of p116Rip consistently and significantly increased myosin phosphorylation as well as stress fiber formation in cells. Based upon these findings, we propose that p116Rip is an important regulatory component that controls the RhoA signaling pathway, thus regulating MLCP activity and myosin phosphorylation in cells.