scholarly journals Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway

2003 ◽  
Vol 374 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Karnam S. MURTHY ◽  
Huiping ZHOU ◽  
John R. GRIDER ◽  
David L. BRAUTIGAN ◽  
Masumi ETO ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Myosin Phosphatase ◽  
Kinase C ◽  
C3 Exoenzyme ◽  
Mlc20 Phosphorylation ◽  
P21 Activated Kinase ◽  
In Cells

Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gβγi3 with activation of phospholipase C-β3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Gαq/11 with activation of phospholipase C-β1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44±5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28±3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities, it could not be used to ascertain the contribution of MYPT1 to inhibition of MLCP activity. m2-dependent phosphorylation and inactivation of MLCK precluded its involvement in sustained MLC20 phosphorylation and contraction.

scholarly journals A Novel Regulatory Mechanism of Myosin Light Chain Phosphorylation via Binding of 14-3-3 to Myosin Phosphatase

2008 ◽  
Vol 19 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
Yasuhiko Koga ◽  
Mitsuo Ikebe
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Regulatory Mechanism ◽  
Kinase Inhibitor ◽  
Myosin Ii ◽  
Critical Role ◽  
Rho Kinase ◽  
Myosin Phosphatase ◽  
Dependent Cell ◽  
Direct Binding

Myosin II phosphorylation–dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3β to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3β overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3β inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3β overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase–dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity.

scholarly journals Increased Phosphorylation of Myosin Light Chain Prevents in Vitro Decidualization

Endocrinology ◽  
2007 ◽  
Vol 148 (7) ◽  
pp. 3176-3184 ◽  
Author(s):  
Ivanna Ihnatovych ◽  
WenYang Hu ◽  
Jody L. Martin ◽  
Asgerally T. Fazleabas ◽  
Primal de Lanerolle ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscle Actin ◽  
Myosin Ii ◽  
Active Form ◽  
Adenoviral Infection ◽  
Decidual Cells ◽  
Cytoskeletal Dynamics ◽  
Mlc20 Phosphorylation

Differentiation of stromal cells into decidual cells, which is critical to successful pregnancy, represents a complex transformation requiring changes in cytoskeletal architecture. We demonstrate that in vitro differentiation of human uterine fibroblasts into decidual cells includes down-regulation of α-smooth muscle actin and β-tubulin, phosphorylation of focal adhesion kinase, and redistribution of vinculin. This is accompanied by varied adhesion to fibronectin and a modified ability to migrate. Cytoskeletal organization is determined primarily by actin-myosin II interactions governed by the phosphorylation of myosin light chain (MLC20). Decidualization induced by cAMP [with estradiol-17β (E) and medroxyprogesterone acetate (P)] results in a 40% decrease in MLC20 phosphorylation and a 55% decline in the long (214 kDa) form of myosin light-chain kinase (MLCK). Destabilization of the cytoskeleton by inhibitors of MLCK (ML-7) or myosin II ATPase (blebbistatin) accelerates decidualization induced by cAMP (with E and P) but inhibits decidualization induced by IL-1β (with E and P). Adenoviral infection of human uterine fibroblast cells with a constitutively active form of MLCK followed by decidualization stimuli leads to a 30% increase in MLC20 phosphorylation and prevents decidualization. These data provide evidence that the regulation of cytoskeletal dynamics by MLC20 phosphorylation is critical for decidualization.

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

2006 ◽  
Vol 396 (1) ◽  
pp. 193-200 ◽  
Author(s):  
Jiean Huang ◽  
Sunila Mahavadi ◽  
Wimolpak Sriwai ◽  
Wenhui Hu ◽  
Karnam S. Murthy
Keyword(s):  
Protein Kinase ◽  
Muscle Contraction ◽  
P38 Mapk ◽  
Light Chain ◽  
Reciprocal Inhibition ◽  
Smooth Muscle Contraction ◽  
Mitogen Activated Protein Kinase ◽  
Mlc20 Phosphorylation ◽  
P21 Activated Kinase ◽  
In Cells

Sustained smooth-muscle contraction or its experimental counterpart, Ca2+ sensitization, by Gq/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 Gi-coupled receptors. Somatostatin acting via Gi1-coupled sstr3 receptor, DPDPE ([D-Pen2,D-Pen5]enkephalin; where Pen is penicillamine) acting via Gi2-coupled δ-opioid receptors, and cyclopentyl adenosine acting via Gi3-coupled adenosine A1 receptors preferentially activated PI3K (phosphoinositide 3-kinase) and ILK (integrin-linked kinase), whereas ACh (acetylcholine) acting via Gi3-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 Gi-coupled receptors is dependent on CPI-17 and MLC20 phosphorylation by ILK.

α1-Adrenoceptor-Gq-RhoA signaling is upregulated to increase myofibrillar Ca2+ sensitivity in failing hearts

2001 ◽  
Vol 281 (2) ◽  
pp. H637-H646 ◽  
Author(s):  
Nobuhiro Suematsu ◽  
Shinji Satoh ◽  
Shintaro Kinugawa ◽  
Hiroyuki Tsutsui ◽  
Shunji Hayashidani ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Expression ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Adrenergic Stimulation ◽  
Regulatory Myosin Light Chain ◽  
Kinase C

α1-Adrenergic stimulation, coupled to Gq, has been shown to promote heart failure. However, the role of α1-adrenergic signaling in the regulation of myocardial contractility in failing myocardium is still poorly understood. To investigate this, we observed 1) the effect of phenylephrine on myofibrillar Ca2+ sensitivity in α-toxin-skinned cardiomyocytes, and 2) protein expression of Gq, RhoA, and myosin light chain phosphorylation using tachypacing-induced canine failing hearts. Phenylephrine significantly increased myofibrillar Ca2+ sensitivity in failing but not in normal cardiomyocytes. Whereas Y-27632 (Rho kinase inhibitor) blocked the phenylephrine-induced Ca2+ sensitization in the failing myocytes, calphostin C (protein kinase C inhibitor) had no effect on Ca2+ sensitization. The protein expression of Gαq and RhoA and the phosphorylation level of regulatory myosin light chain significantly increased in the failing myocardium. Our results suggest that α1-adrenoceptor-Gq signaling is upregulated in the failing myocardium to increase the myofibrillar Ca2+sensitivity mainly through the RhoA-Rho kinase pathway rather than through the protein kinase C pathway.

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

1991 ◽  
Vol 114 (2) ◽  
pp. 277-283 ◽  
Author(s):  
A K Wilson ◽  
G Gorgas ◽  
W D Claypool ◽  
P de Lanerolle
Keyword(s):  
Cell Motility ◽  
Light Chain ◽  
Mammalian Cells ◽  
Inverse Relationship ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Active Form ◽  
Chemotaxis Assay ◽  
Mlc20 Phosphorylation ◽  
Number Of Cells

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.

scholarly journals Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II

2010 ◽  
Vol 5 (1) ◽  
pp. 16 ◽  
Author(s):  
Ailish Murray ◽  
Arifa Naeem ◽  
Sarah H Barnes ◽  
Uwe Drescher ◽  
Sarah Guthrie
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Motor Axon ◽  
Axon Pathfinding

scholarly journals Gq-dependent signalling by the lysophosphatidic acid receptor LPA3 in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

2008 ◽  
Vol 411 (3) ◽  
pp. 543-551 ◽  
Author(s):  
Wimolpak Sriwai ◽  
Huiping Zhou ◽  
Karnam S. Murthy
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Lysophosphatidic Acid ◽  
Light Chain ◽  
Rho Kinase ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Sustained Contraction ◽  
C3 Exoenzyme ◽  
In Cells

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA3 receptors, but not LPA1 and LPA2, 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 Gαq, but not the Gαi, minigene. Initial contraction and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-β (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 MLC20 and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IκBα 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 Thr696 was masked by phosphorylation of MYPT1 at Ser695 by PKA derived from IκB 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-β and MLCK and phosphorylation of MLC20, and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC20. Although Rho kinase was activated, phosphorylation of MYPT1 at Thr696 by Rho kinase was masked by phosphorylation of MYPT1 at Ser695 via cAMP-independent PKA derived from the NF-κB pathway.

Protein kinase C–catalyzed phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet secretion

Blood ◽  
2001 ◽  
Vol 97 (12) ◽  
pp. 3798-3805 ◽  
Author(s):  
Yasuyuki Watanabe ◽  
Masaaki Ito ◽  
Yoshiyuki Kataoka ◽  
Hideo Wada ◽  
Mutsumi Koyama ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Myosin Phosphatase ◽  
Kinase C ◽  
Atp Secretion ◽  
Mlc20 Phosphorylation ◽  
Platelet Secretion

Protein kinase C (PKC)–potentiated inhibitory phosphoprotein of myosin phosphatase (CPI) was detected in human platelets. Like smooth muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC inhibited the activity of myosin phosphatase containing the PP1δ catalytic subunit and the 130-kd myosin-binding subunit (MBS). Treatment of intact platelets with thrombin or the stable thromboxane A2 analog STA2 resulted in increased phosphorylation of both CPI and MBS at Thr-696, whereas phorbol myristate acetate (PMA) and the Ca++ ionophore ionomycin only induced CPI phosphorylation. PMA induced slow adenosine triphosphate (ATP) secretion of fura 2–loaded platelets with no change in cytosolic Ca++. The PMA-induced increase in CPI phosphorylation preceded phosphorylation of 20-kd myosin light chain (MLC20) at Ser-19 and ATP secretion. The PKC inhibitor, GF109203X, inhibited PMA-induced phosphorylation of CPI and MLC20 with similar IC50 values. These findings suggest that the activation of PKC by PMA induces MLC20phosphorylation by inhibiting myosin phosphatase through phosphorylation of CPI. STA2-induced MLC20phosphorylation was also diminished but not abolished by GF109203X, even at high concentrations that completely inhibited STA2-induced CPI phosphorylation. A combination of the Rho-kinase inhibitor Y-27632 and GF109203X led to a further decrease in STA2-induced MLC20 phosphorylation, mainly because of a significant inhibition of MBS phosphorylation at Thr-696. Inhibition of STA2-induced ATP release by Y-27632, GF109203X, or both appeared to correlate with the extent of MLC20 phosphorylation. Thus, CPI phosphorylation by PKC may participate in inhibiting myosin phosphatase, in addition to the Rho-kinase–mediated regulation of myosin phosphatase, during agonist-induced platelet secretion.

scholarly journals Agonist-induced changes in the phosphorylation of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle

2003 ◽  
Vol 369 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Naohisa NIIRO ◽  
Yasuhiko KOGA ◽  
Mitsuo IKEBE
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphatase ◽  
Kinase C ◽  
Agonist Stimulation ◽  
Myosin Binding ◽  
Mlc Phosphorylation

The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI17, thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both α-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203x, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction.

scholarly journals A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

2006 ◽  
Vol 17 (9) ◽  
pp. 4093-4104 ◽  
Author(s):  
Amy Lucero ◽  
Christianna Stack ◽  
Anne R. Bresnick ◽  
Charles B. Shuster
Keyword(s):  
Sea Urchin ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Specific Cell ◽  
Spatial Cues ◽  
Sea Urchin Eggs ◽  
Cortical Contractility

Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase–anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase–anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus.

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