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.

HSP27 in signal transduction and association with contractile proteins in smooth muscle cells

1999 ◽  
Vol 277 (2) ◽  
pp. G445-G454 ◽  
Author(s):  
Adenike I. Ibitayo ◽  
Jeanette Sladick ◽  
Sony Tuteja ◽  
Otto Louis-Jacques ◽  
Hirotaka Yamada ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Muscle Contraction ◽  
Smooth Muscle Contraction ◽  
Contractile Proteins ◽  
Mitogen Activated Protein Kinase ◽  
Signal Transduction Cascade ◽  
Kinase C ◽  
Mitogen Activated Protein

Sustained smooth muscle contraction is mediated by protein kinase C (PKC) through a signal transduction cascade leading to contraction. Heat-shock protein 27 (HSP27) appears to be the link between these two major events, i.e., signal transduction and sustained smooth muscle contraction. We have investigated the involvement of HSP27 in signal transduction and HSP27 association with contractile proteins (e.g., actin, myosin, tropomyosin, and caldesmon) resulting in sustained smooth muscle contraction. We have carried out confocal microscopy to investigate the cellular reorganization and colocalization of proteins and immunoprecipitation of HSP27 with actin, myosin, tropomyosin, and caldesmon as detected by sequential immunoblotting. Our results indicate that 1) translocation of Raf-1 to the membrane when stimulated with ceramide is inhibited by vasoactive intestinal peptide (VIP), a relaxant neuropeptide; 2) PKC-α and mitogen-activated protein kinase translocate and colocalize on the membrane in response to ceramide, and PKC-α translocation is inhibited by VIP; 3) HSP27 colocalizes with actin when contraction occurs; and 4) HSP27 immunoprecipitates with actin and with the contractile proteins myosin, tropomyosin, and caldesmon. We propose a model in which HSP27 is involved in sustained smooth muscle contraction and modulates the interaction of actin, myosin, tropomyosin, and caldesmon.

scholarly journals MSK1 activity is controlled by multiple phosphorylation sites

2005 ◽  
Vol 387 (2) ◽  
pp. 507-517 ◽  
Author(s):  
Claire E. McCOY ◽  
David G. CAMPBELL ◽  
Maria DEAK ◽  
Graham B. BLOOMBERG ◽  
J. Simon C. ARTHUR
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Cascades ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase ◽  
In Cells

MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2.

scholarly journals Activation of p38 Mitogen-Activated Protein Kinase and c-Jun NH2-Terminal Kinase by Double-Stranded RNA and Encephalomyocarditis Virus: Involvement of RNase L, Protein Kinase R, and Alternative Pathways

2000 ◽  
Vol 20 (2) ◽  
pp. 617-627 ◽  
Author(s):  
Mihail S. Iordanov ◽  
Jayashree M. Paranjape ◽  
Aimin Zhou ◽  
John Wong ◽  
Bryan R. G. Williams ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Encephalomyocarditis Virus ◽  
Mitogen Activated Protein Kinase ◽  
Rnase L ◽  
Double Stranded Rna ◽  
Mitogen Activated Protein ◽  
Stimulation Of ◽  
In Cells

ABSTRACT Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH2-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i.e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.

scholarly journals p38 Mitogen-Activated Protein Kinase Mediates Cell Death and p21-Activated Kinase Mediates Cell Survival during Chemotherapeutic Drug-induced Mitotic Arrest

2003 ◽  
Vol 14 (5) ◽  
pp. 2071-2087 ◽  
Author(s):  
Karl Deacon ◽  
Pratibha Mistry ◽  
Jonathan Chernoff ◽  
Jonathan L. Blank ◽  
Rajnikant Patel
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Cell Survival ◽  
P38 Mapk ◽  
Mitogen Activated Protein Kinase ◽  
Chemotherapeutic Drugs ◽  
Chemotherapeutic Drug ◽  
Drug Induced ◽  
Mitogen Activated Protein ◽  
P21 Activated Kinase

Activation of the mitotic checkpoint by chemotherapeutic drugs such as taxol causes mammalian cells to arrest in mitosis and then undergo apoptosis. However, the biochemical basis of chemotherapeutic drug-induced cell death is unclear. Herein, we provide new evidence that both cell survival and cell death-signaling pathways are concomitantly activated during mitotic arrest by microtubule-interfering drugs. Treatment of HeLa cells with chemotherapeutic drugs activated both p38 mitogen-activated protein kinase (MAPK) and p21-activated kinase (PAK). p38 MAPK was necessary for chemotherapeutic drug-induced cell death because the p38 MAPK inhibitors SB203580 or SB202190 suppressed cell death. Dominant-active MKK6, a direct activator of p38 MAPK, also induced cell death by stimulating translocation of Bax from the cytosol to the mitochondria in a p38 MAPK-dependent manner. Dominant active PAK suppressed this MKK6-induced cell death. PAK seems to mediate cell survival by phosphorylating Bad, and inhibition of PAK in mitotically arrested cells reduced Bad phosphorylation and increased apoptosis. Our results suggest that therapeutic strategies that suppress PAK-mediated survival signals may improve the efficacy of current cancer chemotherapies by enhancing p38 MAPK-mediated cell death.

Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27

1998 ◽  
Vol 275 (6) ◽  
pp. G1454-G1462 ◽  
Author(s):  
Pinglang Wang ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Muscle Contraction ◽  
Smooth Muscle Contraction ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Actin Binding ◽  
C3 Exoenzyme ◽  
Mitogen Activated Protein ◽  
Rho A

The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction. However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and C2 ceramide were used. Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60 src , respectively. Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10−7 M) or ceramide (10−7 M) (∼30% increase at 30 s that was sustained at 4 min). The translocation of Rho A to the membrane was confirmed by immunostaining. The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60 src inhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60 src and PKC or may belong to a different pathway than these proteins. Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways. Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.

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 Interleukin-6 Inhibits Fas-Induced Apoptosis and Stress-Activated Protein Kinase Activation in Multiple Myeloma Cells

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Dharminder Chauhan ◽  
Surender Kharbanda ◽  
Atsushi Ogata ◽  
Mitsuyoshi Urashima ◽  
Gerrard Teoh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Kinase ◽  
Dna Fragmentation ◽  
P38 Mapk ◽  
Interleukin 6 ◽  
Early Response ◽  
Mitogen Activated Protein Kinase ◽  
Serum Starvation ◽  
Mapk Activity ◽  
Induced Apoptosis

Abstract Fas belongs to the family of type-1 membrane proteins that transduce apoptotic signals. In the present studies, we characterized signaling during Fas-induced apoptosis in RPMI-8226 and IM-9 multiple myeloma (MM) derived cell lines as well as patient plasma cell leukemia cells. Treatment with anti-Fas (7C11) monoclonal antibody (MoAb) induced apoptosis, evidenced by internucleosomal DNA fragmentation and propidium iodide staining, and was associated with increased expression of c-jun early response gene. We also show that anti-Fas MoAb treatment is associated with activation of stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK); however, no detectable increase in extracellular signal-regulated kinases (ERK1 and ERK2) activity was observed. Because interleukin-6 (IL-6) is a growth factor for MM cells and inhibits apoptosis induced by dexamethasone and serum starvation, we examined whether IL-6 affects anti-Fas MoAb-induced apoptosis and activation of SAPK or p38 MAPK in MM cells. Culture of MM cells with IL-6 before treatment with anti-Fas MoAb significantly reduced both DNA fragmentation and activation of SAPK, without altering induction of p38 MAPK activity. These results therefore suggest that anti-Fas MoAb-induced apoptosis in MM cells is associated with activation of SAPK, and that IL-6 may both inhibit apoptosis and modulate SAPK activity.

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