Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of the present study was to examine the effects of portal hypertension on agonist-induced myosin phosphorylation and RhoA expression in vascular smooth muscle. A possible link to cAMP-dependent events was also examined. Portal hypertension was produced by stenosis of the portal vein. Vessel segments were treated with or without 50 microM of the PKA inhibitor Rp-cAMPS for 30 min and subsequently stimulated with 10(-4) M phenylephrine. Myosin regulatory light-chain phosphorylation was detected by immunoblotting. Total RNA from first-order mesenteric arteries and portal veins was isolated and amplified by RT-PCR using RhoA and GAPDH primers. RhoA protein expression was also measured in first-order mesenteric arteries using Western blot analysis. Myosin phosphorylation in maximally stimulated first-order mesenteric arteries was significantly lower in portal hypertensive animals (19.9 +/- 2.86%) when compared with sham-operated control (43.8 +/- 3.53%). Inhibition of PKA selectively increased myosin phosphorylation to 34.7 +/- 4.18%. Rp-cAMPS did not affect the phosphorylation of the portal veins or superior mesenteric arteries. RhoA mRNA and membrane-associated RhoA protein expression in portal hypertensive first-order mesenteric arteries were significantly lower when compared with controls. Acute inhibition of PKA had no effect on RhoA mRNA expression. However, it restored membrane-associated RhoA protein expression in portal hypertensive vessels to control levels. The results suggest that reductions in membrane-associated RhoA expression, which appear to be regulated by cAMP-dependent events, lead to reduced myosin phosphorylation and may underlie the reduced vasoconstrictor effectiveness in the resistance vasculature of portal hypertensive intestine.
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PMID:Impaired agonist-dependent myosin phosphorylation and decreased RhoA in rat portal hypertensive mesenteric vasculature. 1551 55

In addition to important roles near the actin-rich cell cortex, ample evidence indicates that multiple myosins are also involved in membrane movements in the endomembrane system. Nonmuscle myosin-II has been shown to have roles in anterograde and retrograde trafficking at the Golgi. Myosin-II is present on Golgi stacks isolated from intestinal epithelial cells and has been localized to the Golgi in several polarized and unpolarized cell lines. An understanding of roles of myosin-II in Golgi physiology will be facilitated by understanding the molecular arrangement of myosin-II at the Golgi. Salt-washing removes endogenous myosin-II from isolated Golgi and purified brush border myosin-II can bind in vitro. Brush border myosin-II binds to a tightly bound Golgi peripheral membrane protein with a K(1/2) of 75 nM and binding is saturated at 0.7 pmol myosin/microg Golgi. Binding studies using papain cleavage fragments of brush border myosin-II show that the 120-kDa rod domain, but not the head domain, of myosin heavy chain can bind directly to Golgi stacks. The 120-kDa domain does not bind to Golgi membranes when phosphorylated in vitro with casein kinase-II. These results suggest that phosphorylation in the rod domain may regulate the binding and/or release of myosin-II from the Golgi. These data support a model in which myosin-II is tethered to the Golgi membrane by its tail and actin filaments by its head. Thus, translocation along actin filaments may extend Golgi membrane tubules and/or vesicles away from the Golgi complex.
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PMID:Characterization of myosin-II binding to Golgi stacks in vitro. 1575 58

With the aim to find novel partners of human Cyclin T2a, we performed a two-hybrid screening in yeast using the full-length cDNA of this cyclin as bait, and a human heart cDNA library as preys source. Upon several interesting genes selected, our attention has been focused on the cDNA coding for PKNalpha, a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. Co-immunoprecipitation and in vitro pull-down assays independently confirmed the interaction between the two proteins. Luciferase assays, performed on NIH3T3 cell extracts after transfection with a MyoD-responsive promoter, pointed out that PKNalpha was able to enhance MyoD-dependent transcription, and that this effect was further increased when cyclin T2a was co-overexpressed. Finally, overexpression of both Cyclin T2a and PKNalpha in C2C12 cells strongly enhanced the expression of myogenic differentiation markers, such as Myogenin and Myosin Heavy Chain, during starvation-induced differentiation. Taken together, our data strengthen the hypothesis that Cyclin T2a plays a role in muscle differentiation, and propose PKNalpha as a novel partner of Cyclin T2a in this process.
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PMID:Pkn is a novel partner of cyclin T2a in muscle differentiation. 1633 89

Molluscan catch muscle can maintain tension for a long time with little energy consumption. This unique phenomenon is regulated by phosphorylation and dephosphorylation of twitchin, a member of the titin/connectin family. The catch state is induced by a decrease of intracellular Ca2+ after the active contraction and is terminated by the phosphorylation of twitchin by the cAMP-dependent protein kinase (PKA). Twitchin, from the well-known catch muscle, the anterior byssus retractor muscle (ABRM) of the mollusc Mytilus, incorporates three phosphates into two major sites D1 and D2, and some minor sites. Dephosphorylation is required for re-entering the catch state. Myosin, actin and twitchin are essential players in the mechanism responsible for catch during which force is maintained while myosin cross-bridge cycling is very slow. Dephosphorylation of twitchin allows it to bind to F-actin, whereas phosphorylation decreases the affinity of the two proteins. Twitchin has been also been shown to be a thick filament-binding protein. These findings raise the possibility that twitchin regulates the myosin cross-bridge cycle and force output by interacting with both actin and myosin resulting in a structure that connects thick and thin filaments in a phosphorylation-dependent manner.
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PMID:Twitchin as a regulator of catch contraction in molluscan smooth muscle. 1645 61

Myosin phosphatase targeting subunit 3 (MYPT3) and transforming growth factor-beta-inhibited membrane-associated protein (TIMAP) are two closely related myosin-binding targeting subunits of protein phosphatase 1 (PP1c) with a characteristic CAAX (where AA indicates aliphatic amino acid) box at the C termini. Here we show that MYPT3 can be a substrate for protein kinase A (PKA). We first mapped the multiple phosphorylation sites within a central conserved motif. Deletion or mutations of this motif resulted in enhancement of the associated PP1c activity, suggesting that phosphorylation of MYPT3 may play an important role in regulating PP1c catalytic activity. However, unlike the other known MYPTs, which upon phosphorylation inhibit PP1c, PKA phosphorylation of MYPT3 resulted in PP1c activation, indicating a different mode of action. There is a direct interaction between the central conserved phosphorylated site motif with the N-terminal ankyrin repeat region; this interaction was significantly reduced with MYPT3 phosphorylation or acidic phosphorylation site mutations, with concomitant alterations in biochemical and morphological consequences. We therefore propose a novel mechanism for the phosphorylation of MYPT3 by PKA and activation of the catalytic activity through direct interaction of a central region of MYPT3 with its N-terminal region.
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PMID:Phosphorylation of myosin phosphatase targeting subunit 3 (MYPT3) and regulation of protein phosphatase 1 by protein kinase A. 1692 Jul 2

Atrophy of skeletal muscle is due to a depression in protein synthesis and an increase in degradation. Studies in vitro have suggested that activation of the dsRNA-dependent protein kinase (PKR) may be responsible for these changes in protein synthesis and degradation. In order to evaluate whether this is also applicable to cancer cachexia the action of a PKR inhibitor on the development of cachexia has been studied in mice bearing the MAC16 tumour. Treatment of animals with the PKR inhibitor (5 mg kg(-1)) significantly reduced levels of phospho-PKR in muscle down to that found in non-tumour-bearing mice, and effectively attenuated the depression of body weight, with increased muscle mass, and also inhibited tumour growth. There was an increase in protein synthesis in skeletal muscle, which paralleled a decrease in eukaryotic initiation factor 2alpha phosphorylation. Protein degradation rates in skeletal muscle were also significantly decreased, as was proteasome activity levels and expression. Myosin levels were increased up to values found in non-tumour-bearing animals. Proteasome expression correlated with a decreased nuclear accumulation of nuclear factor-kappaB (NF-kappaB). The PKR inhibitor also significantly inhibited tumour growth, although this appeared to be a separate event from the effect on muscle wasting. These results suggest that inhibition of the autophosphorylation of PKR may represent an appropriate target for the attenuation of muscle atrophy in cancer cachexia.
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PMID:Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase. 1738 45

Previous studies suggest that the activation (autophosphorylation) of dsRNA-dependent protein kinase (PKR) can stimulate protein degradation, and depress protein synthesis in skeletal muscle through phosphorylation of the translation initiation factor 2 (eIF2) on the alpha-subunit. To understand whether these mediators are important in muscle wasting in cancer patients, levels of the phospho forms of PKR and eIF2alpha have been determined in rectus abdominus muscle of weight losing patients with oesophago-gastric cancer, in comparison with healthy controls. Levels of both phospho PKR and phospho eIF2alpha were significantly enhanced in muscle of cancer patients with weight loss irrespective of the amount and there was a linear relationship between phosphorylation of PKR and phosphorylation of eIF2alpha (correlation coefficient 0.76, P=0.005). This suggests that phosphorylation of PKR led to phosphorylation of eIF2alpha. Myosin levels decreased as the weight loss increased, and there was a linear relationship between myosin expression and the extent of phosphorylation of eIF2alpha (correlation coefficient 0.77, P=0.004). These results suggest that phosphorylation of PKR may be an important initiator of muscle wasting in cancer patients.
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PMID:Increased expression of phosphorylated forms of RNA-dependent protein kinase and eukaryotic initiation factor 2alpha may signal skeletal muscle atrophy in weight-losing cancer patients. 1808 77

Activation of beta2 integrins is necessary for neutrophil adhesion and full activation of neutrophil effector functions. We demonstrated previously that inhibition of protein kinase A (PKA) activity in quiescent neutrophils is sufficient to increase beta2-integrin cell surface expression, affinity, and adhesion. Thus, a tonic level of PKA activity prevents inappropriate activation of beta2 integrins in unstimulated neutrophils. Myosin light-chain (MLC) phosphorylation is an important regulator of leukocyte integrin function and adhesion. Moreover, PKA regulates MLC phosphorylation via inhibiting MLC kinase (MLCK) and MLC dephosphorylation via effects on the Rho kinase (ROCK)/MLC phosphatase pathway. We hypothesize that the tonic inhibitory effect of PKA on beta2-integrin activation neutrophils operates via its inhibition of MLC phosphorylation. We demonstrate here that inhibition of PKA activity with KT5720 activated beta2 integrins and adhesion coincident with an increase in MLC serine 19 (Ser 19) phosphorylation. KT5720-induced activation of beta2 integrins, adhesion, and MLC Ser 19 phosphorylation was abolished by pretreatment with the MLCK inhibitor ML-7 and specific MLCK inhibitory peptides but not the ROCK inhibitor Y-27632. These findings demonstrate that tonic PKA activity prevents activation of beta2 integrins and adhesion by inhibiting MLC phosphorylation via a MLCK-dependent but ROCK-independent pathway.
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PMID:Tonic protein kinase A activity maintains inactive beta2 integrins in unstimulated neutrophils by reducing myosin light-chain phosphorylation: role of myosin light-chain kinase and Rho kinase. 1821 60

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser(815). Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-beta, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-beta inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which alpha1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.
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PMID:AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase. 1842 92

Repetitive strain stimulates intestinal epithelial migration across fibronectin via focal adhesion kinase (FAK), Src, and extracellular signal-related kinase (ERK) although how these signals act and interact remains unclear. We hypothesized that PI3K is central to this pathway. We subjected Caco-2 and intestinal epithelial cell-6 cells to 10 cycles/min deformation on flexible fibronectin-coated membranes, assayed migration by wound closure, and signaling by immunoblots. Strain stimulated PI3K, AKT, glycogen synthase kinase (GSK), and p38 phosphorylation. Blocking each kinase prevented strain stimulation of migration. Blocking PI3K prevented strain-stimulated ERK and p38 phosphorylation. Blocking AKT did not. Downstream, blocking PI3K, AKT, or ERK inhibited strain-induced GSK-Ser9 phosphorylation. Upstream of AKT, reducing FAK or Rac1 by siRNA blocked strain-stimulated AKT phosphorylation, but inhibiting Src by PP2 or siRNA did not. Transfection with FAK point mutants at Tyr397, Tyr576/577, or Tyr925 demonstrated that only FAK925 phosphorylation is required for strain-stimulated AKT phosphorylation. Myosin light chain activation by strain required FAK, Rac1, PI3K, AKT, GSK, and ERK but not Src or p38. Finally, blebbistatin, a nonmuscle myosin II inhibitor, blocked the motogenic effect of strain downstream of myosin light chain. Thus strain stimulates intestinal epithelial migration across fibronectin by a complex pathway including Src, FAK, Rac1, PI3K, AKT, GSK, ERK, p38, myosin light chain, and myosin II.
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PMID:Delineating the signals by which repetitive deformation stimulates intestinal epithelial migration across fibronectin. 1917 20


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