Kinase Phosphorylation (kinase + phosphorylation)

Distribution by Scientific Domains

Selected Abstracts

Angiotensin-(1,7) has a dual role on growth-promoting signalling pathways in rat heart in vivo by stimulating STAT3 and STAT5a/b phosphorylation and inhibiting angiotensin II-stimulated ERK1/2 and Rho kinase activity

Jorge F. Giani
Angiotensin (ANG) II contributes to cardiac remodelling by inducing the activation of several signalling molecules, including ERK1/2, Rho kinase and members of the STAT family of proteins. Angiotensin-(1,7) is produced in the heart and inhibits the proliferative actions of ANG II, although the mechanisms of this inhibition are poorly understood. Accordingly, in the present study we examined whether ANG-(1,7) affects the ANG II-mediated activation of ERK1/2 and Rho kinase, STAT3 and STAT5a/b in rat heart in vivo. We hypothesized that ANG-(1,7) inhibits these growth-promoting pathways, counterbalancing the trophic action of ANG II. Solutions of normal saline (0.9% NaCl) containing ANG II (8 pmol kg,1) plus ANG-(1,7) in increasing doses (from 0.08 to 800 pmol kg,1) were administered via the inferior vena cava to anaesthetized male Sprague,Dawley rats. After 5 min, hearts were removed and ERK1/2, Rho kinase, STAT3 and STAT5a/b phosphorylation was determined by Western blotting using phosphospecific antibodies. Angiotensin II stimulated ERK1/2 and Rho kinase phosphorylation (2.3 0.2- and 2.1 0.2-fold increase over basal values, respectively), while ANG-(1,7) was without effect. The ANG II-mediated phosphorylation of ERK1/2 and Rho kinase was prevented in a dose-dependent manner by ANG-(1,7) and disappeared in the presence of the Mas receptor antagonist d -Ala7 -ANG-(1,7). Both ANG II and ANG-(1,7) increased STAT3 and STAT5a/b phosphorylation to a similar extent (130,140% increase). The ANG-(1,7)-stimulated STAT phosphorylation was blocked by the AT1 receptor antagonist losartan and not by d -Ala7 -ANG-(1,7). Our results show a dual action of ANG-(1,7), that is, a stimulatory effect on STAT3 and 5a/b phosphorylation through AT1 receptors and a blocking action on ANG II-stimulated ERK1/2 and Rho kinase phosphorylation through Mas receptor activation. The latter effect could be representative of a mechanism for a protective role of ANG-(1,7) in the heart by counteracting the effects of locally generated ANG II. [source]

Thrombin-mediated impairment of fibroblast growth factor-2 activity

FEBS JOURNAL, Issue 12 2009
Pierangela Totta
Thrombin generation increases in several pathological conditions, including cancer, thromboembolism, diabetes and myeloproliferative syndromes. During tumor development, thrombin levels increase along with several other molecules, including cytokines and angiogenic factors. Under such conditions, it is reasonable to predict that thrombin may recognize new low-affinity substrates that usually are not recognized under low-expression levels conditions. In the present study, we hypothesized that fibroblast growth factor (FGF)-2 may be cleaved by thrombin and that such action may lead to an impairment of its biological activity. The evidence collected in the present study indicates that FGF-2-induced proliferation and chemotaxis/invasion of SK-MEL-110 human melanoma cells were significantly reduced when FGF-2 was pre-incubated with active thrombin. The inhibition of proliferation was not influenced by heparin. Phe-Pro-Arg-chloromethyl ketone, a specific inhibitor of the enzymatic activity of thrombin, abolished the thrombin-induced observed effects. Accordingly, both FGF-2-binding to cell membranes as well as FGF-2-induced extracellular signal-regulated kinase phosphorylation were decreased in the presence of thrombin. Finally, HPLC analyses demonstrated that FGF-2 is cleaved by thrombin at the peptide bond between residues Arg42 and Ile43 of the mature human FGF-2 sequence. The apparent kcat/Km of FGF-2 hydrolysis was 1.1 104 m,1s,1, which is comparable to other known low-affinity thrombin substrates. Taken together, these results demonstrate that thrombin digests FGF-2 at the site Arg42-Ile43 and impairs FGF-2 activity in vitro, indicating that FGF-2 is a novel thrombin substrate. [source]

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

FEBS JOURNAL, Issue 8 2007
Patryk Krzemi
We characterized the expression and functional properties of the ADP-sensitive P2Y1 and P2Y12 nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y12 receptor relative to P2Y1 was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y1 receptor was low, and the P2Y12 receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y12 receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y12 receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y1 receptor, indicating the inhibitory role of P2Y1 in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y1 to P2Y12 would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation. [source]

Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells

FEBS JOURNAL, Issue 8 2004
In Duk Jung
Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3K,, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3K, signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3K, and Rac1/Cdc42. [source]

Aurora-A kinase phosphorylation of Aurora-A kinase interacting protein (AIP) and stabilization of the enzyme-substrate complex

Hiroshi Katayama
Abstract Aurora-A is an oncogenic kinase that plays essential roles in mitosis as well as cell survival. Aurora-A interacting protein (AIP) was identified as a negative regulator of Aurora-A with its ectopic over expression inducing destabilization of Aurora-A protein. Here we present evidence that in human cells, contrary to the earlier report, AIP functions in stabilizing rather than destabilizing Aurora-A. Furthermore, AIP is phosphorylated on Serine 70 by Aurora-A but not Aurora-B and expression of phosphorylation mimic mutant of AIP results in prolonged protein stability compared to unphosphorylatable mutant. We observed that when co-expressed with AIP, protein levels of both Aurora-A and Aurora-B are markedly elevated regardless of their kinase activities and phosphorylation state of AIP. Interaction of Aurora kinases with AIP is necessary for this elevated stability. This phenomenon is commonly detected in several human cancer cell lines used in this study. Depletion of AIP by RNA interference decreased Aurora-A but not Aurora-B in two of the three cell lines analyzed, indicating that under physiological condition, AIP functions in stabilization of Aurora-A but not Aurora-B, though this regulation may be dependent on additional factors as well. Further, AIP siRNA induced cell cycle arrest at G2/M, which is consistent with anticipated loss of function of Aurora-A in these cells. Thus, our study provides the first evidence of a role for AIP in G2/M cell cycle progression by cooperatively regulating protein stabilization of its up-stream regulator, Aurora-A kinase through protein,protein interaction as well as protein phosphorylation. J. Cell. Biochem. 102: 1318,1331, 2007. 2007 Wiley-Liss, Inc. [source]

Diminished contraction-induced intracellular signaling towards mitochondrial biogenesis in aged skeletal muscle

AGING CELL, Issue 4 2009
Vladimir Ljubicic
Summary The intent of this study was to determine whether aging affects signaling pathways involved in mitochondrial biogenesis in response to a single bout of contractile activity. Acute stimulation (1 Hz, 5 min) of the tibialis anterior (TA) resulted in a greater rate of fatigue in old (36 month), compared to young (6 month) F344XBN rats, which was associated with reduced ATP synthesis and a lower mitochondrial volume. To investigate fiber type-specific signaling, the TA was sectioned into red (RTA) and white (WTA) portions, possessing two- to 2.5-fold differences in mitochondrial content. The expression and contraction-mediated phosphorylation of p38, MKK3/6, CaMKII and AMPK, were assessed. Kinase protein expression tended to be higher in fiber sections with lower mitochondrial content, such as the WTA, relative to the RTA muscle, and this was exaggerated in tissues from senescent, compared to young animals. At rest, kinase activation was generally similar between young and old animals, despite the age-related variations in mitochondrial volume. In response to contractile activity, age did not influence the signaling of these kinases in the high-oxidative RTA muscle. However, in the low-oxidative WTA muscle, contraction-induced kinase activation was attenuated in old animals, despite the greater metabolic stress imposed by contractile activity in this muscle. Thus, the reduction of contraction-evoked kinase phosphorylation in muscle from old animals is fiber type-specific, and depends on factors which are, in part, independent of the metabolic milieu within the contracting fibers. These findings imply that the downstream consequences of kinase signaling are reduced in aging muscle. [source]

Manganese potentiates nuclear factor-,B-dependent expression of nitric oxide synthase 2 in astrocytes by activating soluble guanylate cyclase and extracellular responsive kinase signaling pathways

Julie A. Moreno
Abstract Inflammatory activation of glial cells is associated with neuronal injury in several degenerative movement disorders of the basal ganglia, including manganese neurotoxicity. Manganese (Mn) potentiates the effects of inflammatory cytokines on nuclear factor-,B (NF-,B)-dependent expression of nitric oxide synthase 2 (NOS2) in astrocytes, but the signaling mechanisms underlying this effect have remained elusive. It was postulated in the present studies that direct stimulation of cGMP synthesis and activation of mitogen-activated protein (MAP) kinase signaling pathways underlies the capacity of Mn to augment NF-,B-dependent gene expression in astrocytes. Exposure of primary cortical astrocytes to a low concentration of Mn (10 ,M) potentiated expression of NOS2 mRNA and protein along with production of NO in response to interferon-, (IFN,) and tumor necrosis factor-, (TNF,), which was prevented by overexpression of dominant negative I,B,. Mn also potentiated IFN,- and TNF,-induced phosphorylation of extracellular response kinase (ERK), p38, and JNK, as well as cytokine-induced activation of a fluorescent NF-,B reporter construct in transgenic astrocytes. Activation of ERK preceded that of NF-,B and was required for maximal activation of NO synthesis. Independently of IFN,/TNF,, Mn-stimulated synthesis of cGMP in astrocytes and inhibition of soluble guanylate cyclase (sGC) abolished the potentiating effect of Mn on MAP kinase phosphorylation, NF-,B activation, and production of NO. These data indicate that near-physiological concentrations of Mn potentiate cytokine-induced expression of NOS2 and production of NO in astrocytes via activation of sGC, which promotes ERK-dependent enhancement of NF-,B signaling. 2008 Wiley-Liss, Inc. [source]

Eutigoside C inhibits the production of inflammatory mediators (NO, PGE2, IL-6) by down-regulating NF-,B and MAP kinase activity in LPS-stimulated RAW 264.7 cells

Hye-Ja Lee
Eutigoside C, a compound isolated from the leaves of Eurya emarginata, is thought to be an active anti-inflammatory compound which operates through an unknown mechanism. In the present study we investigated the molecular mechanisms of eutigoside C activity in lipopolysacchardide (LPS)-stimulated murine macrophage RAW 264.7 cells. Treatment with eutigoside C inhibited LPS-stimulated production of nitric oxide (NO), prostaglandin E2 (PGE2) and interleukin-6 (IL-6). To further elucidate the mechanism of this inhibitory effect of eutigoside C, we studied LPS-induced nuclear factor (NF)-,B activation and mitogen-activated protein (MAP) kinase phosphorylation. Eutigoside C suppressed NF-,B DNA binding activity, interfering with nuclear translocation of NF-,B. Eutigoside C suppressed the phosphorylation of three MAP kinases (ERK1/2, JNK and p38). These results suggest that eutigoside C inhibits the production of inflammatory mediators (NO, PGE2 and interleukin-6) by suppressing the activation and translocation of NF-,B and the phosphorylation of MAP kinases (ERK1/2, JNK and p38) in LPS-stimulated murine macrophage RAW 264.7 cells. [source]

Neural Stem Cells and Alcohol

ALCOHOLISM, Issue 2 2003
F. T. Crews
This article summarizes the proceedings of a symposium held at the 2002 Research Society on Alcoholism Meeting in San Francisco, California. The aim of this symposium was to review research on the effects of ethanol on neural stems cells and neurogenesis. Ethanol is known to alter neurogenesis during development; however, recent studies indicate that the brain forms new neurons from stem cells throughout life. Furthermore, stem cells can be transplanted into the brain, creating exciting new possibilities to study brain function. The symposium covered these research areas. Dr. Michael W. Miller reviewed knowledge on the effects of ethanol on stem cell proliferation and differentiation during development. Dr. Wu Ma described studies in culture indicating that (1) neural stem cells express functional muscarinic acetylcholine receptors (mAchR), (2) mAchR-mediated proliferation involves Ca2+ signaling and mitogen-activated protein kinase phosphorylation, and (3) phosphoinositol-3 kinase is a downstream effector for mAchR-mediated cell proliferation via activation of Akt. Drs. Kim Nixon and Fulton T. Crews followed with in vivo studies on ethanol's effects on adult neural stem cell proliferation and differentiation. Dr. W. Michael Zawada described studies directed at dopamine neuron cell transplants into mammalian central nervous system. These studies clearly establish that ethanol has significant effects on stem cells. [source]

A snake venom metalloproteinase, kistomin, cleaves platelet glycoprotein VI and impairs platelet functions

Summary.,Background and objectives:,Injuries to the vessel wall and subsequent exposure of the matrix of the subendothelial layer resulted in thrombus formation. Platelet glycoprotein (GP) Ib and VI play a crucial role in matrix-induced activation and aggregation of platelets. Methods and results:,In the present study, we reported that the GPIb-cleaving snake venom metalloproteinase (SVMP), kistomin, inhibited collagen-induced platelet aggregation. Moreover, kistomin inhibited platelet aggregation induced by convulxin (CVX, a GPVI agonist) and a GPVI-specific antibody in a concentration and time-dependent manner. Kistomin treatment decreased platelet GPVI but not integrin ,2,1 and ,IIb,3, accompanied with the formation of GPVI cleavage fragments, as determined by flow cytometric and Western blot analyses. In addition, intact platelet GPVI and recombinant GPVI were digested by kistomin to release 25- and 35-kDa fragments, suggesting that kistomin cleaved GPVI near the mucin-like region. We designed four synthetic peptides ranging from Leu180 to Asn249 as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE205/A206TA and NKV218/F219TT, as analyzed by MALDI-TOF-MS. In addition, GPVI-specific antibody-induced tyrosine kinase phosphorylation in platelets was reduced after kistomin pretreatment, and platelet adhesion to collagen but not to fibrinogen was attenuated by kistomin. Conclusions:,We provided here the first evidence that a P-I snake venom metalloproteinase, kistomin, inhibits the interaction between collagen and platelet GPVI through its proteolytic activity on GPVI, thus providing an alternative strategy for developing new anti-thrombotic agents. [source]

EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturation

Laurence Gall
Abstract EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus,oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell. Mol. Reprod. Dev. 2005 Wiley-Liss, Inc. [source]

Porcine CPEB1 is involved in Cyclin B translation and meiotic resumption in porcine oocytes

ABSTRACT Ovarian immature oocytes accumulate many dormant maternal mRNAs, which have short poly(A) tails. Cytoplasmic-polyadenylation-element binding protein (CPEB) has been reported to play key roles for the elongation of the tails and the translation of these mRNAs in Xenopus oocytes. However, the functions of CPEB in meiotic resumption have not yet been established in mammalian oocytes. The present study examined the roles of porcine CPEB in Cyclin B syntheses and meiotic resumption of porcine oocytes. Porcine CPEB1 (pCPEB1) cDNA was cloned from total RNA of immature oocytes by RT-PCR. The overexpression of pCPEB1 by mRNA injection into immature oocytes increased Cyclin B expression and the rate of meiotic resumption. Conversely, the inhibition of endogenous CPEB by expression of a dominant-negative mutant pCPEB1 (AA-CPEB), which replaced the expected phosphorylation sites with alanines, had the effect of inhibiting Cyclin B synthesis, ribosomal S6 kinase phosphorylation (an indicator of Mos activity), and meiotic resumption. The inhibition of porcine Aurora A by an injection of antisense RNA enhanced the inhibitory effects of AA-CPEB. These results suggest the involvement of mammalian CPEB1 in Cyclin B syntheses and meiotic resumption in mammalian oocytes. In addition, the phosphorylation sites of pCPEB1 were identified and are suggested to be phosphorylated by porcine Aurora A. [source]

Blood,brain barrier breakdown and repair by Src after thrombin-induced injury

Da-Zhi Liu PhD
Objective Thrombin mediates the life-threatening cerebral edema that occurs after intracerebral hemorrhage. Therefore, we examined the mechanisms of thrombin-induced injury to the blood,brain barrier (BBB) and subsequent mechanisms of BBB repair. Methods Intracerebroventricular injection of thrombin (20U) was used to model intraventricular hemorrhage in adult rats. Results Thrombin reduced brain microvascular endothelial cell (BMVEC) and perivascular astrocyte immunoreactivity,indicating either cell injury or death,and functionally disrupted the BBB as measured by increased water content and extravasation of sodium fluorescein and Evans blue dyes 24 hours later. Administration of nonspecific Src family kinase inhibitor (PP2) immediately after thrombin injections blocked brain edema and BBB disruption. At 7 to 14 days after thrombin injections, newborn endothelial cells and astrocytes were observed around cerebral vessels at the time when BBB permeability and cerebral water content resolved. Delayed administration of PP2 on days 2 through 6 after thrombin injections prevented resolution of the edema and abnormal BBB permeability. Interpretation Thrombin, via its protease-activated receptors, is postulated to activate Src kinase phosphorylation of molecules that acutely injure the BBB and produce edema. Thus, acute administration of Src antagonists blocks edema. In contrast, Src blockade for 2 to 6 days after thrombin injections is postulated to prevent resolution of edema and abnormal BBB permeability in part because Src kinase proto-oncogene members stimulate proliferation of newborn BMVECs and perivascular astrocytes in the neurovascular niche that repair the damaged BBB. Thus, Src kinases not only mediate acute BBB injury but also mediate chronic BBB repair after thrombin-induced injury. ANN NEUROL 2010;67:526,533 [source]

Inhibition of Akt induces significant downregulation of survivin and cytotoxicity in human multiple myeloma cells

Teru Hideshima
Summary Akt mediates growth and drug resistance in multiple myeloma (MM) cells in the bone marrow (BM) microenvironment. We have shown that a novel Akt inhibitor Perifosine induces significant cytotoxicity in MM cells in the BM milieu. This study further delineated molecular mechanisms whereby Perifosine triggered cytotoxicity in MM cells. Neither the intensity of Jun NH2 -terminal kinase phosphorylation nor caspase/poly (ADP-ribose) polymerase cleavage correlated with Perifosine-induced cytotoxicity in MM.1S, INA6, OPM1 and OPM2 MM cells. However, survivin, which regulates caspase-3 activity, was markedly downregulated by Perifosine treatment, without changes in other anti-apoptotic proteins. Downregulation of survivin by siRNA significantly inhibited OPM1 MM cell growth, confirming that survivin mediates MM cell survival. Perifosine significantly downregulated both function and protein expression of ,-catenin. Co-culture with BM stromal cells (BMSCs) upregulated both ,-catenin and survivin expression in MM cells, which was blocked by Perifosine. Importantly, Perifosine treatment also downregulated survivin expression in human MM cells grown in vivo in a severe combined immunodeficient mouse xenograft model. Finally, Perifosine inhibited bortezomib-induced upregulation of survivin, associated with enhanced cytotoxicity of combined bortezomib and Perifosine treatment. These preclinical studies provide the framework for clinical trials of bortezomib with Perifosine to improve patient outcome in MM. [source]