CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle

2002 ◽  
Vol 282 (4) ◽  
pp. C754-C761 ◽  
Author(s):  
Roman Ginnan ◽  
Harold A. Singer
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
Protein Receptor

In vascular smooth muscle (VSM) and many other cells, G protein receptor-coupled activation of mitogen-activated protein kinases has been linked, in part, to increases in free intracellular Ca2+. Previously, we demonstrated that ionomycin-, angiotensin II-, and thrombin-induced activation of extracellular signal-regulated kinase (ERK)1/2 in VSM cells was attenuated by pretreatment with KN-93, a selective inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase II). In the present study, we show that the Ca2+-dependent pathway leading to activation of ERK1/2 is preceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation and is attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibits Ca2+-dependent PYK2 activation and EGF receptor tyrosine phosphorylation in response to ionomycin, ATP, and platelet-derived growth factor but has no effect on phorbol 12,13-dibutyrate- or EGF-induced responses. The results implicate CaM kinase II as an intermediate in the Ca2+/calmodulin-dependent activation of PYK2.

Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle

1996 ◽  
Vol 271 (4) ◽  
pp. H1348-H1355 ◽  
Author(s):  
N. Jin ◽  
R. A. Siddiqui ◽  
D. English ◽  
R. A. Rhoades
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Light Chain ◽  
Tyrosine Kinases ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase Pathway

Two separate signal transduction pathways exist in vascular smooth muscle: one for cell growth, proliferation, and differentiation and the other for contraction. Although activation of protein tyrosine kinases is intimately involved in the signaling pathway that induces cell growth, proliferation, and differentiation, activation of myosin light chain kinase (MLCK) is an important step in the pathway leading to smooth muscle contraction. Indirect evidence suggests that “cross talk” exists between these two signaling pathways, but the common intermediates are not well defined. The purpose of this study was to determine whether a vasoconstrictor and a mitogen initiate crossover signaling between the tyrosine kinase pathway and the MLCK pathway in vascular smooth muscle. Rat aorta and pulmonary arteries were isolated and stimulated with either fetal calf serum (FCS) or phenylephrine in the presence or absence of a tyrosine kinase inhibitor (genistein) or tyrosine phosphatase inhibitor [sodium o-vanadate (Na3 VO4)]. Isometric force was recorded as a function of time; myosin light chain phosphorylation, protein tyrosine phosphorylation, and mitogen-activated protein kinase (MAPK) mobility were determined by immunoblotting. The results demonstrate that FCS, which contains a variety of growth factors known to activate tyrosine kinases, induced myosin light chain phosphorylation and contraction in vascular smooth muscle. Phenylephrine, a vasoconstrictor known to activate MLCK, induced tyrosine phosphorylation of a 42-kDa protein identified as MAPK. Tyrosine phosphorylation of this protein was inhibited by genistein and enhanced by vanadate. Genistein significantly inhibited both serum- and phenylephrine-induced myosin light chain phosphorylation as well as the serum- and phenylephrine-induced force generation, whereas vanadate enhanced these responses. These data demonstrate interrelationship between activation of the tyrosine kinase pathway and the MLCK pathway in vascular smooth muscle. These interactions may influence smooth muscle contraction and be important in the regulation of smooth muscle cell proliferation.

Role of insulin-like growth factor 1 receptor and c-Src in endothelin-1- and angiotensin II-induced PKB phosphorylation, and hypertrophic and proliferative responses in vascular smooth muscle cellsThis article is one of a selection of papers published in a special issue on Advances in Cardiovascular Research.

2009 ◽  
Vol 87 (12) ◽  
pp. 1009-1018 ◽  
Author(s):  
Ali Bouallegue ◽  
George Vardatsikos ◽  
Ashok K. Srivastava
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Specific Inhibitor ◽  
Ang Ii ◽  
Vasoactive Peptides ◽  
Pkb Phosphorylation

Endothelin-1 (ET-1) and angiotensin II (Ang II) are vasoactive peptides believed to contribute to the pathogenesis of vascular abnormalities such as hypertension, atherosclerosis, hypertrophy, and restenosis. The concept of transactivation of growth factor receptors, such as epidermal growth factor receptor (EGFR), in triggering vasoactive peptide-induced signaling events has gained much recognition during the past several years. We have demonstrated that insulin-like growth factor type 1 receptor (IGF-1R) plays a role in transducing the effect of H2O2, leading to protein kinase B (PKB) phosphorylation. Since vasoactive peptides elicit their responses through generation of reactive oxygen species, including H2O2, we investigated whether IGF-1R transactivation plays a similar role in ET-1- and Ang II-induced PKB phosphorylation and hypertrophic responses in vascular smooth muscle cells (VSMC). AG1024, a specific inhibitor of IGF-1R protein tyrosine kinase (PTK), attenuated both ET-1- and Ang II-induced PKB phosphorylation in a dose-dependent manner. ET-1 and Ang II treatment also induced the phosphorylation of tyrosine residues in the autophosphorylation sites of IGF-1R, which were blocked by AG1024. In addition, both ET-1 and Ang II evoked tyrosine phosphorylation of c-Src, a nonreceptor PTK, whereas pharmacological inhibition of c-Src PTK activity by PP2, a specific inhibitor of Src-family tyrosine kinase, significantly reduced PKB phosphorylation as well as tyrosine phosphorylation of IGF-1R induced by the 2 vasoactive peptides. Furthermore, protein and DNA synthesis enhanced by ET-1 and Ang II were attenuated by AG1024 and PP2. In conclusion, these data suggest that IGF-1R PTK and c-Src PTK play a critical role in mediating PKB phosphorylation as well as hypertrophic and proliferative responses induced by ET-1 and Ang II in A10 VSMC.

scholarly journals Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

2011 ◽  
Vol 435 (3) ◽  
pp. 609-618 ◽  
Author(s):  
Caroline Pellet-Many ◽  
Paul Frankel ◽  
Ian M. Evans ◽  
Birger Herzog ◽  
Manfred Jünemann-Ramírez ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Tyrosine Phosphorylation ◽  
Muscle Cells ◽  
Immunofluorescent Staining ◽  
Human Coronary Artery ◽  
Neuropilin 1 ◽  
Stimulation Of

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.

PKC-δ and CaMKII-δ2 mediate ATP-dependent activation of ERK1/2 in vascular smooth muscle

2004 ◽  
Vol 286 (6) ◽  
pp. C1281-C1289 ◽  
Author(s):  
Roman Ginnan ◽  
Paul J. Pfleiderer ◽  
Kevin Pumiglia ◽  
Harold A. Singer
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Kinase Inhibitor ◽  
Purinergic Receptor ◽  
Selective Inhibition ◽  
Dominant Negative ◽  
Dependent Protein ◽  
Camkii Activation

ATP, a purinergic receptor agonist, has been shown to be involved in vascular smooth muscle (VSM) cell DNA synthesis and cell proliferation during embryonic and postnatal development, after injury, and in atherosclerosis. One mechanism that ATP utilizes to regulate cellular function is through activation of ERK1/2. In the present study, we provide evidence that ATP-dependent activation of ERK1/2 in VSM cells utilizes specific isoforms of the multifunctional serine/threonine kinases, PKC, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) as intermediates. Selective inhibition of PKC-δ activity with rottlerin, or adenoviral overexpression of kinase-negative PKC-δ, attenuated the ATP- and phorbol 12,13-dibutyrate (PDBu)-stimulated ERK1/2 activation. Inhibition of PKC-α activity with Gö-6976, or adenoviral overexpression of kinase-negative PKC-α, was ineffective. Alternatively, treatment with KN-93, a selective inhibitor of CaMKII activation, or adenoviral overexpression of kinase-negative CaMKII-δ2, inhibited ATP-dependent activation of ERK1/2 but had no effect on PDBu- or PDGF-stimulated ERK1/2. In addition, adenoviral overexpression of dominant-negative ras (Ad.HA-RasN17) partially inhibited the ATP- and PDBu-induced activation of ERK1/2 and blocked ionomycin- and EGF-stimulated ERK1/2, and inhibition of tyrosine kinases with AG-1478, an EGFR inhibitor, or the src family kinase inhibitor PP2 attenuated ATP-stimulated ERK1/2 activation. Taken together, these data indicate that PKC-δ and CaMKII-δ2 coordinately mediate ATP-dependent transactivation of EGF receptor, resulting in increased ERK1/2 activity in VSM cells.

scholarly journals Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

1991 ◽  
Vol 2 (8) ◽  
pp. 663-673 ◽  
Author(s):  
R Campos-González ◽  
J R Glenney
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
C Terminus ◽  
Epidermal Growth

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.

Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells

2000 ◽  
Vol 278 (6) ◽  
pp. L1138-L1145 ◽  
Author(s):  
Barbara Tolloczko ◽  
Florence C. Tao ◽  
Mary E. Zacour ◽  
James G. Martin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Calcium Signaling ◽  
Tyrosine Phosphorylation ◽  
Airway Smooth Muscle ◽  
Tyrosine Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Airway Smooth Muscle Cells ◽  
Mitogen Activated Protein

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 μM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 μM). Genistein and tyrphostin 23 (40 and 10 μM, respectively) significantly decreased 5-HT-evoked peak Ca2+ responses, and the effect of genistein could be observed in the absence of extracellular Ca2+. The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 μM) had no significant effect on peak Ca2+ levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of ∼70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the γ isoform of phospholipase C (PLC-γ). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 μM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-β activity. It is unlikely that PLC-γ or the mitogen-activated protein kinase pathway is involved in Ca2+ signaling to 5-HT.

Attenuation of endothelin-1-induced calcium response by tyrosine kinase inhibitors in vascular smooth muscle cells

1996 ◽  
Vol 270 (6) ◽  
pp. C1825-C1833 ◽  
Author(s):  
C. Y. Liu ◽  
M. Sturek
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Tyrosine Kinase ◽  
Smooth Muscle Cells ◽  
Tyrosine Kinase Inhibitors ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Muscle Cells ◽  
Endothelin 1 ◽  
Fura 2

Although tyrosine kinases play an important role in cell growth and have been implicated in regulation of smooth muscle contraction, their role in agonist-induced myoplasmic Ca2+ responses is unclear. We examined effects of the tyrosine kinase inhibitors genistein and methyl 2,5-dihydroxycinnamate (MDHC) on the endothelin-1 (ET-1)-induced Ca2+ response and determined underlying mechanisms for the effects. Freshly isolated smooth muscle cells from porcine coronary arteries were loaded with fura 2 ester, and myoplasmic free Ca2+ (Ca2+ (m)) concentration was estimated with fura 2 microfluorometry. Both genistein and MDHC inhibited the initial transient Cam2+ response to ET by 54 and 81%, respectively (P < 0.05), in the presence of extracellular Ca2+. Genistein also significantly delayed the Cam2+ response, with the latent period from ET-1 application to the beginning of the Cam2+ response being increased from 1.08 +/- 0.17 to 2.65 +/- 0.52 min (P < 0.05). In the absence of extracellular Ca2+, genistein inhibited the ET-1-induced Cam2+ response by 93% (P < 0.05). The Cam2+ responses to caffeine (5 mM) or inositol trisphosphate (IP3) applied intracellularly via a patch-clamp pipette were not affected by genistein. Both genistein and MDHC also abolished the sustained Cam2+ response to ET-1. However, the Cam2+ response to depolarization by 80 mM K+ was not inhibited by MDHC and only inhibited 22% by genistein (P < 0.05). These results indicate that 1) activation of tyrosine kinases is an important regulatory mechanism for the ET-1-induced Cam2+ response in vascular smooth muscle and 2) tyrosine kinases mediate ET-1-induced Ca2+ release with no direct effect on IP3-mediated Ca2+ release. Thus ET-1-mediated signaling upstream of IP3 interaction with the Ca2+ stores is regulated by tyrosine kinases.

Consequences of postnatal vascular smooth muscle EGFR deletion on acute angiotensin II action

2015 ◽  
Vol 130 (1) ◽  
pp. 19-33 ◽  
Author(s):  
Barbara Schreier ◽  
Mirja Hünerberg ◽  
Sindy Rabe ◽  
Sigrid Mildenberger ◽  
Daniel Bethmann ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Vascular Smooth Muscle ◽  
Egf Receptor ◽  
Ang Ii ◽  
Basal Blood Pressure ◽  
Epidermal Growth

In the present study we demonstrate that the epidermal growth factor (EGF) receptor (EGFR) in vascular smooth muscle cells (VSMC) is involved in basal blood pressure homoeostasis, acute pressure response to angiotensin II (Ang II) but not endothelin-1 and contributes to maturation-related remodelling.

ARG tyrosine kinase activity is inhibited by STI571

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2440-2448 ◽  
Author(s):  
Keiko Okuda ◽  
Ellen Weisberg ◽  
D. Gary Gilliland ◽  
James D. Griffin
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Fusion Partner ◽  
Induced Apoptosis

Abstract The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces hematologic remission in most patients with chronic myeloid leukemia. In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF)β receptor, and c-KIT, but it does not inhibit SRC family kinases, c-FMS, FLT3, the epidermal growth factor receptor, or multiple other tyrosine kinases. ARG is a widely expressed tyrosine kinase that shares substantial sequence identity with c-ABL in the kinase domain and cooperates with ABL to regulate neurulation in the developing mouse embryo. As described here, ARG has recently been implicated in the pathogenesis of leukemia as a fusion partner of TEL. A TEL/ARG fusion was constructed to determine whether ARG can be inhibited by STI571. When expressed in the factor-dependent murine hematopoietic cell line Ba/F3, the TEL/ARG protein was heavily phosphorylated on tyrosine, increased tyrosine phosphorylation of multiple cellular proteins, and induced factor-independent proliferation. The effects of STI571 on Ba/F3 cells transformed with BCR/ABL, TEL/ABL, TEL/PDGFβR, or TEL/ARG were then compared. STI571 inhibited tyrosine phosphorylation and cell growth of Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/PDGFβR, and TEL/ARG with an IC50 of approximately 0.5 μM in each case, but it had no effect on untransformed Ba/F3 cells growing in IL-3 or on Ba/F3 cells transformed by TEL/JAK2. Culture of TEL/ARG-transfected Ba/F3 cells with IL-3 completely prevented STI571-induced apoptosis in these cells, similar to what has been observed with BCR/ABL- or TEL/ABL-transformed cells. These results indicate that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571.

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