Activation of protein tyrosine kinase PYK2 by the m1 muscarinic acetylcholine receptor

Several G protein-coupled receptors are known to direct the tyrosine phosphorylation, and in some cases the activation, of diverse tyrosine kinases. Using a stable cell line approach, we characterize the activation of PYK2, a tyrosine kinase structurally related to focal adhesion kinase, by the G protein-coupled m1 muscarinic acetylcholine receptor. We find that PYK2 tyrosine kinase activity is critical for the m1 receptor-stimulated tyrosine phosphorylation of PYK2. Furthermore, we identify two tyrosine residues that are subject to phosphorylation in response to muscarinic signaling and show that this phosphorylation induces two cytosolic proteins, c-Src and Grb2, to bind to PYK2. This is the first demonstration of the significance played by distinct PYK2 tyrosine residues in G protein-coupled signaling to this kinase. By comparison, though m1 receptors induce the tyrosine phosphorylation of the cytoskeletal protein paxillin, the association of paxillin with PYK2 is unaffected by muscarinic signaling. We also provide evidence that PYK2 specifically phosphorylates the carboxyl-terminal cytosolic portion of the potassium channel Kv1.2 in a manner regulated by the m1 receptor. These results delineate molecular events attending the m1 muscarinic receptor stimulation of this tyrosine kinase and establish PYK2 as an effector of the m1 muscarinic receptor in the regulation of multiple cell functions.     

Activation of AMP-activated protein kinase enhances angiotensin ii-induced proliferation in cardiac fibroblasts

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic regulator of cellular enzymes involved in carbohydrate and fat metabolism, which regulate ATP conservation and synthesis. Here, we investigated whether AMPK signaling has a role in the regulation of angiotensin II (Ang II)-induced proliferation in rat cardiac fibroblasts. Aminoimidazole-4-carboxamide-1-beta-ribofuranoside (AICAR) activated AMPK in rat cardiac fibroblasts and increased Ang II-induced extracellular signal-regulated kinase 1/2 phosphorylation and activity. AICAR also increased Ang II-induced c-fos mRNA expression in the cells. [3H]-thymidine and [3H]-proline incorporation by cardiac fibroblasts treated with Ang II was enhanced when the cells were pretreated with AICAR. Inhibition of AMPK by small interfering RNA for AMPKalpha1 suppressed Ang II-induced extracellular signal-regulated kinase activity, c-fos mRNA expression, and cell proliferation. Treatment of rats with AICAR (1 mg/g body weight per day) for 1 week significantly enhanced Ang II-induced hypertrophy of the myocardium. Our findings indicate that AMPK works as a stimulator of the Ang II-induced proliferative pathway in cardiac fibroblasts. Inhibition of AMPK signaling might serve as a new therapeutic target of remodeling of the hypertrophic myocardium.     

Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases

The Snf1/AMP-activated protein kinase (AMPK) family plays fundamental roles in cellular responses to metabolic stress in eukaryotes. In humans, AMPK regulates lipid and glucose metabolism and has been implicated in such metabolic disorders as diabetes and obesity and in cardiac abnormalities. Snf1 and AMPK are the downstream components of kinase cascades, but the upstream kinase(s) have remained elusive. We have here identified three yeast kinases, Pak1p, Tos3p, and Elm1p, that activate Snf1 kinase in vivo. Triple deletion of the cognate genes causes a Snf- mutant phenotype and abolishes Snf1 catalytic activity. All three kinases phosphorylate recombinant Snf1p on the activation-loop threonine. Moreover, Tos3p phosphorylates mammalian AMPK on the equivalent residue and activates the enzyme, suggesting functional conservation of the upstream kinases between yeast and mammals. We further show that the closely related mammalian LKB1 kinase, which is associated with Peutz-Jeghers cancer-susceptibility syndrome, phosphorylates and activates AMPK in vitro. Thus, the identification of the yeast upstream kinases should facilitate identification of the corresponding, physiologically important mammalian upstream kinases.     

Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor

STI 571 (formerly known as CGP 57148B) is a known inhibitor of the c-abl, bcr-abl, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases. This compound is being evaluated in clinical trials for the treatment of chronic myelogenous leukemia. We sought to extend the activity profile of STI 571 by testing its ability to inhibit the tyrosine kinase activity of c-kit, a receptor structurally similar to PDGFR. We treated a c-kit expressing a human myeloid leukemia cell line, M-07e, with STI 571 before stimulation with Steel factor (SLF). STI 571 inhibited c-kit autophosphorylation, activation of mitogen-activated protein (MAP) kinase, and activation of Akt without altering total protein levels of c-kit, MAP kinase, or Akt. The concentration that produced 50% inhibition for these effects was approximately 100 nmol/L. STI 571 also significantly decreased SLF-dependent growth of M-07e cells in a dose-dependent manner and blocked the antiapoptotic activity of SLF. In contrast, the compound had no effect on MAP kinase activation or cellular proliferation in response to granulocyte-macrophage colony-stimulating factor. We also tested the activity of STI 571 in a human mast cell leukemia cell line (HMC-1), which has an activated mutant form of c-kit. STI 571 had a more potent inhibitory effect on the kinase activity of this mutant receptor than it did on ligand-dependent activation of the wild-type receptor. These findings show that STI 571 selectively inhibits c-kit tyrosine kinase activity and downstream activation of target proteins involved in cellular proliferation and survival. This compound may be useful in treating cancers associated with increased c-kit kinase activity.     

Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity

Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI- 3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.     

Differently spliced cDNAs of human leukocyte tyrosine kinase receptor tyrosine kinase predict receptor proteins with and without a tyrosine kinase domain and a soluble receptor protein.

Leukocyte tyrosine kinase (LTK) is a tyrosine kinase that has been suggested to be specific for hematopoietic cells and neuronal cells and reported as an unusual membrane protein lacking an extracellular domain. Here we report the cloning of a human LTK cDNA clone containing the complete open reading frame of a putative receptor tyrosine kinase protein. The extracellular domain of the receptor protein is larger than previously predicted. Furthermore, we have cloned a set of cDNAs representing differently spliced human LTK mRNAs. These cDNAs predict a truncated receptor protein lacking the tyrosine kinase domain and a soluble receptor protein that has neither a transmembrane nor a tyrosine kinase domain. Our results suggest that the LTK gene produces not only the putative receptor tyrosine kinase for unknown ligand but also multiple protein products that may have different functions.     

Transactivation of EGF receptor and ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis

TNF is a pleiotropic cytokine that activates both anti- and proapoptotic signaling pathways, with cell fate determined by the balance between these two pathways. Activation of ErbB family members, including EGF receptor (EGFR/ErbB1), promotes cell survival and regulates several signals that overlap with those stimulated by TNF. This study was undertaken to determine the effects of TNF on EGFR and ErbB2 activation and intestinal epithelial cell survival. Mice, young adult mouse colon epithelial cells, and EGFR knockout mouse colon epithelial cells were treated with TNF. Activation of EGFR, ErbB2, Akt, Src, and apoptosis were determined in vivo and in vitro. TNF stimulated EGFR phosphorylation in young adult mouse colon epithelial cells, and loss of EGFR expression or inhibition of kinase activity increased TNF-induced apoptosis, which was prevented in WT but not by kinase-inactive EGFR expression. Similarly, TNF injection stimulated apoptosis in EGFR-kinase-defective mice (EGFR^wa2) compared with WT mice. TNF also activated ErbB2, and loss of ErbB2 expression increased TNF-induced apoptosis. Furthermore, Src-kinase activity and the expression of both EGFR and ErbB2 were required for TNF-induced cell survival. Akt was shown to be a downstream target of TNF-activated EGFR and ErbB2. These findings demonstrate that EGFR and ErbB2 are critical mediators of TNF-regulated antiapoptotic signals in intestinal epithelial cells. Given evidence for TNF signaling in the development of colitis-associated carcinoma, this observation has significant implications for understanding the role of EGFR in maintaining intestinal epithelial cell homeostasis during cytokine-mediated inflammatory responses.     

Müllerian inhibiting substance blocks autophosphorylation of the EGF receptor by inhibiting tyrosine kinase

The fetal regressor Müllerian inhibiting substance (MIS), in concentrations as low as picomolar, inhibited the growth of A-431 cells and the autophosphorylation of its epidermal growth factor (EGF) receptor. The inhibition of membrane phosphorylation was due neither to the reduction of the total number of EGF receptor binding sites, nor to stimulation of intrinsic phosphates, but rather to inhibition of tyrosine kinase activity. MIS control of EGF receptor autophosphorylation by tyrosine kinase may be one mechanism by which Müllerian duct regression in the embryo and the inhibition of A-431 proliferation is initiated. In addition, MIS as an inhibitor of phosphorylation may furnish a tool to probe the role of membrane phosphorylation in growth control.     

Selective inhibition of human neutrophil functional responsiveness by erbstatin, an inhibitor of tyrosine protein kinase

The role of tyrosine kinases in the responses of human neutrophils to chemotactic factors was examined using the recently described inhibitor erbstatin. Pre-incubation with erbstatin decreased the amount of tyrosine phosphorylation induced by the formylated oligopeptide formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) without effecting the binding of [3H]-fMet-Leu-Phe. Erbstatin also dose-dependently inhibited the production of superoxide anion induced by fMet-Leu-Phe and platelet-activating factor, but did not affect the oxidative burst induced by either the calcium ionophore A23187 or the phorbol ester phorbol 12-myristate 13-acetate. Furthermore, erbstatin diminished the cytosolic acidification elicited by fMet-Leu-Phe, platelet-activating factor, and leukotriene B4. In contrast, erbstatin was without effect on the increase in the levels of cytoplasmic free calcium and polymerized actin elicited by fMet-Leu-Phe, C5a, leukotriene B4 and platelet-activating factor, whereas the increase in cytoplasmic free calcium elicited by platelet-derived growth factor was inhibited by erbstatin. In addition, erbstatin affected neither the release of elastase stimulated by these agonists nor the release of beta-glucosaminidase, lysozyme or vitamin B12-binding protein induced by fMet-Leu-Phe. These results indicate that tyrosine protein kinases are involved in the signaling pathways employed by chemotactic factors in the stimulation of selective functional responses (and superoxide production in particular) in human neutrophils.     

Activation of the nonreceptor protein tyrosine kinase Ack by multiple extracellular stimuli

Ack/Ack1 is a nonreceptor protein tyrosine kinase that comprises a tyrosine kinase core, an SH3 domain, a Cdc42-binding region, a Ralt homology region, and a proline-rich region. Here we describe a detailed characterization of the Ack protein as well as the chromosomal localization of human Ack (chromosome 3q29) and the primary structure of murine Ack. We demonstrate that Ack is ubiquitously expressed, with highest expression seen in thymus, spleen, and brain. Activation of integrins by cell adhesion on fibronectin leads to strong tyrosine phosphorylation and activation of Ack. Upon cell stimulation with EGF or PDGF, Ack is tyrosine-phosphorylated and recruited to activated EGF or PDGF receptors, respectively. A pool of endogenous Ack molecules is constitutively tyrosine-phosphorylated, even in starved cells. Moreover, tyrosine-phosphorylated Ack forms a stable complex with the adapter protein Nck via its SH2 domain. Finally, we have characterized a membrane-targeting sterile alpha motif-like domain in the amino terminus of Ack. Using several Ack mutants, we show that the amino-terminal and CRIB domains are necessary for Ack autophosphorylation, whereas the SH3 domain appears to have an autoinhibitory role. These experiments suggest a functional role for Ack as an early transducer of multiple extracellular stimuli.     

Thromboxane receptor activates the AMP-activated protein kinase in vascular smooth muscle cells via hydrogen peroxide

Thromboxane A2 receptor (TPr) stimulation induces cellular hypertrophy in vascular smooth muscle cells (VSMCs); however, regulation of VSMC hypertrophy remains poorly understood. Here we show that TPr stimulation activates AMP-activated kinase (AMPK), which in turn limits TPr-induced protein synthesis in VSMCs. Exposure of cultured VSMCs to either TPr agonists, IBOP and U46619, or exogenous hydrogen peroxide (H2O2) caused time- and dose-dependent AMPK activation, as evidenced by increased phosphorylation of both AMPK-Thr172 and acetyl-coenzyme A carboxylase-Ser79, a downstream enzyme of AMPK, whereas SQ29548, a selective TPr antagonist, significantly attenuated TPr-enhanced AMPK activation. In parallel, both IBOP and U46619 significantly increased the production of reactive oxygen species such as H2O2. Furthermore, adenoviral overexpression of catalase (an H2O2 scavenger) abolished, whereas superoxide dismutase (which catalyzes H2O2 formation) enhanced, IBOP-induced AMPK activation, suggesting that TPr-activated AMPK was mediated by H2O2. Consistently, exposure of VSMCs to either TPr agonists or exogenous H2O2 dose-dependently increased the phosphorylation of LKB1 (at serines 428 and 307), an AMPK kinase, as well as coimmunoprecipitation of AMPK with LKB1. In addition, direct mutagenesis of either Ser428 or Ser307 of LKB1 into alanine, like the kinase-dead LKB1 mutant, abolished both TPr-stimulated AMPK activation and coimmunoprecipitation. Finally, genetic inhibition of AMPK significantly accentuated IBOP-enhanced protein synthesis, whereas adenoviral overexpression of constitutively active AMPK abolished IBOP-enhance protein synthesis in VSMCs. We conclude that TPr stimulation triggers reactive oxygen species-mediated LKB1-dependent AMPK activation, which in return inhibits cellular protein synthesis in VSMCs.     

Mechanics of EGF receptor/ErbB2 kinase activation revealed by luciferase fragment complementation imaging

Binding of EGF to its receptor induces dimerization of the normally monomeric receptor. Activation of its intracellular tyrosine kinase then occurs through the formation of an asymmetric kinase dimer in which one subunit, termed the "receiver" kinase, is activated by interaction with the other subunit, termed the "activator" kinase [Zhang, et al. (2006) Cell 125: 1137-1149]. Although there is significant experimental support for this model, the relationship between ligand binding and the mechanics of kinase activation are not known. Here we use luciferase fragment complementation in EGF receptor (EGFR)/ErbB2 heterodimers to probe the mechanics of ErbB kinase activation. Our data support a model in which ligand binding causes the cis-kinase (the EGFR) to adopt the receiver position in the asymmetric dimer and to be activated first. If the EGF receptor is kinase active, this results in the phosphorylation of the trans-kinase (ErbB2). However, if the EGF receptor kinase is kinase dead, the ErbB2 kinase is never activated. Thus, activation of the kinases in the EGFR/ErbB2 asymmetric dimer occurs in a specific sequence and depends on the kinase activity of the EGF receptor.     

Phosphorylation of the hepatic EGF receptor with cAMP-dependent protein kinase

The 170 000 dalton hepatic epidermal growth factor (EGF) receptor is phosphorylated on serine and tyrosine residues. The evidence indicates that distinct protein kinases are involved. Since EGF and agents that elevate cAMP are believed to participate in the regulation of liver regeneration, we tested whether or not the catalytic subunit of cAMP-dependent protein kinase (catalytic subunit), a known serine kinase, would utilize the EGF receptor as a substrate. The catalytic subunit increased phosphorylation of the EGF receptor in purified rat liver plasma membranes. The serine specificity of the catalytic subunit was established by phosphoamino acid analysis of electrophoretically purified EGF receptor. The result was confirmed by catalytic subunit phosphorylation of affinity purified preparations of the EGF receptor. The rates of dephosphorylation of the membrane-associated EGF receptor phosphorylated on different residues were compared. Dephosphorylation of serine residues (after catalytic subunit phosphorylation) was considerably slower (t1/2 greater than 120 sec) than the removal of phosphotyrosine after stimulation with EGF (t1/2 less than 30 sec).     

Peroxisome proliferator-activated receptor gamma ligands inhibit Rho/Rho kinase pathway by inducing protein tyrosine phosphatase SHP-2

Although peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have an antihypertensive effect in vivo, the precise mechanism has not been fully elucidated. We examined their effects on Rho/Rho kinase pathway, a key regulator of vascular tone. In cultured rat aortic smooth muscle cells (RASMC), Rho kinase stimulated by angiotensin II was suppressed by the pretreatment with pioglitazone and troglitazone, and these effects were explained by the inhibition of the Rho translocation to the cell membrane. We evaluated the role of Vav, a GTP/GDP exchange factor upregulating Rho kinase activity, and Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2), a protein tyrosine phosphatase that dephosphorylated Vav and subsequently inactivated Rho kinase. Both pioglitazone and troglitazone upregulated SHP-2, particularly in the cytosolic fraction, and the SHP-2-bound Vav, and reduced the phosphorylation of Vav. Furthermore, 4-week treatment with pioglitazone lowered systolic blood pressure in spontaneously hypertensive rats (SHR) and suppressed the Rho/Rho kinase activity in aortic tissues isolated from SHR. Consistently, the expression of SHP-2 was upregulated in vascular tissues from pioglitazone-treated SHR. The phosphorylated Vav was increased in SHR, compared with that in normotensive Wistar-Kyoto rats (WKY), which was mitigated by pioglitazone. Finally, both basal and angiotensin II-stimulated levels of Rho kinase activity were greater in RASMC from SHR than those from WKY, and the enhanced Rho kinase activity was blocked by pioglitazone or troglitazone in both strains. Collectively, PPARgamma ligands inhibit the Rho/Rho kinase pathway through upregulation of cytosolic SHP-2 expression and inactivation of Vav, and may contribute to the hemodynamic, in addition to metabolic, action in hypertensive metabolic syndrome. The full text of this article is available online at http://circres.ahajournals.org.     

The Tie receptor tyrosine kinase is expressed by human hematopoietic progenitor cells and by a subset of megakaryocytic cells

Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting- gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation.