P-Tyr Content Research Articles

Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells.

Activation of ERK1/2 implies the phosphorylation of tyrosine (pTyr) and threonine (pThr) by MEK1/2; both reactions were thought to be cytoplasmic, promoting ERK to reach the nucleus where it activates several transcription factors. In addition, H2O2 concentrations are known to modulate ERK intracellular translocation, which impacts on cellular proliferation. In this context, the objective of this work was to study the sequence of ERK phosphorylation under two redox conditions and to analyze a putative mitochondrial contribution to this process, in LP07 murine lung cells. A time-course of H2O2 administration was used and ERK phosphorylation was analyzed in cytosol, mitochondria and nuclei. At 1μM H2O2, a proliferative redox stimulus, immunoblot revealed a fast and transient increase in cytosol pTyr and a sustained increase in mitochondrial pTyr content. The detection for pThr/pTyrERK (2pERK) showed in cytosol a marked increase at 5 minutes with a fast dephosphorylation after that time, for both H2O2 concentrations. However, at 50 μM H2O2, an anti-proliferative condition, 2pERK was gradually retained in mitochondria. Interestingly, these results were confirmed by in vivo experiments using mice treated with a highly oxidizing agent [H2O2]. By the use of two ERK2 mutant constructions, where Tyr and Thr were replaced by alanine, we confirmed that 2pERK relied almost completely on pThr183. Confocal microscopy confirmed ERK subcellular distribution dependence on the incidence of cytosolic pTyr and mitochondrial pThr at 1μM H2O2. This work shows for the first time, both in vitro and in vivo, an ERK cycle involving a cross-talk between cytosol and mitochondria phosphorylation events, which may play a significant role in cell cycle progression, proliferation or differentiation under two different redox conditions.

Poly(colloid)s: "Polymerization" of Poly(l-tyrosine)-silica Composite Particles through the Photoinduced Cross-Linking of Unmodified Proteins Method.

Photoinduced cross-linking of unmodified proteins, PICUP, was extended to core-shell silica-polypeptide composite particles to produce poly(colloid)s. Silica particles coated with poly(l-tyrosine), PTYR-SiO2, served as the monomer units. The PICUP reaction accomplished the formation of dityrosil linkages between the tyrosine units by illumination of photo-oxidizing ruthenium(II) bipyridyl catalyst under physiological conditions. The PICUP method was compared with an enzymatic route intermediated by horseradish peroxidase as catalyst. The PTYR-SiO2 particles feature high PTYR content in the shell, which facilitated the formation of heavily cross-linked but unstructured aggregates. After magnetic alignment of superparamagnetic PTYR-SiO2-cobalt composite particles, only the PICUP approach enabled the preparation of isolated chain-like poly(colloid)s. The cross-linking products were confirmed by FTIR. The native secondary structure of poly(l-tyrosine) is preserved in these poly(colloid)s. Because the PICUP reaction does not require the modification of the polypeptide structure, the cross-linked PTYR will retain its characteristic functions as a poly(amino acid). The PICUP method opens the door to a variety of PTYR-based poly(colloid) architectures.

Abstract 230: A novel quantitative phosphoproteomic approach in mouse xenograft models to identify Src oncogenic signaling in colorectal cancer

Abstract The non receptor tyrosine kinase (TyrK) Src mediates signal transduction induced by growth factors and integrins. Src also exhibits oncogenic activity when deregulated, a situation frequently observed in colorectal cancer (CRC). Moreover, the extent of kinase deregulation often correlates with malignant potential, in agreement with an important role Src in this tumorigenic process. However, the exact role of Src in CRC is only partially elucidated. Src has been implicated in growth and invasiveness of various CRC cells. A previous SILAC quantitative phosphoproteomic analysis allowed the characterization of Src signaling leading to CRC cells growth and invasion (Leroy et al, Cancer Res, 2009, 15:2279-86). Interestingly, Src also increased tumor growth in nude mice engraft with human SW620 CRC cells. This biological effect correlated with a strong increase of tyrosine phosphorylation (pTyr) content in Src-positive tumors. Here, we wished to characterize Src oncogenic signaling in these xenograft tumor models. To this end, we have developed an innovative quantitative phosphoproteomic analysis coupled to stable isotope labeling in mice named SILAX for Stable Isotope Labeling in mouse Xenografts. This method is based on tumor heavy isotope labeling in nude mice by addition of [13C]-Lysine into the food. An incorporation level >87% was obtained in subcutaneous tumors after 30 days of heavy lysine diet. This approach allowed the identification of 61 proteins which exhibited a significant increase in and/or association with pTyr upon Src expression in tumors. These mainly include vesicular trafficking, signaling and RNA binding proteins. Interestingly, we identified the novel vesicular trafficking protein TOM1L1 (Franco et al, Mol Cell Biol, 2006, 26:1932-47) as a specific regulator of Src oncogenic signaling of CRC cells in vivo. While TOM1L1 depletion poorly affected Src-induced CRC proliferation in vitro, it significantly inhibited tumor growth in nude mice. This data suggests that vesicular trafficking plays an important role in Src-induced CRC tumor growth and validate SILAX as a valuable approach to decipher TyrK signaling in mouse xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 230. doi:1538-7445.AM2012-230

Muscle contractions, AICAR, and insulin cause phosphorylation of an AMPK-related kinase

We hypothesized that AMP-activated protein kinase-related kinase 5 (ARK5)/novel kinase family 1 (NUAK1), an AMP-activated protein kinase (AMPK)-related kinase that has been found to be stimulated by protein kinase B (Akt), would be expressed in rat skeletal muscle and activated by electrically elicited contractions, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), or insulin. We verified expression of ARK5 in muscle through RT-PCR and Western blot. Cross-reactivity of ARK5 immunoprecipitates with antibodies against phospho-AMPK was increased by approximately 30% by muscle contractions and approximately 60% by incubation of muscle with AICAR. AMPK was not detected in the ARK5 immunoprecipitates. Despite the apparent increase in phosphorylation of ARK5 at a site essential to its activation, neither contractions nor AICAR increased ARK5 activity. For muscles from animals injected with saline or insulin, we probed nonimmunoprecipitated samples in sequence for phosphotyrosine (P-Tyr), ARK5, and phosphorylated substrates of Akt (P-AS) and found that the ARK5 band could be precisely superimposed on phosphoprotein bands from the P-Tyr and P-AS blots. In the band corresponding to ARK5, insulin increased P-Tyr content by approximately 45% and cross-reactivity with the antibody against P-AS by approximately threefold. We also detected ARK5 in phosphotyrosine immunoprecipitates. Our data suggest that increased phosphorylation of ARK5 by muscle contractions or exposure to AICAR is insufficient to activate ARK5 in skeletal muscle, suggesting that some other modification (e.g., phosphorylation on tyrosine or by Akt) may be necessary to its activity in muscle.

SHP-2-Dependent Mitogen-Activated Protein Kinase Activation Regulates EGFRvIII but not Wild-Type Epidermal Growth Factor Receptor Phosphorylation and Glioblastoma Cell Survival

In human glioblastomas, the most common mutation of epidermal growth factor receptor (EGFR) is an in-frame deletion of an 801-bp sequence in the extracellular domain of EGFR termed EGFRvIII. The EGFRvIII does not bind ligand EGF but has constitutive tyrosine phosphorylation (pTyr) content and kinase activity that result in enhanced transformation, reduced apoptosis, and resistance to therapy. Here we report that the protein tyrosine phosphatase SHP-2 modulates a mitogen-activated protein kinase (MAPK) kinase (MEK)-mediated signaling pathway that regulates EGFRvIII pTyr and cell survival in U87MG.EGFRvIII cells. Overexpression of the phosphatase-inactive form of SHP-2 inhibited EGFRvIII pTyr by decreasing MAPK phosphorylation. Consistent with this, we observed that the MEK inhibitor PD98059, but not the phosphatidylinositol 3'-kinase inhibitor LY294002, inhibited EGFRvIII pTyr. Furthermore, constitutive EGFRvIII pTyr content observed in U87MG, LN229, and U373MG glioblastoma cells, but not in NR6.EGFRvIII fibroblasts, correlated with elevated MAPK levels in these cells. Interestingly, LY294002, but not PD98059, inhibited wild-type EGFR pTyr in response to EGF treatment in U87MG parental cells and in wild-type EGFR-overexpressing U87MG cells. Inhibition of EGFRvIII pTyr by PD98059 was not observed to be phosphorylation site specific. However, LY294002 more specifically inhibited wild-type EGFR pTyr at residues Tyr(992) and Tyr(1068) in the COOH terminus. Treatment of U87MG.EGFRvIII cells with PD98059, but not LY294002, also resulted in increased cell death in response to cisplatin. Collectively, a distinct MEK-mediated pathway in human glioblastoma cells appears to differentially modulate EGFRvIII and wild-type EGFR pTyr, and inhibition of the MAPK pathway sensitizes EGFRvIII-containing human glioblastoma cells to cisplatin-induced cell death.

Insulin Stimulates PKCζ-mediated Phosphorylation of Insulin Receptor Substrate-1 (IRS-1): A SELF-ATTENUATED MECHANISM TO NEGATIVELY REGULATE THE FUNCTION OF IRS PROTEINS

Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKCzeta fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKCzeta in Fao cells, by infection of the cells with adenovirus-based PKCzeta construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR.IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKCzeta was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC alpha, delta, or eta. Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKCzeta, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKCzeta inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKCzeta itself. These findings implicate PKCzeta as a key element in a multistep negative feedback control mechanism of IRS-1 functions.

Tyrosyl‐phosphorylated proteins are involved in regulation of meiosis in the rat egg

Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P-Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P-Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P-Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P-Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl-phosphorylated, mitogen-activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P-Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin-induced Ca2+ rise is sufficient to imitate fertilization-induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg. Mol. Reprod. Dev. 49:176–185, 1998. © 1998 Wiley-Liss, Inc.

Tyrosyl-phosphorylated proteins are involved in regulation of meiosis in the rat egg.

Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P-Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P-Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P-Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P-Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl-phosphorylated, mitogen-activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P-Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin-induced Ca2+ rise is sufficient to imitate fertilization-induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg.

Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells.

In src- and ras-transformed cells, tyrosine phosphorylation of adherens junction (AJ) components is related to impairment of cell-cell adhesion. In this paper we report that in human endothelial cells (EC), tyrosine phosphorylation of AJ can be a physiological process regulated by cell density. Immunofluorescence analysis revealed that a phosphotyrosine (P-tyr) antibody could stain cell-cell junctions only in sparse or loosely confluent EC, while the staining was markedly reduced in tightly confluent cultures. This process was reversible, since on artificial wounding of EC monolayers, the cells at the migrating front reacquired P-tyr labelling at cell contacts. In EC, the major cadherin at intercellular AJ is the cell-type-specific VE-cadherin. We therefore analyzed whether this molecule was at least in part responsible for the changes in P-tyr content at cell junctions. Tyrosine phosphorylation of VE-cadherin, beta-catenin and p120, occurred in looser AJ, i.e. in recently confluent cells, and was notably reduced in tightly confluent cultures. Changes in P-tyr content paralleled changes in the molecular organization of AJ. VE-cadherin was mostly associated with beta-catenin and p120 in loose EC monolayers, while in long-confluent cells, these two catenins were largely replaced by plakoglobin. Inhibition of P-tyr phosphatases (PTPases) by PV markedly augmented the P-tyr content of VE-cadherin, which bound p120 and beta-catenin more efficiently, but not plakoglobin. Transfection experiments in CHO cells showed that p120 could bind to a VE-cadherin cytoplasmic region different from that responsible for beta-catenin binding, and PV stabilized this association. Overall these data indicate that endothelial AJ are dynamic structures that can be affected by the state of confluence of the cells. Tyrosine phosphorylation of VE-cadherin and its association to p120 and beta-catenin characterizes early cell contacts, while the formation of mature and cytoskeleton-connected junctions is accompanied by dephosphorylation and plakoglobin association.

Protein tyrosine phosphorylation in the cyanobacterium Anabaena sp. strain PCC 7120.

Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [gamma-32P]ATP but not [alpha-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density.

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH2Cl: synergistic role of phosphotyrosine and intracellular Ca2+ concentration.

The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not the non-membrane-permeating chloramine, taurine-NHCl) induced detachment of fetal mouse cardiac myocytes and other cell types (fibroblasts, epithelial cells, and endothelial cells) from the culture dish, concomitant with cell shrinkage ("peeling off"). Stimulated human neutrophils also induced peeling off of cultured mouse cardiac myocytes when the latter were pretreated with inhibitors of .OH and elastase. Immunofluorescence microscopy revealed that the NH2Cl-induced peeling off of WI-38 fibroblasts is accompanied by disorganization of integrin alpha 5 beta 1, vinculin, stress fibers, and phosphotyrosine (p-Tyr)-containing proteins. Decrease in the content of the p-Tyr-containing proteins of the NH2Cl-treated cells was analyzed by immunoblotting techniques. Coating of fibronectin on the culture dish prevented both NH2Cl-induced peeling off and a decrease in p-Tyr content. Preincubation with a protein-tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), also prevented NH2Cl-induced peeling off, suggesting that dephosphorylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling off was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiac myocytes and WI-38 fibroblasts. The absence of extracellular Ca2+ prevented both NH2Cl-induced peeling off and increased [Ca2+]i, both of which did occur on subsequent incubation of the cells in Ca2+-containing medium. These observations suggest that an increase in [Ca2+]i is also necessary for peeling off. Depletion of microsomal and cytosolic Ca2+ by incubation with the microsomal Ca2+-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ) plus EGTA prevented both NH2Cl-induced increases in [Ca2+]i and peeling off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl-induced [Ca2+]i increment. A combination of p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinase) and an increase in [Ca2+]i by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphorylation and increased [Ca2+]i in NH2Cl-induced peeling off.

Nicotinic Agonists, Phorbol Esters, and Growth Factors Activate Two Extracellular Signal‐Regulated Kinases, ERK1 and ERK2, in Bovine Chromaffin Cells

Treatment of bovine chromaffin cells with nicotinic agonists, phorbol esters, and growth factors increases protein kinase activity toward microtubule-associated protein-2 and myelin basic protein (MBP) in vitro. To characterize the kinases that are activated by these agents, we separated chromaffin cell proteins by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels into which MBP had been incorporated, allowed the proteins to renature, and then assayed MBP kinase activity by incubating the gels with [gamma-32P]ATP. Chromaffin cells contain a family of kinases that phosphorylate MBP in vitro. Two of these kinases, of M(r) 46,000 and 42,000 (PK46 and PK42), were activated by treatment of the cells with dimethylphenylpiperazinium (DMPP), phorbol 12,13-dibutyrate (PDBu), or insulin-like growth factor I (IGF-I). Activation of PK46 and PK42 by DMPP was dependent on extracellular Ca2+, whereas the effects of PDBu and IGF-I were Ca2+ independent. Down-regulation of protein kinase C by incubation of the cells with PDBu abolished the activation of PK46 and PK42 by DMPP, PDBu, and IGF-I. Staurosporine, a protein kinase C inhibitor, prevented the activation of PK46 and PK42 by DMPP and PDBu but did not block the activation of these kinases by IGF-I. Immunoblotting experiments with antiphosphotyrosine (anti-PTyr) antibodies demonstrated that agents that increased the kinase activities of PK46 and PK42 also increased the apparent PTyr content of M(r) 46,000 and 42,000 proteins. PK46 and PK42 comigrated with proteins that reacted with antibodies against extracellular signal-regulated kinases (ERKs). Thus, PK46 and PK42 appear to be the bovine homologues of ERK1 and ERK2.(ABSTRACT TRUNCATED AT 250 WORDS)

Components of signaling pathways for insulin and insulin-like growth factor-I in muscle myoblasts and myotubes.

To survey and compare the signaling pathways from the insulin and insulin-like growth factor-I (IGF-I) receptors in undifferentiated and differentiated muscle cells, we examined the phosphotyrosine (Ptyr)-containing polypeptides elicited in L6 and Sol8 myoblasts and myotubes by the combination of insulin and IGF-I. These polypeptides were detected by immunoblotting with antibodies against Ptyr. In the L6 myoblasts and myotubes and the Sol8 myoblasts, Ptyr polypeptides of approximately 240, 175, 115, 100, 41, and 37 kilodaltons (kDa) appeared in response to insulin-IGF-I. With the Sol8 myotubes, the 240-, 175-, and 37-kDa Ptyr polypeptides were detected in basal cells, and only the Ptyr content of the 175-kDa one increased in response to insulin-IGF-I. The polypeptides of 175, 41, and 37 kDa were tentatively identified as the insulin receptor substrate 1 (IRS1) and extracellular signal-regulated kinases 1 and 2 (ERK1 and -2), respectively, by immunoblotting with antibodies specific for these proteins, and the 115- and 100-kDa polypeptides are probably the beta-subunits of the insulin and IGF-I receptors. The amounts of IRS1, ERK1, and ERK2 were roughly the same in the L6 and Sol8 myoblasts and myotubes. Thus, differentiation of the myoblasts to myotubes was not accompanied by the detectable appearance of new insulin-IGF-I-elicited Ptyr polypeptides or marked changes in the amounts of known participants in their signaling pathways.

Angiotensin II Stimulates Protein-Tyrosine Phosphorylation in a Calcium-Dependent Manner

Cellular responses to epidermal growth factor (EGF) are dependent on the tyrosine-specific protein kinase activity of the cell-surface EGF receptor. Previous studies using WB rat liver epithelial cells have detected at least 10 proteins whose phosphotyrosine (P-Tyr) content is increased by EGF. In this study, we have examined alternate modes of activating tyrosine phosphorylation. Treatment of WB cells with hormones linked to Ca2+ mobilization and protein kinase C (PKC) activation, including angiotensin II, [Arg8]vasopressin, or epinephrine, stimulated rapid (less than or equal to 15-s) and transient increases in the P-Tyr content of several proteins (p120/125, p75/78, and p66). These proteins, detected by anti-P-Tyr immunoblotting, were similar in molecular weight to a subset of EGF-sensitive P-Tyr-containing proteins (P-Tyr-proteins). The increased P-Tyr content was confirmed by [32P]phosphoamino acid analysis of proteins recovered by anti-P-Tyr immunoprecipitation. Elevating intracellular [Ca2+] with the ionophore A23187 or ionomycin or with the tumor promoter thapsigargin mimicked the effects of hormones on tyrosine phosphorylation, whereas treatment with a PKC-activating phorbol ester did not. In addition, responses to angiotensin II were not diminished in PKC-depleted cells. Ca2+ mobilization, measured by fura-2 fluorescence, was coincident with the increase in tyrosine phosphorylation in response to angiotensin II or thapsigargin. Loading cells with the intracellular Ca2+ chelator bis-(o-aminophenoxy)ethane-N ,N ,N' , N'-tetraacetic acid (BAPTA) inhibited the appearance of all P-Tyr-proteins in response to angiotensin II, thapsigargin, or ionophores, as well as two EGF-stimulated P-Tyr-proteins. The majority of EGF-stimulated P-Tyr-proteins were not affected by BAPTA. These studies indicate that angiotensin II can alter protein-tyrosine phosphorylation in a manner that is secondary to, and apparently dependent on, Ca2+ mobilization. Thus, ligands such as EGF and angiotensin II, which act through distinct types of receptors, may activate secondary pathways involving tyrosine phosphorylation. These results also raise the possibility that certain growth-promoting effects of Ca2+ -mobilizing agents such as angiotensin II may be mediated via tyrosine phosphorylation.

Angiotensin II stimulates protein-tyrosine phosphorylation in a calcium-dependent manner.

Cellular responses to epidermal growth factor (EGF) are dependent on the tyrosine-specific protein kinase activity of the cell-surface EGF receptor. Previous studies using WB rat liver epithelial cells have detected at least 10 proteins whose phosphotyrosine (P-Tyr) content is increased by EGF. In this study, we have examined alternate modes of activating tyrosine phosphorylation. Treatment of WB cells with hormones linked to Ca2+ mobilization and protein kinase C (PKC) activation, including angiotensin II, [Arg8]vasopressin, or epinephrine, stimulated rapid (less than or equal to 15-s) and transient increases in the P-Tyr content of several proteins (p120/125, p75/78, and p66). These proteins, detected by anti-P-Tyr immunoblotting, were similar in molecular weight to a subset of EGF-sensitive P-Tyr-containing proteins (P-Tyr-proteins). The increased P-Tyr content was confirmed by [32P]phosphoamino acid analysis of proteins recovered by anti-P-Tyr immunoprecipitation. Elevating intracellular [Ca2+] with the ionophore A23187 or ionomycin or with the tumor promoter thapsigargin mimicked the effects of hormones on tyrosine phosphorylation, whereas treatment with a PKC-activating phorbol ester did not. In addition, responses to angiotensin II were not diminished in PKC-depleted cells. Ca2+ mobilization, measured by fura-2 fluorescence, was coincident with the increase in tyrosine phosphorylation in response to angiotensin II or thapsigargin. Loading cells with the intracellular Ca2+ chelator bis-(o-aminophenoxy)ethane-N ,N ,N' , N'-tetraacetic acid (BAPTA) inhibited the appearance of all P-Tyr-proteins in response to angiotensin II, thapsigargin, or ionophores, as well as two EGF-stimulated P-Tyr-proteins. The majority of EGF-stimulated P-Tyr-proteins were not affected by BAPTA. These studies indicate that angiotensin II can alter protein-tyrosine phosphorylation in a manner that is secondary to, and apparently dependent on, Ca2+ mobilization. Thus, ligands such as EGF and angiotensin II, which act through distinct types of receptors, may activate secondary pathways involving tyrosine phosphorylation. These results also raise the possibility that certain growth-promoting effects of Ca2+ -mobilizing agents such as angiotensin II may be mediated via tyrosine phosphorylation.

The insulinomimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells.

H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate, or H2O2 alone. Employing Western blotting with anti-P-Tyr antibodies, we have identified in Fao cells at least four proteins (pp180, 150, 114, and 100) whose P-Tyr content is rapidly increased upon treatment of the cells with 3 mM H2O2. Tyrosine phosphorylation of these and additional proteins was markedly potentiated (6-10-fold) when 100 microM sodium orthovanadate was added together with H2O2. The effects of H2O2 and vanadate on protein tyrosine phosphorylation were rapid and specific. The enhanced tyrosine phosphorylation was accompanied by a concomitant inhibition of a cytosolic protein tyrosine phosphatase activity. The latter was inhibited by 50% in 3 mM H2O2-treated cells. The inhibitory effect was augmented in the combined presence of H2O2 and vanadate. Half- and maximal effects of vanadate were obtained at 15 microM and 1 mM, respectively. Vanadate (1 mM) alone, added to the cells, had only a trivial effect on protein tyrosine phosphatase activity. A 45-s challenge with insulin (10(-7) M) of cells pretreated with H2O2 largely mimicked the potentiating effects of vanadate on protein tyrosine phosphorylation but not on protein tyrosine phosphatase activity. Our results suggest the involvement of multiple tyrosine-phosphorylation proteins in mediating the biological effects of H2O2/vanadate. Their enhanced phosphorylation can be attributed at least in part, to the inhibitory effects exerted by H2O2 alone, or in combination with vanadate, on protein tyrosine phosphatase activity. The similarity between proteins phosphorylated in Fao cells in response to H2O2/vanadate or H2O2/insulin, suggests that either treatment stimulates protein tyrosine kinases having similar substrate specificities. The insulin receptor kinase is a likely candidate as its activity is markedly enhanced either by insulin (plus H2O2) or by H2O2/vanadate.

The epidermal growth factor receptor tyrosine kinase in liver epithelial cells: The effect of ligand-dependent changes in cellular location

Abstract Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.