N-acetylcysteine inhibits angiotensin ii-mediated activation of extracellular signal-regulated kinase and epidermal growth factor receptor

Angiotensin II (Ang II) is known to stimulate reactive oxygen species (ROS) generation and epidermal growth factor (EGF) receptor transactivation to mediate growth-promoting signals such as extracellular signal-regulated kinase (ERK) in vascular smooth muscle cells (VSMCs). However, how ROS and EGF receptor interact to orchestrate these signals in VSMCs remains unclear. Here we found that an antioxidant, N-acetylcysteine, inhibited ERK activation and EGF receptor tyrosine phosphorylation induced by Ang II. Moreover, H(2)O(2) stimulates EGF receptor tyrosine phosphorylation and EGF receptor inhibitors attenuated H(2)O(2)-induced ERK activation. These data indicate that ROS mediate Ang II-induced EGF receptor transactivation, a critical mechanism for ERK-dependent growth in VSMCs.     

H2O2-induced activation of transcription factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2

Reactive oxygen species initiate multiple signal transduction pathways including tyrosine kinase signaling. Here, we demonstrate tyrosine phosphorylation of EGF receptor, STAT3, and, to a lesser extent, STAT1 upon H2O2 treatment of HER14 cells (NIH3T3 fibroblasts transfected with full-length EGF receptor). Maximum phosphorylation levels were observed in 5 min of stimulation at 1-2 mM H2O2. It has been shown that the intrinsic EGF-receptor tyrosine kinase is responsible for the receptor phosphorylation upon H2O2 stimulation. STAT3 and STAT1 activation in HER14 cells was demonstrated to depend on EGF receptor kinase activity, rather than JAK2 activity, while in both K721A and CD126 cells (NIH3T3 transfected with kinase-dead EGF receptor, and EGF receptor lacking major autophosphorylation sites, respectively) STAT1 and STAT3 tyrosine phosphorylation requires JAK2 kinase activity. Furthermore, STAT3 is constitutively phosphorylated in K721A and CD126 cells, and STAT1 H2O2-stimulated activation in these cells is much more prominent than in HER14. In all the cell lines used, Src-kinase activity was demonstrated to be unnecessary for ROS-initiated phosphorylation of STATs. Herein, we postulate that EGF receptor plays a role in H2O2-induced STAT activation in HER14 cells. Our data also prompted a hypothesis of constitutive inhibition of JAK2-dependent STAT activation in this cell line.     

Metalloprotease-mediated HB-EGF release regulates EGF receptor transactivation in A431 cells at oxidative stress

Previously, we showed that hydrogen peroxide (H₂O₂) induces the ligand-independent activation (transactivation) of EGF receptor in various cells overexpressing EGF receptor. In the present work, the mechanism of H₂O₂-induced EGF receptor transactivation was studied in A431 human epidermoid carcinoma cells. The autophosphorylation of the EGF receptor at tyrosine residues 1045, 1068, 1148, 1173, as well as the phosphorylation of tyrosine 845, was demonstrated. It has been shown that the tyrosine phosphorylation of the EGF receptor does not involve autophosphorylation at tyrosine 992. The blockage of the function of metalloproteases with broad-spectrum inhibitor GM6001 suppressed H₂O₂-induced phosphorylation of EGF receptor, which suggests the dependence of the transactivation on metalloprotease activity. To elucidate the possible role of EGF receptor agonists in its activation, antibodies against HB-EGF and TGF-α were used. H₂O₂-induced EGF receptor phosphorylation was inhibited by HB-EGF, but not TGF-α, a neutralizing antibody. We believe that the mechanism of transactivation of EGF receptor during oxidative stress is realized via autophosphorylation and includes HB-EGF as a necessary component of signal transduction mediated by metalloprotease activity.     

Cyclic strain activates redox-sensitive proline-rich tyrosine kinase 2 (PYK2) in endothelial cells

Proline-rich tyrosine kinase 2 (PYK2), structurally related to focal adhesion kinase, has been shown to play a role in signaling cascades. Endothelial cells (ECs) under hemodynamic forces increase reactive oxygen species (ROS) that modulate signaling pathways and gene expression. In the present study, we found that bovine ECs subjected to cyclic strain rapidly induced phosphorylation of PYK2 and Src kinase. This strain-induced PYK2 and Src phosphorylation was inhibited by pretreating ECs with an antioxidant N-acetylcysteine. Similarly, ECs exposed to H(2)O(2) increased both PYK2 and Src phosphorylation. An increased association of Src to PYK2 was observed in ECs after cyclic strain or H(2)O(2) exposure. ECs treated with an inhibitor to Src (PPI) greatly reduced Src and PYK2 phosphorylation, indicating that Src mediated PYK2 activation. Whereas the protein kinase C (PKC) inhibitor (calphostin C) pretreatment was shown to inhibit strain-induced NADPH oxidase activity, ECs treated with either calphostin C or the inhibitor to NADPH oxidase (DPI) reduced strain-induced ROS levels and then greatly inhibited the Src and PYK2 activation. In contrast to the activation of PYK2 and Src with calcium ionophore (ionomycin), ECs treated with a Ca(2+) chelator inhibited both phosphorylation, indicating that PYK2 and Src activation requires Ca(2+). ECs transfected with antisense to PKCalpha, but not antisense to PKCepsilon(,) reduced cyclic strain-induced PYK2 activation. These data suggest that cyclic strain-induced PYK2 activity is mediated via Ca(2+)-dependent PKCalpha that increases NADPH oxidase activity to produce ROS crucial for Src and PYK2 activation. ECs under cyclic strain thus activate redox-sensitive PYK2 via Src and PKC, and this PYK2 activation may play a key role in the signaling responses in ECs under hemodynamic influence.     

Unique regulation of c-Jun N-terminal kinase by PYK2/CAK-beta in angiotensin II-stimulated vascular smooth muscle cells

Activation of tyrosine kinases is believed to play a central role in angiotensin II (AngII) signaling. Here, we have investigated whether a tyrosine kinase, PYK2, is functionally involved in AngII-induced c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells (VSMCs). Adenovirus expressing PYK2 kinase-inactive mutant K457A or a tyrosine phosphorylation site mutant Y402F was transfected in VSMCs. AngII-induced JNK phosphorylation was markedly enhanced by K457A, whereas it was suppressed by Y402F. Protein synthesis induced by AngII was also enhanced by K457A and inhibited by Y402F. In this regard, K457A suppressed PYK2 kinase activation by AngII, whereas it enhanced AngII-induced PYK2 Tyr(402) phosphorylation. By contrast, Y402F inhibited PYK2 Tyr(402) phosphorylation, whereas it markedly enhanced AngII-induced PYK2 kinase activation. Thus, we conclude that PYK2 kinase activity negatively regulates JNK activation and protein synthesis, whereas Tyr(402) phosphorylation positively regulates these events in AngII-stimulated VSMCs, suggesting a unique role of PYK2 in mediating vascular remodeling.     

Nitric oxide inhibits angiotensin II-induced activation of the calcium-sensitive tyrosine kinase proline-rich tyrosine kinase 2 without affecting epidermal growth factor receptor transactivation.

In a previous study, we showed that nitric oxide donors and N-acetylcysteine, either alone or in combination, inhibited the activation of several mitogen-activated protein kinases by angiotensin II in rat cardiac fibroblasts (Wang, D., Yu, X., and Brecher, P. (1998) J. Biol. Chem. 273, 33027-33034). In the present study, we have focused on the mechanism by which nitric oxide exerts this effect on the activation of extracellular signal-regulated kinase (ERK). We contrasted the effects of nitric oxide on ERK activation by angiotensin II and epidermal growth factor (EGF), since the transactivation of the EGF receptor has been implicated as a response to angiotensin II. We found that nitric oxide inhibited ERK activation by angiotensin II but did not inhibit the relatively slight but significant transactivation of the EGF receptor by angiotensin II. The tyrphostin AG1478, known to inhibit EGF receptor phosphorylation, also inhibited the angiotensin II and EGF-induced activation of ERK, the phosphorylation of the EGF receptor, and the subsequent association of Shc and Grb2. Nitric oxide did not affect either EGF receptor phosphorylation or Shc-Grb2 activation induced by either Ang II or EGF. However, the activation of the calcium-sensitive tyrosine kinase PYK2, which occurred in response to angiotensin II, but not EGF, was inhibited by nitric oxide. The data suggested that PYK2 activation may be an important inhibitory site in signaling pathways affected by nitric oxide.     

Dependence of EGF receptor and STAT factor activation on redox of A431 cells

Reactive oxygen species (ROS) were established to play an important role in cellular signaling as second messengers by integrating different pathways. Recently, we showed that EGF initiated a rapid tyrosine phosphorylation of both EGF-receptor and STAT factors with simultaneous increase in the intracellular ROS level. Now, we have investigated the effect of intracellular red-ox state on EGF- and H2O2-induced activation of EGF receptor, STAT1 and STAT3. We demonstrated that the pretreatment of A431 cells with antioxidant N-acetyl-L-cysteine (NAC) partly reduced the level of EGF-induced phosphorylation of proteins under investigation. Besides, H2O2-induced activation of EGF receptor, and STAT factors was fully prevented by NAC pretreatment. The inhibition of ROS generation by DPI declined EGF-dependent activation of EGF receptor and STAT factors to basal level. Our results demonstrate the essential role of cellular red-ox status in the modulation of EGF-mediated activation of receptor and STAT factors. We have postulated that EGF-induced ROS generation is a very important initial event promoting physiological activation of EGF receptor and subsequent STAT factor activation.     

Cholecystokinin-stimulated protein kinase C-delta kinase activation,tyrosine phosphorylation,and translocation are mediated by Src tyrosine kinases in pancreatic acinar cells

Protein kinase C-delta (PKC-delta) is involved in growth, differentiation, tumor suppression, and regulation of other cellular processes. PKC-delta activation causes translocation, tyrosine phosphorylation, and serine-threonine kinase activity. However, little is known about the ability of G protein-coupled receptors to activate these processes or the mediators involved. In the present study, we explored the ability of the neurotransmitter/hormone, CCK, to stimulate these changes in PKC-delta and explored the mechanisms. In rat pancreatic acini under basal conditions, PKC-delta is almost exclusively located in cytosol. CCK and TPA stimulated a rapid PKC-delta translocation to membrane and nuclear fractions, which was transient with CCK. CCK stimulated rapid tyrosine phosphorylation of PKC-delta and increased kinase activity. Using tyrosine kinase (B44) and a tyrosine phosphatase inhibitor (orthovanadate), changes in both CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation were shown to correlate with changes in its kinase activity but not translocation. Both PKC-delta tyrosine phosphorylation and activation occur exclusively in particulate fractions. The Src kinase inhibitors, SU6656 and PP2, but not the inactive related compound, PP3, inhibited CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation and activation. In contrast, PP2 also had a lesser effect on CCK- but not TPA-stimulated PKC-delta translocation. CCK stimulated the association of Src kinases with PKC-delta, demonstrated by co-immunoprecipitation. These results demonstrate that CCKA receptor activation results in rapid translocation, tyrosine phosphorylation, and activation of PKC-delta. Stimulation of PKC-delta translocation precedes tyrosine phosphorylation, which is essential for activation to occur. Activation of Src kinases is essential for the tyrosine phosphorylation and kinase activation to occur and plays a partial role in translocation.     

Growth hormone signalling and apoptosis in neonatal rat cardiomyocytes

Growth hormone (GH) has been reported to be useful to treat heart failure. To elucidate whether GH has direct beneficial effects on the heart, we examined effects of GH on oxidative stress-induced apoptosis in cardiac myocytes. TUNEL staining and DNA ladder analysis revealed that hydrogen peroxide (H₂O₂)-induced apoptosis of cardiomyocytes was significantly suppressed by the pretreatment with GH. GH strongly activated extracellular signal-regulated kinases (ERKs) in cardiac myocytes and the cardioprotective effect of GH was abolished by inhibition of ERKs. Overexpression of dominant negative mutant Ras suppressed GH-stimulated ERK activation. Overexpression of Csk that inactivates Src family tyrosine kinases also inhibited ERK activation evoked by GH. A broad-spectrum inhibitor of protein tyrosine kinases (PTKs), genistein, strongly suppressed GH-induced ERK activation and the cardioprotective effect of GH against apoptotic cell death. GH induced tyrosine phosphorylation of EGF receptor and JAK2 in cardiac myocytes, and an EGF receptor inhibitor tyrphostin AG1478 and a JAK2 inhibitor tyrphostin B42 completely inhibited GH-induced ERK activation. Tyrphostin B42 also suppressed the phosphorylation of EGF receptor stimulated by GH. These findings suggest that GH has a direct protective effect on cardiac myocytes against apoptosis and that the effect of GH is attributed at least in part to the activation of ERKs through Ras and PTKs including JAK2, Src, and EGF receptor tyrosine kinase.     

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

In vascular smooth muscle (VSM) and manyother cells, G protein receptor-coupled activation of mitogen-activatedprotein kinases has been linked, in part, to increases in freeintracellular Ca²⁺. Previously, we demonstrated thationomycin-, angiotensin II-, and thrombin-induced activation ofextracellular signal-regulated kinase (ERK)1/2 in VSM cells wasattenuated by pretreatment with KN-93, a selective inhibitor of themultifunctional Ca²⁺/calmodulin-dependent protein kinase(CaM kinase II). In the present study, we show that theCa²⁺-dependent pathway leading to activation of ERK1/2 ispreceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation andis 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 inhibitsCa²⁺-dependent PYK2 activation and EGF receptor tyrosinephosphorylation in response to ionomycin, ATP, and platelet-derivedgrowth factor but has no effect on phorbol 12,13-dibutyrate- orEGF-induced responses. The results implicate CaM kinase II as anintermediate in the Ca²⁺/calmodulin-dependent activation of PYK2.

     

STAT1 and STAT3 activation by oxidative stress in A431 cells involves Src-dependent EGF receptor transactivation

Different cellular signal transduction cascades are affected by environmental stressors (UV-radiation, gamma-irradiation, hyperosmotic conditions, oxidants). In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of STATs in A431 carcinoma cells. Oxidative stress, initiated by addition of H2O2 (1-2 mM) to A431 cells, activates STAT3 and, to a lesser extent, STAT1 in dose- and time-dependent manner. Maximum phosphorylation levels were observed after a 2 minutes stimulation at 1-2 mM H2O2. Phosphorylation was blocked by AG1478, a pharmacological inhibitor of the epidermal growth factor receptor tyrosine kinase, implicating intrinsic EGF receptor tyrosine kinase in this process. Consistent with this observation, H2O2-stimulated EGFR tyrosine phosphorylation was abolished by specific Src kinase family inhibitor CGP77675, implicating Src in H2O2-induced EGFR activation. An essential role for Src and JAK2 in STATs activation was suggested by three findings. 1. Src kinase family inhibitor CGP77675 blocked STAT3 and STAT1 activation by H2O2 in a concentration-dependent manner. 2. In Src-/-fibroblasts, activation of both STAT3 and STAT1 by H2O2 was significantly attenuated. 3. Inhibiting JAK2 activity with the specific inhibitor AG490 reduced the level of H2O2-induced STAT3 phosphorylation, but not STAT1 in A431 cells. These data show essential roles for Src and JAK2 inactivation of STAT3. In contrast, H2O2-mediated activation of STAT1 requires only Src kinase activity. Herein, we postulate also that H2O2-induced STAT activation in carcinoma cells involves Src-dependent EGFR transactivation.     

Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor

Numerous external stimuli, including G protein-coupled receptor agonists, cytokines, growth factors, and steroids activate mitogen-activated protein kinases (MAPKs) through phosphorylation of the epidermal growth factor receptor (EGF-R). In immortalized hypothalamic neurons (GT1-7 cells), agonist binding to the gonadotropin-releasing hormone receptor (GnRH-R) causes phosphorylation of MAPKs that is mediated by protein kinase C (PKC)-dependent transactivation of the EGF-R. An analysis of the mechanisms involved in this process showed that GnRH stimulation of GT1-7 cells causes release/shedding of the soluble ligand, heparin binding epidermal growth factor (HB-EGF), as a consequence of metalloprotease activation. GnRH-induced phosphorylation of the EGF-R and, subsequently, of Shc, ERK1/2, and its dependent protein, p90RSK-1 (p90 ribosomal S6 kinase 1 or RSK-1), was abolished by metalloprotease inhibition. Similarly, blockade of the effect of HB-EGF with the selective inhibitor CRM197 or a neutralizing antibody attenuated signals generated by GnRH and phorbol 12-myristate 13-acetate, but not those stimulated by EGF. In contrast, phosphorylation of the EGF-R, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and metalloprotease activity. The signaling characteristics of HB-EGF closely resembled those of GnRH and EGF in terms of the phosphorylation of EGF-R, Shc, ERK1/2, and RSK-1 as well as the nuclear translocation of RSK-1. However, neither the selective Src kinase inhibitor PP2 nor the overexpression of negative regulatory Src kinase and dominant negative Pyk2 had any effect on HB-EGF-induced responses. In contrast to GT1-7 cells, human embryonic kidney 293 cells expressing the GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation. These data indicate that the GnRH-induced transactivation of the EGF-R and the subsequent ERK1/2 phosphorylation result from ectodomain shedding of HBEGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells.     

The molecular mechanism of EGF receptor activation in pancreatic beta-cells by thyrotropin-releasing hormone

Thyrotropin-releasing hormone (TRH) and its receptor subtype TRH receptor-1 (TRHR1) are found in pancreatic beta-cells, and it has been shown that TRH might have potential for autocrine/paracrine regulation through the TRHR1 receptor. In this paper, TRHR1 is studied to find whether it can initiate multiple signal transduction pathways to activate the epidermal growth factor (EGF) receptor in pancreatic beta-cells. By initiating TRHR1 G protein-coupled receptor (GPCR) and dissociated alphabetagamma-complex, TRH (200 nM) activates tyrosine residues at Tyr845 (a known target for Src) and Tyr1068 in the EGF receptor complex of an immortalized mouse beta-cell line, betaTC-6. Through manipulating the activation of Src, PKC, and heparin-binding EGF-like growth factor (HB-EGF), with corresponding individual inhibitors and activators, multiple signal transduction pathways linking TRH to EGF receptors in betaTC-6 cell line have been revealed. The pathways include the activation of Src kinase and the release of HB-EGF as a consequence of matrix metalloproteinase (MMP)-3 activation. Alternatively, TRH inhibited PKC activity by reducing the EGF receptor serine/threonine phosphorylation, thereby enhancing tyrosine phosphorylation. TRH receptor activation of Src may have a central role in mediating the effects of TRH on the EGF receptor. The activation of the EGF receptor by TRH in multiple circumstances may have important implications for pancreatic beta-cell biology.     

Cholecystokinin activates PYK2/CAKbeta by a phospholipase C-dependent mechanism and its association with the mitogen-activated protein kinase signaling pathway in pancreatic acinar cells.

PYK2/CAKbeta is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125(FAK)) that can be activated by a number of stimuli including growth factors, lipids, and some G protein-coupled receptors. Studies suggest PYK2/CAKbeta may be important for coupling various G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) cascade. The hormone neurotransmitter cholecystokinin (CCK) is known to activate both phospholipase C-dependent cascades and MAPK signaling pathways; however, the relationship between these remain unclear. In rat pancreatic acini, CCK-8 (10 nM) rapidly stimulated tyrosine phosphorylation and activation of PYK2/CAKbeta by both activation of high affinity and low affinity CCK(A) receptor states. Blockage of CCK-stimulated increases in protein kinase C activity or CCK-stimulated increases in [Ca(2+)](i), inhibited by 40-50% PYK2/CAKbeta but not p125(FAK) tyrosine phosphorylation. Simultaneous blockage of both phospholipase C cascades inhibited PYK2/CAKbeta tyrosine phosphorylation completely and p125(FAK) tyrosine phosphorylation by 50%. CCK-8 stimulated a rapid increase in PYK2/CAKbeta kinase activity assessed by both an in vitro kinase assay and autophosphorylation. Total PYK2/CAKbeta under basal conditions was largely localized (77 +/- 7%) in the membrane fraction, whereas total p125(FAK) was largely localized (86 +/- 3%) in the cytosolic fraction. With CCK stimulation, both p125(FAK) and PYK2/CAKbeta translocated to the plasma membrane. Moreover CCK stimulation causes a rapid formation of both PYK2/CAKbeta-Grb2 and PYK2/CAKbeta-Crk complexes. These results demonstrate that PYK2/CAKbeta and p125(FAK) are regulated differently by CCK(A) receptor stimulation and that PYK2/CAKbeta is probably an important mediator of downstream signals by CCK-8, especially in its ability to activate the MAPK signaling pathway, which possibly mediates CCK growth effects in normal and neoplastic tissues.     

PYK2/CAKbeta represents a redox-sensitive tyrosine kinase in vascular smooth muscle cells

In vascular smooth muscle cells (VSMCs), the focal adhesion kinase-related tyrosine kinase PYK2/CAKbeta is activated by vascular mitogens. Because reactive oxygen species (ROS) are assumed to mediate mitogenic signals by these agonists, we examined the possible link between ROS and PYK2 in cultured rat VSMCs. Here we present several lines of evidence showing that PYK2 is activated by ROS in VSMCs. The inhibitory effect of an antioxidant, N-acetyl-cysteine, on PYK2 activation by its specific agonists further suggests the pivotal role of PYK2 in vascular remodeling associated with enhanced ROS production.     

Fruit-juice concentrate of Asian plum inhibits growth signals of vascular smooth muscle cells induced by angiotensin II

Bainiku-ekisu, the fruit-juice concentrate of the Oriental plum (Prunus mume) has recently been shown to improve human blood fluidity. We have shown that angiotensin II (AngII) stimulates growth of vascular smooth muscle cells (VSMCs) through epidermal growth factor (EGF) receptor transactivation that involves reactive oxygen species (ROS) production. To better understanding the possible cardiovascular protective effect of Bainiku-ekisu, we have studied whether Bainiku-ekisu inhibits AngII-induced growth promoting signals in VSMCs. Bainiku-ekisu markedly inhibited AngII-induced EGF receptor transactivation. H(2)O(2)-induced EGF receptor transactivation was also inhibited by Bainiku-ekisu. Thus, Bainiku-ekisu markedly inhibited AngII-induced extracellular signal-regulated kinase (ERK) activation. However, EGF-induced ERK activation was not affected by Bainiku-ekisu. AngII stimulated leucine uptake in VSMCs that was significantly inhibited by Bainiku-ekisu. Also, Bainiku-ekisu possesses a potent antioxidant activity. Since the activation of EGF receptor, ERK and the production of ROS play central roles in mediating AngII-induced vascular remodeling, these data suggest that Bainiku-ekisu could exert a powerful cardiovascular protective effect with regard to cardiovascular diseases.     

Role of metalloproteinase-dependent EGF receptor activation in alpha-adrenoceptor-stimulated MAP kinase phosphorylation in GT1-7 neurons

Adrenoceptors (ARs) are involved in the regulation of gonadotropin-releasing hormone (GnRH) release from native and immortalized hypothalamic (GT1-7) neurons. However, the AR-mediated signaling mechanisms and their functional significance in these cells are not known. Stimulation of GT1-7 cells with the alpha1-AR agonist, phenylephrine (Phe), causes phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinases that is mediated by protein kinase C (PKC)-dependent transactivation of the epidermal growth factor receptor (EGF-R). Phe stimulation causes shedding of the soluble ligand, heparin-binding EGF (HB-EGF), as a consequence of matrix metalloproteinase (MMP) activation. Phe-induced phosphorylation of the EGF-R, and subsequently of Shc and ERK1/2, was attenuated by inhibition of MMP or HB-EGF with the selective inhibitor, CRM197, or by a neutralizing antibody. In contrast, phosphorylation of the EGF-R, Shc and ERK1/2 by EGF and HB-EGF was independent of PKC and MMP activity. Moreover, inhibition of Src attenuated ERK1/2 responses by Phe, but not by HB-EGF and EGF, indicating that Src acts upstream of the EGF-R. Consistent with a potential role of reactive oxygen species (ROS), Phe-induced phosphorylation of EGF-R was attenuated by the antioxidant, N-acetylcysteine. These data suggest that activation of the alpha1-AR causes phosphorylation of ERK1/2 through activation of PKC, ROS and Src, and shedding of HB-EGF, which binds to and activates the EGF-R.