Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation

Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.     

Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation

The aryl hydrocarbon receptor (AhR) mediates many toxic effects of environmental pollutants. AhR also interacts with multiple growth factor-driven signaling pathways. In the course of examining effects of growth factors on proliferation of human colon cancer cells, we identified cross talk between AhR and the epidermal growth factor receptor (EGFR). In the present work, we explored underlying signal transduction mechanisms and functional consequences of this interaction. With the use of two human colon cancer cell lines, H508 and SNU-C4, we examined the effects of AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell proliferation and activation of EGFR, ERK1/2, and Src kinases. In colon cancer cells, 5-day incubation with TCDD stimulated a twofold dose-dependent increase in cell proliferation that was detectable with 1 nM and maximal with 30 nM TCDD. TCDD induced dose- and time-dependent phosphorylation of EGFR (Tyr845) and ERK1/2; maximal phosphorylation was observed 5 to 10 min after addition of 30 nM TCDD. Both TCDD-induced ERK1/2 phosphorylation and cell proliferation were abolished by AhR small interfering RNA, AhR-specific inhibitor CH223191, Src kinase inhibitor PP2, neutralizing antibodies against matrix metalloproteinase 7, heparin-binding-EGF-like growth factor and EGFR, EGFR inhibitors (AG1478 and PD168393), and MEK1 inhibitor PD98059. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527). These data support novel observations that, in human colon cancer cells, Src-mediated cross talk between aryl hydrocarbon and EGFR results in ERK1/2 activation, thereby stimulating cell proliferation.     

Src-mediated RGS16 tyrosine phosphorylation promotes RGS16 stability

The amplitude of signaling evoked by stimulation of G protein-coupled receptors may be controlled in part by the GTPase accelerating activity of the regulator of G protein signaling (RGS) proteins. In turn, subcellular targeting, protein-protein interactions, or post-translational modifications such as phosphorylation may shape RGS activity and specificity. We found previously that RGS16 undergoes tyrosine phosphorylation on conserved tyrosine residues in the RGS box. Phosphorylation on Tyr(168) was mediated by the epidermal growth factor receptor (EGFR). We show here that endogenous RGS16 is phosphorylated after epidermal growth factor stimulation of MCF-7 cells. In addition, p60-Src or Lyn kinase phosphorylated recombinant RGS16 in vitro, and RGS16 underwent phosphorylation in the presence of constitutively active Src (Y529F) in EGFR(-) CHO-K1 cells. Blockade of endogenous Src activity by selective inhibitors attenuated RGS16 phosphorylation induced by pervanadate or receptor stimulation. Furthermore, the rate of RGS16 degradation was reduced in cells expressing active Src or treated with pervanadate or a G protein-coupled receptor ligand (CXCL12). Induction of RGS16 tyrosine phosphorylation was associated with increased RGS16 protein levels and enhanced GAP activity in cell membranes. These results suggest that Src mediates RGS16 tyrosine phosphorylation, which may promote RGS16 stability.     

Src-mediated inter-receptor cross-talk between the Na+/K+-ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases

Binding of ouabain to Na(+)/K(+)-ATPase activates tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), Src, and p42/44 mitogen-activated protein kinases (MAPKs) in both cardiac myocytes and A7r5 cells. Here, we explored the roles of Src and the EGFR in the ouabain-invoked pathways that lead to the activation of MAPKs. Exposure of A7r5 and LLC-PK1 cells to ouabain caused a dose-dependent inhibition of Na(+)/K(+)-ATPase activity, which correlated well with ouabain-induced activation of Src and MAPKs in these cells. Immunoprecipitation experiments showed that ouabain stimulated Src binding to Na(+)/K(+)-ATPase in a dose- and time-dependent manner and increased phosphorylation of Src at Tyr(418) but had no effect on Tyr(529) phosphorylation. Ouabain failed to activate MAPKs in A7r5 cells that were pretreated with the Src inhibitor PP2 and in SYF cells in which Src family kinases are knocked out. Preincubation with AG1478, but not AG1295, also blocked the effects of ouabain on p42/44 MAPKs in A7r5 cells. Significantly, both herbimycin A and PP2 abrogated ouabain-induced but not epidermal growth factor-induced Src binding to the EGFR and the subsequent EGFR tyrosine phosphorylation. Ouabain also failed to affect tyrosine phosphorylation of the EGFR in SYF cells. In addition, unlike epidermal growth factor, ouabain did not increase EGFR autophosphorylation at Tyr(1173). These findings clearly indicate that ouabain transactivates the EGFR by activation of Src and stimulation of Src binding to the EGFR. Furthermore, we found that the transactivated EGFR was capable of recruiting and phosphorylating the adaptor protein Shc. This resulted in increased binding of another adaptor protein Grb2 to the Src-EGFR complex and the subsequent activation of Ras and MAPKs. Taken together, these new findings suggest that Src mediates the inter-receptor cross-talk between Na(+)/K(+)-ATPase and the EGFR to transduce the signals from ouabain to the Ras/MAPK cascade.     

Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 microg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak 2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.     

Activation of downstream signals by the long form of the leptin receptor

The adipocyte-derived hormone leptin signals the status of body energy stores by activating the long form of the leptin receptor (LRb). Activation of LRb results in the activation of the associated Jak2 tyrosine kinase and the transmission of downstream phosphotyrosine-dependent signals. We have investigated the signaling function of mutant LRb intracellular domains under the control of the extracellular erythropoietin (Epo) receptor. By using this system, we confirm that two tyrosine residues in the intracellular domain of murine LRb become phosphorylated to mediate LRb signaling; Tyr(985) controls the tyrosine phosphorylation of SHP-2, and Tyr(1138) controls STAT3 activation. We furthermore investigated the mechanisms by which LRb controls downstream ERK activation and c-fos and SOCS3 message accumulation. Tyr(985)-mediated recruitment of SHP-2 does not alter tyrosine phosphorylation of Jak2 or STAT3 but results in GRB-2 binding to tyrosine-phosphorylated SHP-2 and is required for the majority of ERK activation during LRb signaling. Tyr(985) and ERK activation similarly mediate c-fos mRNA accumulation. In contrast, SOCS3 mRNA accumulation requires Tyr(1138)-mediated STAT3 activation. Thus, the two LRb tyrosine residues that are phosphorylated during receptor activation mediate distinct signaling pathways as follows: SHP-2 binding to Tyr(985) positively regulates the ERK --> c-fos pathway, and STAT3 binding to Tyr(1138) mediates the inhibitory SOCS3 pathway.     

Ligand-independent phosphorylation of Y869 (Y845) links mutant EGFR signaling to stat-mediated gene expression

Activating mutants of EGFR have been identified in a subset of non-small-cell lung cancers. To investigate mutant-driven signaling, we focused on Y869, a residue in the same activation loop where the L858R and L861Q mutations are located. We observed ligand-independent phosphorylation of Y869 in 32D cells EGFR(L858R) and EGFR(L861Q). The EGFR tyrosine kinase inhibitor (TKI) erlotinib inhibited Y869 P-EGFR in intact cells as well as in a cell-free kinase reaction. Expression of kinase domain of EGFR(L858R) and EGFR(L861Q) exhibited auto-phosphorylation of Y869; this was inhibited by EGFR TKIs but not by Src kinase inhibitor. P-Y859 of EGFR-mediated downstream component, STAT5, was also analyzed. Y694 P-STAT5 was eliminated by erlotinib treatment. Analysis of immune-complexes showed constitutive association of mutant EGFRs with STAT5 and Src which was unaffected by erlotinib or PP1. On the other hand, 32D-EGFR(WT) exhibited constitutive STAT5 phosphorylation and association of EGFR with JAK2. In these cells, a JAK2 inhibitor abrogated P-STAT5 whereas mutant EGFRs did not associate with JAK2. Expression of c-myc was regulated by EGFR/STAT5 signaling in cells expressing EGFR(L858R) and EGFR(L861Q). Our results suggest that ligand-independent and Src activity-independent phosphorylation of Y869 in mutant EGFR regulates STAT5 activation and c-myc expression.     

Mechanisms of SOCS3 phosphorylation upon interleukin-6 stimulation. Contributions of Src- and receptor-tyrosine kinases

The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine signal transduction. SOCS3 is a key negative regulator of interleuking-6 (IL-6) signal transduction. Furthermore, SOCS3 was shown to be phosphorylated upon treatment of cells with IL-2, and this has been reported to regulate its function and half-life. We set out to investigate whether SOCS3 phosphorylation may play a role in IL-6 signaling. Tyrosine-phosphorylated SOCS3 was detected upon treatment of mouse embryonic fibroblasts with IL-6. Interestingly, the observed SOCS3 phosphorylation does not require SOCS3 recruitment to phosphotyrosine (Tyr(P)) 759 of gp130, and the kinetics of SOCS3 phosphorylation do not match the activation kinetics of the Janus kinases. This suggests that other kinases may be involved in SOCS3 phosphorylation. Using Src and Janus kinase inhibitors as well as Src kinase-deficient mouse embryonic fibroblasts, we provide evidence that Src kinases, which we found to be constitutively active in these cells, are involved in the phosphorylation of IL-6-induced SOCS3. In addition, we found that receptor-tyrosine kinases such as platelet-derived growth factor receptor or epidermal growth factor receptor can very potently phosphorylate IL-6-induced SOCS3. Taken together, these results suggest that SOCS3 phosphorylation is not a JAK-mediated phenomenon but is dependent on the activity of other kinases such as Src kinases or receptor-tyrosine kinases, which can either be constitutively active or activated by an additional stimulus.     

RGS16 function is regulated by epidermal growth factor receptor-mediated tyrosine phosphorylation

Galpha(i)-coupled receptor stimulation results in epidermal growth factor receptor (EGFR) phosphorylation and MAPK activation. Regulators of G protein signaling (RGS proteins) inhibit G protein-dependent signal transduction by accelerating Galpha(i) GTP hydrolysis, shortening the duration of G protein effector stimulation. RGS16 contains two conserved tyrosine residues in the RGS box, Tyr(168) and Tyr(177), which are predicted sites of phosphorylation. RGS16 underwent phosphorylation in response to m2 muscarinic receptor or EGFR stimulation in HEK 293T or COS-7 cells, which required EGFR kinase activity. Mutational analysis suggested that RGS16 was phosphorylated on both tyrosine residues (Tyr(168) Tyr(177)) after EGF stimulation. RGS16 co-immunoprecipitated with EGFR, and the interaction did not require EGFR activation. Purified EGFR phosphorylated only recombinant RGS16 wild-type or Y177F in vitro, implying that EGFR-mediated phosphorylation depended on residue Tyr(168). Phosphorylated RGS16 demonstrated enhanced GTPase accelerating (GAP) activity on Galpha(i). Mutation of Tyr(168) to phenylalanine resulted in a 30% diminution in RGS16 GAP activity but completely eliminated its ability to regulate G(i)-mediated MAPK activation or adenylyl cyclase inhibition in HEK 293T cells. In contrast, mutation of Tyr(177) to phenylalanine had no effect on RGS16 GAP activity but also abolished its regulation of G(i)-mediated signal transduction in these cells. These data suggest that tyrosine phosphorylation regulates RGS16 function and that EGFR may potentially inhibit Galpha(i)-dependent MAPK activation in a feedback loop by enhancing RGS16 activity through tyrosine phosphorylation.     

Suppressor of cytokine signaling 7 inhibits prolactin, growth hormone, and leptin signaling by interacting with STAT5 or STAT3 and attenuating their nuclear translocation

We report here the role of one of the less studied members of the family of suppressors of cytokine signaling (SOCS), namely SOCS-7, in cytokine signaling. We demonstrate that SOCS-7 inhibits prolactin (PRL), growth hormone (GH), or leptin (LEP) signaling mediated through STAT3 and STAT5 in a dose-dependent manner. SOCS-7 also attenuated STAT3 and STAT5 signaling induced by overexpression of JH1, the catalytic subdomain of JAK2. Since SOCS-7 interacted with phosphorylated STAT3 or STAT5, we assumed that SOCS-7 acts at the level of STAT proteins. Indeed, we showed that SOCS-7 inhibits PRL- and leptin-induced STAT5 and STAT3 phosphorylation and prevented the nuclear translocation of activated STAT3. Taken together, our results indicate that SOCS-7 is a physiological dysregulator of PRL, leptin, and probably also GH signaling and that its mode of action is a novel variation of SOCS protein inhibition of cytokine-inducible STAT-mediated signal transduction.     

Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22-Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22-Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.     

Ouabain-induced signaling and vascular smooth muscle cell proliferation

The hypothesis of this study is that the sodium pump complex acts as an intracellular signal-transducing molecule in canine vascular smooth muscle cells through its interaction with other membrane and cytoskeletal proteins. We have demonstrated that 1 nm ouabain induced transactivation of the epidermal growth factor receptor (EGFR), resulting in increased proliferation and bromodeoxyuridine (BrdUrd) uptake. Immunoprecipitation and Western blotting showed that the EGFR and Src were phosphorylated within 5 min of 10(-9) m ouabain stimulation. Both ouabain-induced DNA synthesis (BrdUrd uptake) and MAPK42/44 phosphorylation were inhibited by the Src inhibitor PP2, the EGFR kinase inhibitor AG1478, the tyrosine kinase inhibitor genistein, and the MEK1 inhibitor PD98059. Ouabain concentrations higher than 1 nm had little or no stimulating effect on proliferation or BrdUrd uptake but did minimally activate ERK1/2. Thus, low concentrations of ouabain, which do not inhibit the sodium pump sufficiently to perturb the resting cellular ionic milieu, initiate a transactivational signaling cascade leading to vascular smooth muscle cell proliferation.     

Desensitization of the JAK2/STAT5 GH signaling pathway associated with increased CIS protein content in liver of pregnant mice

Chronic exposure to growth hormone (GH) was related to the desensitization of the JAK2/STAT5 signaling pathway in liver, as demonstrated in cells, female rats, and transgenic mice overexpressing GH. The cytokine-induced suppressor (CIS) is considered a major mediator of this desensitization. Pregnancy is accompanied by an increment in GH circulating levels, which were reported to be associated with hepatic GH resistance, although the molecular mechanisms involved in this resistance are not clearly elucidated. We thus evaluated the JAK2/STAT5b signaling pathway and its regulation by the suppressors of cytokine signaling (SOCS)/CIS family and the JAK2-interacting protein SH2-Bbeta in pregnant mouse liver, a model with physiological prolonged exposure to high GH levels. Basal tyrosyl phosphorylation levels of JAK2 and STAT5b in pregnant mice were similar to values obtained for virgin animals, in spite of the important increment of GH they exhibit. Moreover, these signaling mediators were not phosphorylated upon GH stimulation in pregnant mice. A 3.3-fold increase of CIS protein content was found for pregnant mice, whereas the abundance of the other SOCS proteins analyzed and SH2-Bbeta did not significantly change compared with virgin animals. The desensitization of the JAK2/STAT5b GH signaling pathway observed in pregnant mice would then be mainly related to increased CIS levels rather than to the other regulatory proteins examined.