Bradykinin B2 Receptor-Mediated Mitogen-Activated Protein Kinase Activation in COS-7 Cells Requires Dual Signaling via Both Protein Kinase C Pathway and Epidermal Growth Factor Receptor Transactivation

The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase gamma (PI3Kgamma), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B(2) receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the alpha subunits of a G(q/11) protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kgamma or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms alpha and epsilon and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.     

A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.     

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.     

ATF1 phosphorylation by the ERK MAPK pathway is required for epidermal growth factor-induced c-jun expression

Epidermal growth factor induction of c-jun expression requires ATF1 and MEF2 sites in the c-jun promoter. We find that activation of the c-jun promoter through the ATF1 site requires phosphorylation of ATF1 at serine 63. A serine 63 to alanine mutation of ATF1 acts to block epidermal growth factor (EGF) induction of a transfected c-jun gene. ATF1 can be phosphorylated by mitogen- and stress-activated protein kinase 1 (MSK1), which is activated by EGF and ERK1/2. Kinase-dead MSK1 mutants blocked EGF induction of a transfected c-jun gene suggesting that MSK1 or a similar family member is required for induced c-jun expression. Use of the MEK1 inhibitor U0126 and dominant negative MEK1 further showed that MSK1 activation and c-jun induction require the ERK pathway. In contrast, a JNK inhibitor blocked EGF induction of c-jun expression but not ATF1 phosphorylation. These results show that the two MAPK pathways, ERK and JNK, are required for EGF-induced c-jun expression and that the ERK pathway acts through downstream phosphorylation of ATF1.