Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16.

We have identified a rapid protein phosphorylation event at residue serine 16 of stathmin using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry in combination with post-source decay analysis, which is induced by the epidermal growth factor receptor. Phosphorylation is specifically mediated by the small GTPases Rac and Cdc42 and their common downstream target, the serine/threonine kinase p65PAK. Both GTPases have previously been shown to regulate the dynamics of actin polymerization. Because stathmin destabilizes microtubules, and this process is inhibited by phosphorylation at residue 16, Rac and Cdc42 can potentially regulate both F-actin and microtubule dynamics.

Distinct calcium-dependent pathways of epidermal growth factor receptor transactivation and PYK2 tyrosine phosphorylation in PC12 cells.

Recently, we have demonstrated that in PC12 cells activation of the Ras/extracellular signal-regulated kinase pathway in response to membrane depolarization or bradykinin is mediated by calcium-dependent transactivation of the epidermal growth factor receptor (EGFR). Here we address the question whether Ca(2+)-calmodulin-dependent protein kinase (CaM kinase) has a role in the EGFR transactivation signal. Using compounds that selectively interfere with either CaM kinase activity or calmodulin function, we show that KCl-mediated membrane depolarization-triggered, but not bradykinin-mediated signals involve CaM kinase function upstream of the EGFR. Although both depolarization-induced calcium influx and bradykinin stimulation of PC12 cells were found to induce c-fos transcription through EGFR activation, the former signal is CaM kinase-dependent and the latter was shown to be independent. As PYK2 is also activated upon elevation of intracellular calcium, we investigated the potential involvement of this cytoplasmic tyrosine kinase in EGFR transactivation. Interestingly, we observed that inhibition of CaM kinase activity in PC12 cells abrogated tyrosine phosphorylation of PYK2 upon KCl but not bradykinin treatment. Nevertheless, PYK2 activation in response to both stimuli appeared to be mediated by pathways parallel to EGFR transactivation. Our data demonstrate the existence of two distinct calcium-dependent mechanisms leading either to EGFR-mediated extracellular signal-regulated activation or to PYK2 tyrosine phosphorylation. Both pathways either in concert or independently might contribute to the definition of biological responses in neuronal cell types.

EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF.

Cross-communication between different signalling systems allows the integration of the great diversity of stimuli that a cell receives under varying physiological situations. The transactivation of epidermal growth factor receptor (EGFR)-dependent signalling pathways upon stimulation of G-protein-coupled receptors (GPCRs), which are critical for the mitogenic activity of ligands such as lysophosphatidic acid, endothelin, thrombin, bombesin and carbachol, provides evidence for such an interconnected communication network. Here we show that EGFR transactivation upon GPCR stimulation involves proHB-EGF and a metalloproteinase activity that is rapidly induced upon GPCR-ligand interaction. We show that inhibition of proHB-EGF processing blocks GPCR-induced EGFR transactivation and downstream signals. The pathophysiological significance of this mechanism is demonstrated by inhibition of constitutive EGFR activity upon treatment of PC3 prostate carcinoma cells with the metalloproteinase inhibitor batimastat. Together, our results establish a new mechanistic concept for cross-communication among different signalling systems.

Ligand-independent activation of platelet-derived growth factor receptor is a necessary intermediate in lysophosphatidic, acid-stimulated mitogenic activity in L cells.

Growth factor-derived mitogenic signals from the cell surface are transmitted to the nucleus via receptor tyrosine kinases (RTKs), the adaptor proteins Shc and Grb2, and a Ras-dependent protein kinase cascade that activates the extracellular signal regulated kinase (ERK) subfamily of mitogen-activated protein kinases. ERKs also are activated by hormones that stimulate G protein-coupled receptors (GPCRs). We report here that, in agreement with previous data, the epidermal growth factor receptor (EGFR) is a signaling intermediate in ERK activation by GPCRs. Of import, we show that cross-talk between two classes of surface receptors, RTKs and GPCRs, is a general feature. Lysophosphatidic acid not only induces ligand-independent tyrosine autophosphorylation of EGFR but also of platelet-derived growth factor beta receptor (PDGF-beta-R) as shown by detection of tyrosine phosphorylation and by the use of specific inhibitors of RTKs. The cross-talk appears to be cell type-specific: In L cells that lack EGFR, lysophosphatidic acid-induced Shc and ERK activation is prevented completely by specific inhibition of PDGFR, whereas in COS-7 cells expressing only EGFR, the pathway via EGFR is chosen. In Rat-1 cells, however, that express both EGFR and PDGFR, the EGFR pathway dominates.

Critical role of calcium- dependent epidermal growth factor receptor transactivation in PC12 cell membrane depolarization and bradykinin signaling.

PC12 cells respond to a variety of external stimuli such as growth factors, neurotransmitters, and membrane depolarization by activating the Ras/mitogen-activated protein kinase pathway. Here we demonstrate that both depolarization-induced calcium influx and treatment with bradykinin stimulate tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Using a tetracycline-controlled expression system in conjunction with a dominant-negative EGFR mutant, we demonstrate that depolarization and bradykinin triggered signals involve EGFR function upstream of SHC and MAP kinase. Furthermore, bradykinin-stimulated EGFR transactivation is critically dependent on the presence of extracellular calcium, and when triggered by ionophore treatment, calcium influx is already sufficient to induce EGFR tyrosine phosphorylation. Taken together, our results establish calcium-dependent EGFR transactivation as a signaling mechanism mediating activation of the Ras/mitogen-activated protein kinase pathway in neuronal cell types.

Novel mechanisms of RTK signal generation.

Recent findings shed new light on the process of receptor tyrosine kinase (RTK) activation and signal definition. In extension to the established mechanism of ligand-induced homodimeric receptor complex formation, recent findings highlight heterodimeric receptor aggregation as a powerful means of signal diversification. Promiscuous receptor interactions involve different ligand binding kinetics and generate divergent receptor phosphorylation sites that could allow enhanced or modified signal generation. Besides activation by a specific ligand, a newly defined RTK function involves signal integration of a variety of stimuli, including calcium-dependent responses in neuronal cells, activation of G-protein-coupled receptors or cellular stress such as UV irradiation. On the basis of existing evidence for such crossactivation pathways, RTKs must be considered as representing critical foci and switch points for multiple environmental and internal stimuli.

Signal characteristics of G protein-transactivated EGF receptor.

The epidermal growth factor receptor (EGFR) tyrosine kinase recently was identified as providing a link to mitogen-activated protein kinase (MAPK) in response to G protein-coupled receptor (GPCR) agonists in Rat-1 fibroblasts. This cross-talk pathway is also established in other cell types such as HaCaT keratinocytes, primary mouse astrocytes and COS-7 cells. Transient expression of either Gq- or Gi-coupled receptors in COS-7 cells allowed GPCR agonist-induced EGFR transactivation, and lysophosphatidic acid (LPA)-generated signals involved the docking protein Gab1. The increase in SHC tyrosine phosphorylation and MAPK stimulation through both Gq- and Gi-coupled receptors was reduced strongly upon selective inhibition of EGFR function. Inhibition of phosphoinositide 3-kinase did not affect GPCR-induced stimulation of EGFR tyrosine phosphorylation, but inhibited MAPK stimulation, upon treatment with both GPCR agonists and low doses of EGF. Furthermore, the Src tyrosine kinase inhibitor PP1 strongly interfered with LPA- and EGF-induced tyrosine phosphorylation and MAPK activation downstream of EGFR. Our results demonstrate an essential role for EGFR function in signaling through both Gq- and Gi-coupled receptors and provide novel insights into signal transmission downstream of EGFR for efficient activation of the Ras/MAPK pathway.

Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors.

Transduction of a mitogenic signal from the cell membrane to the nucleus involves the adapter proteins SHC and Grb2, which mediate activation of the Ras/mitogen-activated protein (MAP) kinase pathway. In contrast to receptor tyrosine kinases (RTKs), the signalling steps leading to Ras/MAP kinase activation by G-protein-coupled receptors (GPCRs) are still poorly characterized but appear to include beta gamma subunits of heterotrimeric G-proteins and as-yet unidentified tyrosine kinases. We report here that the epidermal growth factor receptor (EGFR) and the neu oncoprotein become rapidly tyrosine-phosphorylated upon stimulation of Rat-1 cells with the GPCR agonists endothelin-1, lysophosphatic acid and thrombin, suggesting that there is an intracellular mechanism for transactivation. Specific inhibition of EGFR function by either the selective tyrphostin AG1478 or a dominant-negative EGFR mutant suppressed MAP kinase activation and strongly inhibited induction of fos gene expression and DNA synthesis. Our results demonstrate a role for RTKs as downstream mediators in GPCR mitogenic signalling and suggest a ligand-independent mechanism of RTK activation through intracellular signal crosstalk.