Ron tyrosine kinase receptor synergises with EGFR to confer adverse features in head and neck squamous cell carcinoma.

BACKGROUND: Although EGFR inhibitors have shown some success in the treatment of head and neck squamous cell carcinomas (HNSCCs), the results are not dramatic. Additional molecular targets are urgently needed. We previously showed that the loss of Ron receptor activity significantly slowed squamous tumour growth and progression in a murine model. Based on these data, we hypothesised that Ron expression confers an aggressive phenotype in HNSCCs. We prospectively collected and evaluated 154 snap-frozen, primary HNSCCs for Ron and EGFR expression/phosphorylation. Biomarker correlation with clinical, pathological and outcome data was performed. The biological responses of HNSCC cell lines to Ron knockdown, its activation and the biochemical interaction between Ron and EGFR were examined. RESULTS: We discovered that 64.3% (99 out of 154) HNSCCs expressed Ron. The carcinomas expressed exclusively mature functional Ron, whereas the adjacent nonmalignant epithelium expressed predominantly nonfunctional Ron precursor. There was no significant association between Ron and sex, tumour differentiation, perineural/vascular invasion or staging. However, patients with Ron+HNSCC were significantly older and more likely to have oropharyngeal tumours. Ron+HNSCC also had significantly higher EGFR expression and correlated strongly with phosphorylated EGFR (pEGFR). Newly diagnosed HNSCC with either Ron/pEGFR or both had lower disease-free survival than those without Ron and pEGFR. Knocking down Ron in SCC9 cells significantly blunted their migratory response to not only the Ron ligand, MSP, but also EGF. Stimulation of Ron in SCC9 cells significantly augmented the growth effect of EGF; the synergistic effect of both growth factors in SCC9 cells was dependent on Ron expression. Activated Ron also interacted with and transactivated EGFR. CONCLUSION: Ron synergises with EGFR to confer certain adverse features in HNSCCs.

Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway.

The c-Jun NH2-terminal kinase (JNK) is activated when cells are exposed to ultraviolet (UV) radiation. However, the functional consequence of JNK activation in UV-irradiated cells has not been established. It is shown here that JNK is required for UV-induced apoptosis in primary murine embryonic fibroblasts. Fibroblasts with simultaneous targeted disruptions of all the functional Jnk genes were protected against UV-stimulated apoptosis. The absence of JNK caused a defect in the mitochondrial death signaling pathway, including the failure to release cytochrome c. These data indicate that mitochondria are influenced by proapoptotic signal transduction through the JNK pathway.

Crystal structure of the Dbl and pleckstrin homology domains from the human Son of sevenless protein.

Proteins containing Dbl homology (DH) domains activate Rho-family GTPases by functioning as specific guanine nucleotide exchange factors. All known DH domains have associated C-terminal pleckstrin homology (PH) domains that are implicated in targeting and regulatory functions. The crystal structure of a fragment of the human Son of sevenless protein containing the DH and PH domains has been determined at 2.3 A resolution. The entirely alpha-helical DH domain is unrelated in architecture to other nucleotide exchange factors. The active site of the DH domain, identified on the basis of sequence conservation and structural features, lies near the interface between the DH and PH domains. The structure suggests that ligation of the PH domain will be coupled structurally to the GTPase binding site.

Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells.

The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors.

Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos.

The Son of Sevenless (Sos) proteins control receptor-mediated activation of Ras by catalyzing the exchange of guanosine diphosphate for guanosine triphosphate on Ras. The NH2-terminal region of Sos contains a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. In COS-1 cells, the DH domain of Sos stimulated guanine nucleotide exchange on Rac but not Cdc42 in vitro and in vivo. The tandem DH-PH domain of Sos (DH-PH-Sos) was defective in Rac activation but regained Rac stimulating activity when it was coexpressed with activated Ras. Ras-mediated activation of DH-PH-Sos did not require activation of mitogen-activated protein kinase but it was dependent on activation of phosphoinositide 3-kinase. These results reveal a potential mechanism for coupling of Ras and Rac signaling pathways.