 EGFR signaling: finding new partners
Knockdown experiments targeting the human kinome have identified new roles for kinases in modulating the generation, amplitude and duration of EGFR signals.
The sequence of events induced by epidermal growth factor (EGF) binding to the EGF receptor (EGFR) is well studied, as is the consequence — uncontrolled cellular proliferation — of dysregulation of this signaling pathway. However, how other molecules modulate this pathway and how it is connected to other pathways is not well understood. Kakajan Komurov and colleagues have now systematically interrogated the EGFR-induced mitogenic signaling network, and report their findings in the Journal of Biological Chemistry.
They first transfected the squamous cell carcinoma-derived A431 cells, which express high levels of EGFR, with 672 synthetic short interfering (si)RNA pools to knockdown the elements of the human kinome before stimulating the cells with EGF. Lysates were printed onto nitrocellulose slides, which were then incubated with active-site phosphorylation-specific antibodies against extracellular signal-regulated kinase 1/2 (ERK1/2) or signal transducer and activator of transcription 3 (STAT3) — both of which are activated in response to EGFR stimulation. Antibody binding was quantified using fluorescent quantum dot-coupled secondary antibodies.
Komurov and colleagues then investigated siRNA pools that had reproducible effects (at least a 20% change) on ERK1/2 and/or STAT3 activation. Of the 52 candidate EGFR signal modulators identified, 31 were previously reported as somatic mutations in tumor tissues or cell lines. Interestingly, the components required for EGF-induced ERK1/2 activation in A431 cells were also involved in the survival of Mnt-1 melanoma cells, in which mutations in the ERK1/2 upstream activators often cause transformation.
In a series of experiments, Komurov and colleagues showed that ERK1/2 and STAT3 pathways are differentially sensitive to EGFR signaling. The resultant data indicates that maximal ERK1/2 activation can be induced by minimal numbers of EGFRs, whereas STAT3 activation is more dependent on the number of EGFR phosphorylation events.
Some siRNA pools abrogated EGF-mediated ERK1/2 and/or STAT3 activation, implicating the targeted gene products in EGF signal propagation. Others were implicated in signal desensitization or termination, as their depletion enhanced EGF responsiveness. Selective knockdown of the nucleotide kinase UCK1 or the receptor tyrosine kinase MERTK considerably reduced the accumulation of EGFR in response to EGF, suggesting that these kinases may influence EGFR trafficking and/or signal desensitization. The researchers also identified deoxyguanosine kinase (DGUOK) with the potential to regulate EGFR signaling. In breast cancer patients with high, but not low, ERBB2 expression (and potentially high EGFR signaling), high expression of UCK1 or DGUOK correlated with poor outcome, indicating that these nucleotide kinases might sustain aberrant EGFR signaling in vivo, thereby highlighting their role as potential targets for therapeutic intervention.
This large-scale strategy has uncovered kinases that modulate EGFR signal generation, amplitude and duration, and could be applied more widely across the human genome to further investigate cell-autonomous signaling networks.
Katrin Legg
Reference:
Komurov K. et al. Comprehensive mapping of the human kinome to epidermal growth factor receptor signaling.
J. Biol. Chem., 285, 21134-21142 (2010) Full text
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