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)
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Collective cell migration: Fine tuning Rac

Photoactivation of Rac in a single cell is sufficient to drive collective migration of a group of cells.

Collective cell migration is important during morphogenesis and in tumor metastasis. In Drosophila migratory border cells, the small GTPase Rac controls collective cell migration in vivo in response to guidance signals through platelet-derived growth factor/vascular endothelial growth factor receptor (PVR) and the epidermal growth factor receptor (EGFR). In Nature Cell Biology, Denise Montell and colleagues now report that localized activation of Rac in a single cell is sufficient for collective migration of cells in vivo and that guidance signals finely tune Rac activity, whereas Jun N-terminal kinase (JNK) signaling maintains communication between the cells.

Using transgenic flies with fluorescently labeled photoactivatable Rac, the authors first show that local activation of Rac in a single cell next to the leading cell is sufficient to redirect migration of the rest of the cells in a cluster. Photoactivation of Rac at the rear was also sufficient to stop forward protrusion and promote a slower rearward movement. Furthermore, the cells recovered forward movement when illumination was halted, showing that Rac activity is sufficient to polarize a group of cells and guide their collective movement.

But, why was Rac-induced rearward movement slower than normal? Experiments in border cells expressing dominant-negative forms of PVR and EGFR showed that in the absence of guidance signals, random protrusions formed in all directions and the cells moved very little. Photoactivation of Rac rescued these defects and restored direction sensing. Interestingly, illuminating the rescued cells at the front or the rear resulted in forward or reverse migration, respectively, now with similar speeds. Moreover, they could not recover forward movement when rear illumination stopped. This suggests that the guidance signals normally produce directional Rac activity. Consistently, time-lapse imaging of a Rac FRET biosensor revealed that Rac activity is normally asymmetric in border cells — highest at the front and lowest at the back — and this asymmetry was lost in the absence of PVR and EGFR.

Next, they tested what happens when Rac is locally inhibited. Photoinactivation of Rac in the leading cell stopped forward migration and promoted rearward protrusion, whereas photoinhibition of Rac at the rear enhanced forward protrusion. High magnification images revealed that local photoactivation or photoinactivation of Rac in a single cell is sufficient to cause morphological changes of all cells in the cluster.

So, what is the signaling mechanism downstream of Rac? As JNK signaling controls directional protrusions and collective movement of border cells, they tested whether JNK is important for the effects of Rac. Rac activity was sufficient to extend protrusions in the direction of light even in the absence of JNK signaling; however, retraction of protrusions in other directions was inhibited when JNK activity was reduced. This indicates that JNK is not required downstream of Rac to promote protrusion, but maintains the communication from the cell with highest Rac activity to other cells within the cluster.

Together, this study identifies a key role for Rac and JNK in collective cell migration, which has implications for tumor metastasis. It also demonstrates the power of using light-controlled activation of a Rho GTPase to control migration in vivo.

Iley Ozerlat

Reference:
Wang X. et al.
Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo.
Nature Cell Biology 12, 591-597 (2010)
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