CXCL12 regulates HER1 Y1068 and Y1173 phosphorylation via G-proteins.

<p>5637 or HeLa cells were stimulated with 25 ng/mL HB-EGF or with 200 ng/mL CXCL12 or N33A]CXCL12 alone or followed after 1 minute by 25 ng/mL HB-EGF. HER1 Y1068 or ERK1/2 TY185/187 phosphorylation was evaluated at the indicated time-points by using specific mAbs and was expressed as percentages of phosphorylation at the specified times after normalization as phosphorylated molecule/total molecule ratios. (A) HER1 phosphorylation at Y1068 in HeLa cells induced by HB-EGF alone (black) was abolished by prestimulation with [N33A]CXCL12 (blue) and modified by prestimulation with CXCL12 (red): maximum phosphorylation was reached at 4 minutes, and the plateau after the initial spike was around 50% of the maximum at 10 minutes. No phosphorylation was induced by CXCL12 alone. (B) Phosphorylation at Y1173 in 5637 cells displayed the same kind of pattern. (C) Prestimulation with CXCL12 did not modify the mitogenic effect of HB-EGF. (D) Stimulation with CXCL12 induced ERK1/2 phosphorylation (red) resulting from two spikes: a G-protein-dependent (blue) and a β-arrestin-dependent (red) phosphorylation spike. By using the PKC inhibitor Ro-31 the G-protein-dependent spike was abolished, whereas the β-arrestin-dependent spike persisted. [N33A]CXCL12 induced only the G-protein-dependent spike (blue), which was abolished by Ro-31. (E) Ro-31 abolished the effects of prestimulation with CXCL12 or [N33A]CXCL12 at Y1068 in 5637 cells. (F) The same pattern at Y1173 in HeLa cells. The means ±SD of 10 experiments are depicted.</p

Receptors involved in the crosstalk between CXCL12 and HER1.

<p>5637 or HeLa cells were cultured alone, or in the presence of 200 ng/mL CXCL12 or 25 ng/mL HB-EGF. After 20 minutes, the cells were collected and evaluated by flow cytometry for the expression of CXCR4 and HER1. (A) Stimulation with CXCL12 led to downregulation of CXCR4 due to its internalization. (B) Stimulation with HB-EGF induced HER1 internalization. In contrast, no changes in the surface expression of HER1 were detected after stimulation with CXCL12. Thus, CXCL12 binding to CXCR4 did not transactivate HER1 in these cells <i>via</i> shedding of HER1 ligands. Representative flow cytometry patterns and the means ±SD of 10 experiments are depicted.</p

Regulation of HER1 Y1068 or Y1173 phosphorylation downstream of G-proteins requires calmodulin/calcineurin activity.

<p>(A) and (B) Time-course. HeLa cells were stimulated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034432#pone-0034432-g003" target="_blank">Figure 3</a>. Treatment with W-13 (a calmodulin inhibitor) or CsA (a calcineurin inhibitor) restored HB-EGF-dependent HER1 phosphorylation at Y1068, whereas KN-62 (a CaMKII inhibitor) had no effect. (C) Following prestimulation with [N33A]CXCL12 the HB-EGF-dependent HER1 phosphorylation at Y1068 and Y1173 was abolished at the plateau (15 minutes) and restored by W-13 or CsA; KN-62 had no effect. The means ±SD of 10 experiments are depicted.</p

Knockdown of β-arrestin 2 protein levels allows for transinhibition of HER1.

<p>5637 or HeLa cells were transfected with control siRNA or β-arrestin 2 siRNA, prestimulated for 1 minute with 200 ng/mL CXCL12 and subsequently stimulated with 25 ng/mL HB-EGF for 2, 4, 5, 7, 10 or 15 minutes. In knockdown cells either Y1068 or Y1173 phosphorylation was inhibited as opposed to non-silenced cells at each time observed, as determined by ELISA (p<0.05). This shows that CXCL12 signaling transinhibits HER1 phosphorylation via G-protein-pathways in the absence of β-arrestin 2 activation and further supports that [N33A]CXCL12, which strongly transhinibits HER1, is a G-protein-biased ligand. The means ±SD out of 4 experiments are shown.</p

Phosphorylation of HER1 and ERK1/2, and cell proliferation following treatment with HB-EGF and CXCL12.

<p>(A) 5637 and HeLa cells were stimulated with 25 ng/mL HB-EGF for 20 minutes, and cell lysates were analyzed by mass spectrometry following trypsin digestion. Y1068 and Y1173, sites of autophosphorylation coupled to the activation of Ras, MEK and ERK1/2, were evaluated in the following experiments with specific phosphotyrosine mAbs. (B) HB-EGF induced phosphorylation of both HER1 Y1068 and ERK1/2 TY185/187 in 5637 or HeLa cells. In contrast, stimulation with 200 ng/mL CXCL12 for 20 minutes, which induced phosphoERK1/2, led to no phosphorylation of HER1. (C) In dose-response experiments, exposures of CXCL12 ranging from 6 to 400 ng/mL for 20 minutes did not induce HER1 phosphorylation in either cells. (D) No proliferation was induced with 200 ng/mL CXCL12. The means ±SD of 10 experiments are depicted.</p

CXCL12 and [N33A]CXCL12 transinhibit HER1 in 5637 or HeLa cells.

<p>Both chemokines signal <i>via</i> G-proteins to calmodulin/calcineurin and modulate the ligand-dependent phosphorylation of HER1. CXCL12 induces a delay in the phosphorylation. [N33A]CXCL12, which is a G-protein-biased ligand, inhibits the HER1 phosphorylation.</p