Hierarchical Organization of Multi-Site Phosphorylation at the CXCR4 C Terminus
Figure 4
Heterologous and homologous phosphorylation of CXCR4.
A, Stably CXCR4-transfected HEK293 cells were stimulated for 30 min as indicated to produce maximal effects. Note that CXCL12 and PMA but not EGF or forskolin induce CXCR4 phosphorylation. B, When indicated, cells were pre-incubated for 30 min with the non-selective PKC inhibitor staurosporine and stimulated for 15 min with CXCL12 or PMA in the presence or absence of staurosporine. Staurosporine reduces CXCL12-promoted phosphorylation at S324/325 and reduces PMA-induced phosphorylation at all three sites. A,B, Images are representative for three independent experiments yielding similar results. C,D, Cells were pre-treated for 72 h with control siRNA (scr) or siRNAs for GRK2 and GRK3 as indicated. Lysates were generated 15 min after CXCL12 stimulation and detected in an immunoblot with anti-pS346/347 and anti-HA. Combined knockdown of GRK2 and GRK3 reduces the CXCL12-promoted signal increase of anti-pS346/347 indicating attenuated phosphorylation at S346/347. D, Quantification of 3 independent experiments analyzing the effect of GRK2 and GRK3 knockdown on CXCL12-promoted S346/347 phosphorylation. *: p<0.05; Student's t test vs. scr-group. E, Western Blot analysis of the siRNA-mediated knockdown of GRK2 and GRK3.