jetPRIME: BLM melanoma cell line; HEK-293T, MDA-MB-231, A375 INTERFERin: BLM melanoma cell line

Activation of the GTPase RhoA linked to cell invasion can be tightly regulated following Galpha13 stimulation. We have used a cellular model displaying Galpha13-dependent inhibition of RhoA activation associated with defective cell invasion to the chemokine CXCL12 to characterize the molecular players regulating these processes. Using both RNAi transfection approaches and protein overexpression experiments here we show that the Src kinase Blk is involved in Galpha13-activated tyrosine phosphorylation of p190RhoGAP, which causes RhoA inactivation and ultimately leads to deficient cell invasion. Characterization of molecular interplays between Galpha13, Blk and p190RhoGAP revealed that Blk binds Galpha13, and that Blk-mediated p190RhoGAP phosphorylation upon Galpha13 activation correlates with weakening of Galpha13-Blk association connected to increased Blk-p190RhoGAP assembly. These results place Blk upstream of the p190RhoGAP-RhoA pathway in Galpha13-activated cells, overall representing an opposing signaling module during CXCL12-triggered invasion. In addition, analyses with Blk- or Galpha13-knockdown cells indicated that Blk can also mediate CXCL12-triggered phosphorylation of p190RhoGAP independently of Galpha13. However, even if CXCL12 induces the Blk-mediated GAP phosphorylation, the simultaneous stimulation of the guanine-nucleotide exchange factor Vav1 by the chemokine, as earlier reported, leads to a net increase in RhoA activation. Therefore, when Galpha13 is concurrently stimulated with CXCL12 there appears to be sufficient Blk activity to promote adequate levels of p190RhoGAP tyrosine phosphorylation to inactivate RhoA and to impair cell invasiveness.