Identification of Jak PTK-regulated rho-specific GEFs involved in activation of lymphocyte adhesion

The rapid integrin affinity up-regulation is a crucial dynamic process in leukocyte recruitment that is controlled by a complex inside-out signalling pathway induced by chemokines. Small GTP binding proteins of rap and rho family are certainly the most studied signaling molecules involve in this pathway; in addition our recent data identified Jak PTKs as new upstream regulator of these small GTPases. Considering that Guanosine Exchange Factors (GEFs) are the main direct activators of small GTPases, they represent obvious molecule candidates to fill out the functional gap between Jak PTKs and rho-module. In this study we show the concurrent regulatory role of four rho specific GEFs Vav1, Sos1, Arhgef1 and Dock2 in CXCL12-induced LFA-1 affinity triggering and mediated-adhesion in human T lymphocytes. A reduced expression of these four molecules resulted in an impaired chemokine-induced LFA-1 affinity up-regulation and in a reduced cell adhesion to ICAM-1 in static and under-flow conditions. Importantly, CXCL12-activation of these four proteins is mediated by Jak PTKs and occurs in a time frame coherent with LFA-1 affinity triggering by chemokine. Moreover the activation of RhoA and Rac1 is strictly dependent on Vav1, Sos1, Arhgef1 and Dock2 activity. Collectively in this study we identified and fully characterized the role of four rho-GEFs in CXCL12-induced LFA-1 mediated adhesion providing a comprehensive signalling link between Jak PTKs and rho-module. Considering our results from a quantitative point of view, we observed some variability in the relative regulatory role of these proteins, with a major role for Vav1 and Sos1 with respect to Arhgef1 and Dock2 activity. This variable involvement of multiple rho-GEFs with apparently the same function may support the new emergent quantitative-concurrency view of signal transduction in which this complexity in mechanisms controlling integrin activation is essential to generate a very flexible signalling system able to efficiently respond to a variety of environmental conditions.