Given the importance of the Rho GTPase family member Rad and the Rac1/Cdc42
effector PAK1 in T-cell activation, we investigated the requirements for t
heir activation by the T-cell receptor (TCR), Rad and PAK1 activation requi
red the tyrosine kinases ZAP-70 and Syk, but not the cytoplasmic adaptor Sl
p-76, Surprisingly, PAK1 was activated in the absence of the transmembrane
adaptor LAT while Rad was not. However, efficient PAK1 activation required
its binding sites for Rho GTPases and for PIX, a guanine nucleotide exchang
e factor for Rho GTPases, The overexpression of beta PIX that either cannot
bind PAK1 or lacks GEF function blocked PAK1 activation. These data sugges
t that PAK1-PIX complex is recruited to appropriate sites for activation an
d that PIX is required for Rho family GTPase activation upstream of PAK1, F
urthermore, we detected a stable trimolecular complex of PAK1, PIX and the
paxillin kinase linker p95PKL, Taken together, these data show that PAK1 co
ntained in this trimolecular complex is activated by a novel LAT-and Slp-76
-independent pathway following TCR stimulation.