The role of Rac and Pak signaling in human tumor cell invasion

Abstract

The Rho-GTPase, Rac1 and its direct effector the group I p21-associated kinases (PAKs 1 - 3) have recently been shown to play a role in cell migration and invasion, however there is very little evidence available for specific functions of the different Rac and Pak isoforms. Here I show using siRNA, that Rac1 is required for the generation of heregulin-stimulated lamellipodia and invasion as well as signaling to Pak1, Pak2 and the JNK, cofilin and myosin light chain (MLC) pathways. Inhibition of the JNK pathway or cofilin significantly inhibits heregulin-mediated invasion, demonstrating that these pathways play a positive role in mediating invasion signaling downstream of Rac1. In the analysis of the Pak proteins in breast carcinoma cell invasion, we find that both Pak1 and Pak2 contribute to invasion stimulated by heregulin, and their roles are mediated by distinct signaling mechanisms. Silencing of Pak1 has a more robust inhibitory effect on lamellipodial protrusion and interferes with the dephosphorylation of cofilin, whereas Pak2 does not. Interestingly, Pak1 and Pak2 play opposite roles in regulating the phosphorylation of the myosin light chain subunit of non-muscle myosin II. Whereas depletion of Pak1 decreases phospho-MLC levels in heregulin-stimulated cells, depletion of Pak2 enhances phospho-MLC levels. Consistent with their opposite effects on MLC, we observed that Pak1 and Pak2 differentially modulate focal adhesions. Pak2-depleted cells fail to generate new focal adhesions in response to heregulin. On the other hand, in Pak1-depleted cells focal adhesions fail to mature in response to heregulin. Interestingly, we found that depletion of Pak2, but not Pak1, enhances RhoA activity, suggesting that downregulation of RhoA contributes to the roles of Pak2 in the regulation of MLC phosphorylation, focal adhesions and invasion. In support of this, inhibition of the RhoA/ROCK/MLC signaling axis in Pak2-depleted cells decreases MLC phosphorylation and restores cell invasion. In addition, we found the Rac/Pak axis to play a critical role during invasion of a medulloblastoma cell line. In summary this work demonstrates a widespread role for Rac/Pak signaling in human tumor cell invasion.