Pak1 and PIP2
both play roles in the regulation of the actin cytoskeleton, maintenance of focal contacts, and cytokinesis (van den Bout and Divecha, 2009
), and our results suggest that the regulation of Pak1 by PIP2
may provide an underlying mechanism for these observations. While we cannot exclude that other phosphoinositides might also regulate Pak1 under physiological conditions, we find that PIP2
, the most abundant phosphoinositide at the plasma membrane, is also the most potent phosphoinositide activator of Pak1 under the conditions tested.
The potentiation of Pak1 activation by phosphoinositides and Rho GTPases in vitro
begs the question of the relative importance of these two inputs for Pak1 activation in vivo
. Overexpression of constitutively active forms of Rac1 or Cdc42 alone in cultured cells is sufficient to activate Pak1 (Knaus et al., 1998
). Our findings suggest the possibility that this common experimental protocol may overestimate the importance of the Rho GTPase input on Pak1 activation by producing non-physiological concentrations of active Rac1/Cdc42. Rac1/Cdc42 overexpression might, in fact, promote PIP2
synthesis as Rac1 interacts directly with phosphatidylinositol 4-phosphate 5-kinases (PIP5K), and both Rac1 and Cdc42 enhance PIP5K activity (van den Bout and Divecha, 2009
). Similarly, local synthesis of PIP2
has been shown to promote Rho GTPase recruitment via interactions with a C-terminal basic region (Heo et al., 2006
). These interactions may therefore provide a positive feedback loop to promote synergistic enrichment of both active Rho GTPase and PIP2,
which would ultimately promote Pak1 activation.
Our data indicate a critical role for PIP2
-Pak1 binding in vitro
, in cell extracts, and in cultured mammalian cells suggesting that Pak1 activation may be phosphoinositide-dependent in many, if not all, physiological contexts. Our findings also provide an explanation for previous observations that Pak1 lysine residues Lys66-68 (mutated in both 4T and 8T Pak1) play a role in Pak1 activation independent of GTPase binding (Knaus et al., 1998
). Furthermore, our demonstration of Pak1 activation solely by PIP2
liposomes raises the possibility that pathologically elevated levels of phosphoinositides on their own might lead to Pak1 activation. Elevated Pak1 kinase activity is associated with cancers of the breast and other tissues, leading to significant interest in the therapeutic potential of Pak1 inhibitors (Deacon et al., 2008
; Molli et al., 2009
). However, unlike the situation with Ras-induced neoplasia, activating mutations in the upstream GTPases Rac1 and Cdc42 are not commonly found in human tumors. Thus, perturbations in phosphoinositide metabolism could account for the Pak1 hyperactivity associated with various cancers.
In electronic circuits, coincidence detectors reduce stochastic noise by requiring cotemporaneous signals from multiple inputs to produce a positive signal. We postulate that the cooperative regulation of Pak1 by PIP2 and Rho GTPases may be functioning in an analogous manner in the cell to ensure Pak1 activation only at sites where PIP2 and activated Rho GTPase overlap. The evolutionary conservation of basic regions in the related Paks 2 and 3 suggests that phosphoinositide-dependent activation may be a hallmark of all three Group I Pak isoforms. Interestingly, a number of Rac1/Cdc42 effectors also have basic amino acid-enriched sequences adjacent to their GTPase binding domains including myotonic dystrophy kinase-related Cdc42-binding kinase, BORG1/2, and the mixed lineage kinases, suggesting that phophoinositide binding may be a common regulatory mechanism. In striking contrast, other Rac/Cdc42 effectors, such as Ack, lack identifiable basic regions in this position, implying that the combinatorial activation by Rac1/Cdc42 and phosphoinositides may provide a mechanism for selectively activating a subset of effectors downstream of Rho GTPases.