Endogenous Protein Inhibitors of Pak1 Bind the Autoregulatory Domain

In addition to the intramolecular interactions that regulate Pakl activity, there are a number of other proteins that interact with and modulate Pakl activity. Interestingly, many of these proteins bind within the Pakl autoregulatory region. For example, human Pakl-interacting protein (hPIPl) is a WD repeat containing protein that was shown to inhibit Pakl kinase activity as well as the signaling of Pakl effectors JNK and NFkB in human cells. hPIPl binds within the autoregulatory region of Pakl, suggesting that it could inhibit Pakl activity by stabilizing the autoinhibited conformation (Xia et al. 200l).

The product of the tumor suppressor gene NF2, termed merlin, is another cellular protein with the ability to inhibit Pakl activity. While the full spectrum of merlin functions are still under investigation, it has been demonstrated that overexpression of merlin inhibited Pakl activation as well as Pakl interaction with the focal adhesion protein paxillin (Kissil et al. 2003; Xiao et al. 2005). Conversely, loss of merlin expression resulted in an increase in Pakl activation (Kissil et al. 2003). Merlin binds Pakl within the autoregulatory region and inhibits the interaction between Pakl and Rac (Kissil et al. 2003), indicating that this protein functions by disrupting a protein interaction required for Pakl activation. In addition to acting as an inhibitor of Pak activity, merlin is also a Pak substrate, as it was shown that Pak phosphorylates merlin on serine 5l8 (Kissil et al. 2002; Xiao et al. 2002). Phosphorylation on this residue alters merlin localization and anti-proliferation activity (Shaw et al. 200l; Kissil et al. 2002; Xiao et al. 2002), suggesting a feedback signaling relationship between these two proteins.

The recently identified protein Cysteine Rich-Inhibitor of Pakl (CRIPak) was shown to inhibit Pakl activity both in vitro and in cells, and blocked the activation of the Pakl effector LIM kinase (Talukder et al. 2006). CRIPak also binds within the autoregulatory region of Pakl, and therefore could also act to by stabilizing autoinhibited Pakl dimers. However, this region of interaction overlaps with the Pix-binding region of Pakl, raising the possibility that CRIPak could antagonize Pakl activity by inhibiting the binding of Pix (Talukder et al. 2006).

Interestingly, CIBl, an EF-hand domain-containing protein, also binds to the Pakl autoregulatory domain of Pakl; however, this binding results in activation of Pakl catalytic activity (Leisner et al. 2005). Thus, activation and inhibition of Pakl activity through the binding of proteins to the Pakl autoregulatory domain are a biologically relevant regulatory mechanism.

The studies discussed above suggest that some endogenous Pakl protein inhibitors may act by stabilizing the autoinhibited conformation of Pakl. Thus, the binding of proteins other than Rac or Cdc42 to the autoregulatory domain may be a physiological mechanism for negatively regulating Pakl kinase activity. It may therefore be possible for a small molecule to exploit this inherent regulatory mechanism in a similar way. In the following section, we will discuss screening strategies that could be employed to specifically identify such an inhibitor for members of the group I Pak family.

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