Relevance of an Allosteric Pakl Inhibitor

Current methods used to eliminate Pak1 function include classical genetic knockout (Hofmann et al. 2004) as well as transfection of interfering reagents, such as siRNAs (Tang et al. 2005), constructs encoding the IS and KI sequence elements (Beeser and Chernoff 2005), or catalytically inactive, full-length versions of the kinase (Adam et al. 2000). These approaches, while useful in abrogating Pak1 activity, are limited in their degree of control of Pak1 activity, as well as by a lack of control over the time required to achieve Pak1 inhibition. In addition, overexpression of kinase-dead or truncated Pak1 constructs may titrate away relevant Pak1 binding partners, such as the guanine nucleotide exchange factor Pak interacting exchange factor (Pix) or Nck (Bokoch et al. 1996; Galisteo et al. 1996; Bagrodia et al. 1998; Manser et al. 1998), leading to complications in the interpretation of observed phenotypes. Small-molecule inhibitors can address some of the limitations associated with these currently used methods of inhibiting Pak1 activity. Indeed, kinase inhibitors in general have had a tremendous impact on our understanding of the functions of specific kinases (Knight and Shokat 2005). The advantages of using chemical inhibitors make small-molecule inhibitors of Pak1 a potentially important and complementary tool in the study of Pak1 function in cells.

The vast majority of existing kinase inhibitors binds to the kinase active site in a manner competitive with ATP. Since the ATP-binding pocket of kinases is highly conserved in sequence and structure, most inhibitors target multiple related kinases in addition to their intended target (Davies et al. 2000; Fabian et al. 2005). Indeed, a chemical inhibitor of Pak1 that is based on the ATP-competitive inhibitor CEP-1347 (Nheu et al. 2002) is also a potent inhibitor of mixed-lineage kinases 1-3 (Maroney et al. 2001; Nheu et al. 2002). In contrast, an allosteric inhibitor that stabilizes the autoinhibited Pak conformation might exhibit significantly greater kinase specificity due to the fact that the autoregulatory domain of group I Paks is not conserved in other protein kinases. Thus, allosteric inhibitors that target this domain to stabilize the inactive Pak1 dimer would not be expected to inhibit other kinases. Furthermore, since autoinhibition is not conserved in group II Pak family members, one would expect such inhibitors to target only the group I Paks, despite significant sequence conservation in the catalytic domains of these two groups. As such, allosteric inhibitors of Pakl could serve as highly specific tools to elucidate group I Pak functions.

Small-molecule Pakl inhibitors may also have clinical relevance. Pakl expression and activity have been linked to tumor development in a number of different cancers including colorectal, ovarian, and breast (Kumar and Vadlamudi 2002; Schraml et al. 2003; Carter et al. 2004), making identification of clinically relevant Pakl inhibitors of great interest (Kumar et al. 2006). Paks involvement in tumorigenesis is likely due in part to its ability to promote activation of the mitogen-activated protein kinase (MAPK) pathway. Group I Paks directly phosphorylate two kinases in this pathway, c-Raf and MEK1, and Pakl phosphorylation of these kinases is thought to be required for their full activation by the Ras oncogene (Frost et al. l997; King et al. l998; Chaudhary et al. 2000; Eblen et al. 2002; Zang et al. 2002; Slack-Davis et al. 2003; Tran and Frost 2003). Indeed, expression of dominant negative Pakl constructs inhibits Ras-mediated transformation in Ratl fibroblasts, rat Schwann cells, and in a neurofibrosarcoma cell line (Tang et al. l997, l998). Conversely, expression of an activated form of Pakl stimulates the MAPK pathway in HEK 293 cells (Lu et al. l997) and is tumorigenic in the mammary glands of transgenic mice (Wang et al. 2006), again underscoring the importance of this kinase in cell proliferation signaling.

A role for Pakl in tumorigenesis is also supported by the interaction of Pak with a number of proteins involved in cell cycle progression. Pakl phosphorylates the DNA scaffolding protein histone H3 on serine l0 both in vitro and in human breast cancer cells, and phosphorylation at this site has been linked to mitotic chromosome assembly, segregation, and movement to the metaphase plate (Cheung et al. 2000; Li et al. 2002). Additionally, Pakl is localized to centrosomes (Banerjee et al. 2002) and was recently shown to phosphorylate and activate the mitotic kinase Aurora A. Inhibition of Pakl in this study led to a delay in both recruitment of Aurora-A to centrosomes as well as centrosome duplication (Zhao et al. 2005). Finally, Pakl is known to induce the expression of cyclin Dl (Balasenthil et al. 2004), a key regulator of Gl cell cycle progression.

Elevated Pak activity has been observed in a number of highly invasive cancer cells, suggesting that Pakl may also play a role in the cell motility underlying metastasis. Expression of constitutively active Pakl increases the motility of breast cancer-derived MCF7 cells (Vadlamudi et al. 2000). Conversely, expression of a kinase-dead Pakl mutant resulted in a three-fold decrease in migration of MDA-MB-435 melanoma cells (Adam et al. 2000). The role of Pakl in mediating cell migration may involve Pakl phosphorylation of the cytoskeletal signaling protein LIM kinase (LIMK), which increases LIMK activity ten-fold (Edwards et al. l999). Activated LIMK phosphorylates and deactivates the F-actin severing protein cofilin, leading to changes in actin cytoskeletal dynamics (Arber et al. l998; Yang et al. l998). Indeed, LIMK activity has been shown to be required for migration in breast cancer-derived cells (Yoshioka et al. 2003), highlighting the role of this Pakl substrate in tumor cell motility.

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