Identification and Characterization of a NEF-Associated Kinase
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The nef gene encoded by primate lentiviruses is a major determinant of virulence in vivo. It is expressed early in the viral life cycle; its importance likely stems from its ability to prime the host environment for efficient viral replication. Reasonable models by which cellular phenotypes associated with Nef expression could enhance viral replication in vivo have been proposed. The molecular mechanisms by which Nef executes its functions, however, are poorly understood. The work presented here investigates the regulation of cellular proteins by Nef. I first identified a previously described Nef-associated kinase as the cellular kinase p21 activated kinase 2 (Pak2). This was done using proteolytic digestion of the Nef-associated kinase in multiple systems as well as by demonstrating the presence of active, ectopically expressed Pak2 associated with Nef in a cellular expression system. I further demonstrated that Nef induces Pak2 activation in vivo using multiple systems. First, Nef dependent activation of ectopically expressed tagged Pak2 was demonstrated in vivo. Second, an in gel renaturable kinase activity assay showed in cell extracts a Nef dependent kinase activity I subsequently demonstrated to be Pak2 by proteolytic digestion. Third, I showed that in vivo Nef expression induces the phosphorylation of Merlin at S518, a known and specific Pak2 substrate. The mechanism by which Nef leads to Pak2 activation was also addressed. Rho family GTPases are well-described endogenous activators of Pak2. Inhibition of Rho family GTPase activity in vivo also blocked Nef mediated activation of Pak2 as did mutation of the Rho GTPase binding site in Pak2. Thus, Nef induced Pak2 activation is dependent on endogenous Rho family GTPases. No Nef dependent effect on Rho GTPase activity levels, however, was detected. Instead, biochemical separation and cellular localization suggested that Nef mediates Pak2 activation by recruiting Pak2 to membranes where it encounters high concentrations of constituitively active Rho family GTPases. In summary, this work conclusively demonstrated that Pak2 associates with Nef and is activated by Nef via a endogenous Rho GTPase. Lastly, the potential roles of these molecular events in mediating Nef's pathogenic effects are discussed.