Identification and Characterization of a NEF-Associated Kinase

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Title: Identification and Characterization of a NEF-Associated Kinase
Author: Arora, Vivek Kumar
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .5 /219
Date: 2004-05-04

Citation

Identification and Characterization of a NEF-Associated Kinase. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /219 .

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