Browsing by Subject "Genes, nef"

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Identification and Characterization of a NEF-Associated Kinase
(2004-05-04) Arora, Vivek Kumar; Bennett, Michael
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.
Structural and Functional Analysis of HIV-1 Nef Activation of PAK-2
(2009-06-19) Kuo, Lillian S.; Garcia-Martinez, J. Victor
Nef is an accessory protein encoded by HIV-1 that activates the host cellular p21 activated protein kinase 2 (PAK-2). Previous work has characterized the structural plasticity of Nef with regard to PAK-2 activation. Residues 89 and 191 were identified to be components of an effector domain required for Nef mediating PAK-2 activation with lesser contributions from position 85 and 188. H89 and F191 are highly conserved in subtype B Nefs (LHKF), however in subtype E Nef F89 and R191 predominate. Subtype E Nefs also activate PAK-2, therefore it appeared at least two different structural variants are present in HIV-1 Nefs. Substitution of all four residues in a subtype B Nef with subtype E-like residues (F85, F89, A188 and R191, FFAR) generated a fully functional subtype E PAK-2 effector domain in a subtype B background. A third effector domain found in subtype C Nefs (F85, F89, H188, and H191, FFHH) was also investigated. The contribution of residues 187 and 188 in these alternative Nef structural variants (LHKF, FFAR, and FFHH) to activate PAK-2 was determined. Surprisingly, the L188 substitution in the LHKF structure resulted in PAK-2 hyperactivation. While the I187 substitution in LHKF completely ablated PAK-2 activity. In stark contrast, I187 in the FFHH variant resulted in hyperactivation. Thus, subtle changes in amino acid composition can dramatically affect kinase activation levels. The work in this thesis has characterized a PAK-2 effector domain on Nef constituted by amino acid position 85, 89, 187, 188 and 191. The results indicate that this is not the only Nef region mediating PAK-2 activation. The highly conserved polyproline helix also plays a role in the activation of PAK-2. Conservative mutations of this SH3 binding region completely abrogated PAK-2 activation suggesting SH3 binding is necessary, however this binding appears to be weak. My data suggest a model where activation of PAK-2 by Nef requires a ternary, or higher order, complex containing SH3/Nef/PAK-2. Synergistic interactions between the two Nef effector domains investigated here and a host cell protein, or proteins, could explain the specific activation of PAK-2 by Nef.