Structural and Functional Analysis of HIV-1 Nef Activation of PAK-2




Kuo, Lillian S.

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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.

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