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

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Title: Structural and Functional Analysis of HIV-1 Nef Activation of PAK-2
Author: Kuo, Lillian S.
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .5 /678
Date: 2009-06-19

Citation

Structural and Functional Analysis of HIV-1 Nef Activation of PAK-2. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /678 .

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