{"id":{"repo_id":"utswmed","oai_identifier":"oai:utswmed-ir.tdl.org:2152.5/678"},"canonical_url":"https://search.dev.ndltd.org/etd/utswmed/oai:utswmed-ir.tdl.org:2152.5/678","repository":{"repo_id":"utswmed","name":"University of Texas Southwestern Medical Center","base_url":"https://utswmed-ir.tdl.org/server/oai/request"},"display":{"title":"Structural and Functional Analysis of HIV-1 Nef Activation of PAK-2","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Kuo, Lillian S."],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Garcia-Martinez, J. Victor"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2010,"date_issued":"2010-07-12T18:49:19Z","date_published":"2010-07-12T18:49:19Z","updated_at":"2026-07-24T05:52:06Z","subjects":["HIV-1","Genes, nef","Protein-Serine-Threonine Kinases"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["754616865"],"render_values":[{"text":"754616865","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/2152.5/678","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Garcia-Martinez, J. Victor"]},{"key":"dc:creator","label":"Author","values":["Kuo, Lillian S."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2010-07-12T18:49:19Z","2009-06-19"]},{"key":"dc:type","label":"Dc Type","values":["Thesis","Text","dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["HIV-1","Genes, nef","Protein-Serine-Threonine Kinases"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2152.5/678","754616865"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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."]},{"key":"dc:format","label":"Dc Format","values":["Electronic","application/pdf","born digital"]},{"key":"dc:title","label":"Title","values":["Structural and Functional Analysis of HIV-1 Nef Activation of PAK-2"]}]}],"canonical_facts":{"dc:contributor":["Garcia-Martinez, J. Victor"],"dc:creator":["Kuo, Lillian S."],"dc:date":["2010-07-12T18:49:19Z","2009-06-19"],"dc:description":["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."],"dc:format":["Electronic","application/pdf","born digital"],"dc:identifier":["https://hdl.handle.net/2152.5/678","754616865"],"dc:language":["en"],"dc:subject":["HIV-1","Genes, nef","Protein-Serine-Threonine Kinases"],"dc:title":["Structural and Functional Analysis of HIV-1 Nef Activation of PAK-2"],"dc:type":["Thesis","Text","dissertation"]},"updated_at":"2026-07-24T05:52:06Z"}