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Journal of Virology, October 2005, p. 12732-12741, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12732-12741.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Reconstitution and Molecular Analysis of an Active Human Immunodeficiency Virus Type 1 Nef/p21-Activated Kinase 2 Complex

Alexa Raney, Lillian S. Kuo, Laura L. Baugh, John L. Foster, and J. Victor Garcia*

Department of Internal Medicine, Division of Infectious Diseases, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390

Received 20 June 2005/ Accepted 29 July 2005

Human immunodeficiency virus type 1 (HIV-1) Nef activation of p21-activated kinase 2 (PAK-2) was recapitulated in a cell-free system consisting of in vitro-transcribed RNA, rabbit reticulocyte lysate, and microsomal membranes on the basis of the following observations: (i) Nef associated with a kinase endogenous to the rabbit reticulocyte lysate that was identified as PAK-2, (ii) Nef-associated kinase activity was detected with Nefs from HIV-1SF2, HIV-1YU2, and SIVmac239, (iii) kinase activation was not detected with a myristoylation-defective Nef (HIV-1SF2NefG2A) or with a Nef defective in PAK-2 activation but fully competent in other Nef functions (HIV-1SF2NefF195I), and (iv) Nef-associated kinase activation required activated endogenous p21 GTPases (Rac1 or Cdc42). The cell-free system was used to analyze the mechanism of Nef activation of PAK-2. First, studies suggest that the p21 GTPases may act transiently to enhance Nef activation of PAK-2 in vitro. Second, addition of wortmannin to the cell-free system demonstrated that Nef activation of PAK-2 does not require PI 3-kinase activity. Third, ultracentrifugation analysis revealed that whereas the majority of Nef and PAK-2 partitioned to the supernatant, Nef-associated PAK-2 activity partitioned to the membrane-containing pellet as a low-abundance complex. Lastly, Nef activation of PAK-2 in vitro requires addition of microsomal membranes either during or after translation of the Nef RNA. These results are consistent with a model in which activation of PAK-2 by Nef occurs by recruiting PAK-2 to membranes. As demonstrated herein, the cell-free system is a new and important tool in the investigation of the mechanism of PAK-2 activation by Nef.

* Corresponding author. Mailing address: Department of Internal Medicine, Division of Infectious Diseases, University of Texas Southwestern Medical Center at Dallas, Y9.206, 5323 Harry Hines Boulevard, Dallas, TX 75390-9113. Phone: (214) 648-9970. Fax: (214) 648-0231. E-mail: victor.garcia{at}utsouthwestern.edu.

Journal of Virology, October 2005, p. 12732-12741, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12732-12741.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Rauch, S., Pulkkinen, K., Saksela, K., Fackler, O. T. (2008). Human Immunodeficiency Virus Type 1 Nef Recruits the Guanine Exchange Factor Vav1 via an Unexpected Interface into Plasma Membrane Microdomains for Association with p21-Activated Kinase 2 Activity. J. Virol. 82: 2918-2929 [Abstract] [Full Text]  
  • Wei, B. L., Arora, V. K., Raney, A., Kuo, L. S., Xiao, G.-H., O'Neill, E., Testa, J. R., Foster, J. L., Garcia, J. V. (2005). Activation of p21-Activated Kinase 2 by Human Immunodeficiency Virus Type 1 Nef Induces Merlin Phosphorylation. J. Virol. 79: 14976-14980 [Abstract] [Full Text]  


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