3rd Conference on Retroviruses and Opportunistic Infections Washington, DC - January 28-February 1, 1996

Conf Retroviruses Opportunistic Infect 1996 Jan 28-Feb 1; 3rd:60 (abstract no. 41)

Nunn M, Marsh J
National Institute of Mental Health, NIH, Bethesda, MD.

We have modified the nef gene of HIV-1 SF2 to include an eight amino acid epitope at the carboxy-terminus in order to facilitate the purification of the expressed gene product. The modification should not disrupt myristilation of the N-terminus of Nef which is known to be essential for Nef function. Expression of the mutated Nef protein in either the human T-cell line, VB, or U937 cells results in the down-regulation of cell surface CD4 molecules, as has been shown previously for the wild type nef.

Analysis of ³⁵S-radiolabeled proteins immunoprecipitated from VB cells lysed with l% Triton X-100 identifies several candidate Nef-associated proteins at 65KDa, 79KDa and 97KDa. One of these, p65, is the predominant substrate for phosphorylation in an in vitro kinase assay when coimmunoprecipitated with Nef. P65 can be labelled with the ATP analogue FSBA identifying p65 as an ATP-binding protein. P65 kinase can be inhibited with 10μM staurosporine, but is resistant to 100μM genistein, consistent with its identification as a protein serine/threonine kinase.

The p65 kinase preferentially recognizes myelin basic protein and histone H4 as substrates, but does not readily phosphorylate casein or other histones. P65 is not significantly affected by inhibitors specific for PKA, PKC, PKG, PKR or myosin light chain kinase.

Labeled p65 can be immunoprecipitated using antisera directed against peptides of PAK65, a neutrophil histone H4 kinase. P65 is also immunoprecipitated by antisera to the rat PAK-1 and PAK-2 but not antisera recognizing other kinases. Thus, Nef associates with an active form of p65, a member of the PAK family of histone H4 kinases which are known to play central roles in the transduction of extracellular signals.

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Copyright © 1996 - Foundation for Retrovirology and Human Health . Reproduction of this abstract (other than one copy for personal reference) must be cleared through the Foundation for Retrovirology and Human Health.