Abstract
Expression of the human immunodeficiency virus (HIV) Nef protein has been linked to both decreased cell surface expression of CD4 and an impairment of signal transduction. The recently reported association of Nef with an unidentified serine kinase provides a clue as to how Nef might exert its effects. Considering the key role of protein kinase C (PKC) in T cell activation, we investigated the possibility that Nef interacts with PKC. Our results, using two approaches for detecting interactions between Nef and PKC isozymes in Jurkat cells, show that Nef interacts preferentially with thetaPKC. The interaction of Nef and thetaPKC is independent of calcium, enhanced by phospholipid activators of PKC and not affected by a PKC pseudosubstrate peptide. Phorbol 12-myristate 13-acetate and phytohemagglutinin stimulation of Jurkat cells expressing Nef fails to produce the usual translocation of thetaPKC from the cytosol to the particulate fraction; translocation of betaPKC and epsilonPKC was unaffected. Indeed, there appears to be a net loss of thetaPKC in Nef-expressing cells following stimulation. The loss of thetaPKC, which may be a result of inhibition of its binding to RACKs due to Nef binding, could contribute to the various impairments of T cell function associated with HIV infection and Nef expression.
Highlights
To test for an interaction between Nef and protein kinase C (PKC), Nef was expressed as a GST fusion protein in E. coli, purified by adsorption on glutathione-agarose beads, and incubated with the cytosolic fraction of Jurkat cells
PKC associated with the GSTNef fusion protein was assayed by measuring the phosphorylation of a specific PKC substrate peptide (Fig. 1)
Maximal phosphorylation activity occurred in the presence of phosphatidylserine, diacylglycerol, and EGTA, suggesting that a calcium-independent PKC isozyme binds Nef
Summary
From the Departments of Biochemistry and ‡Molecular Pharmacology, Beckman Center and Stanford University School of Medicine, Stanford, California 94305. Expression of the human immunodeficiency virus functions attributed to different PKC isozymes as well as the (HIV) Nef protein has been linked to both decreased cell specific intracellular localization characteristic of individual surface expression of CD4 and an impairment of signal isozymes suggests that endogenous anchoring proteins or retransduction. Considering the key role of protein kinase C (PKC) in T cell activation, we investigated the possibility that Nef interacts with PKC. Our results, using two approaches for detecting interactions between Nef and PKC isozymes in Jurkat cells, show that Nef interacts preferentially with PKC. We have explored the possibility that Nef interacts with PKC and with PKC. Our results suggest that Nef may interfere with the interaction of PKC with its endogenous anchoring proteins and that this inhibition may result in the net loss of the isozyme
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