Figure 5

The VGF region is important for Nef-PAK2 association, cofilin hyper- phopsorylation and inhibition of F-actin rich circumferential ring formation by Nef. (A) Jurkat TAg cells were transfected arrows with WT, AxxA, VGF→AAA, VGF→AGF, VGF→VAF or VGF→VGA mutant Nef.GFP fusion proteins, plated onto poly-L-lysine (PLL)–coated cover glasses, fixed and stained for phospho-cofilin (p-cofilin). Images show intense staining for p-cofilin (red-orange) only in cells expressing Nef (green) of wild-type, but not of mutated sequence. Scale bar = 10 μm (B) Frequency of the cells from cultures as shown in panel A that were scored high in p-cofilin. Values are the means of 3 independent experiments, and error bars represent SD from the mean; ≥ 100 cells were analyzed per transfection; * indicates P < 0.01, ** indicates P<0.001 and *** indicates P < 0.0001. (C) Lysates of Jurkat TAg cells expressing eGFP or WT, VGF→AAA or AxxA SF2 Nef.GFP fusion proteins. Lower panels (input) show Western blot for Nef and loading control (transferrin receptor). These lysates were immunoprecipitated for Nef.GFP (rαGFP-IP), and subsequently used for in vitro kinase assay (IVKA). Nef-PAK2 association is detected in the IVKA only with WT Nef, by the presence of autophosphorylated PAK2 (62 kDa; p-PAK2) as well as of a yet unindentified 72 kDa substrate (p72). (D) Jurkat TAg cells were transfected with WT, AxxA, VGF→AAA, VGF→AGF, VGF→VAF or VGF→VGA mutant Nef.GFP fusion proteins, plated onto anti-CD3–coated cover glasses, fixed and stained with phalloidin to reveal F-actin (red). Scale bar = 10 μm (E) Frequency of the cells from cultures as shown in panel A that to form F-actin–rich circumferential rings. Values are the means of 3 independent experiments, and error bars represent SD from the mean; ≥ 100 cells were analyzed per transfection, * indicate P < 0.01, ** indicates P<0.001 and *** indicates P < 0.0001.