Fig. 1. PAK1 activation requires ZAP-70 but not LAT. The cells indicated were stimulated for 3 min with C305 (+) or a buffer control (–) at 37°C. Endogenous PAK1 was immunoprecipitated with an anti-PAK1 antibody and subjected to an in vitro kinase assay as described in Materials and methods. The fold increase in H4 phosphorylation after TCR stimulation was normalized to the fold activation of parental Jurkat cells in the same experiment. This percentage of wild-type Jurkat PAK1 activation was averaged over n experiments and plotted above. Error bars indicate the range or standard error over n experiments. P116 (n = 2) is a ZAP-70-deficient Jurkat cell. P116 C39 (n = 2) is a ZAP-70 reconstituted P116 cell. J.CaM2 (n = 11) is a LAT-deficient cell. J.CaM2 LAT3 (n = 3) and J.CaM2 LAT9 (n = 2) are two J.CaM2 cells independently reconstituted with LAT.

Fig. 2. TCR-mediated PAK1 activation is independent of Slp-76 and Nck. (A) Parental Jurkat cells and the Slp-76-deficient Jurkat subline, J14, were tested for PAK1 kinase activity as in Figure 1 (n = 4). (B) Jurkat T cells were transfected with either 5 µg of pEF-HA-PAK1 (WT), 12 µg pEF-HA-PAK1P13A (P13A) or 5 µg pEFBos (vector) to achieve equal expression levels. Fourteen hours later, the cells were stimulated for 3 min with C305 (+) or a buffer control (–) at 37°C. Anti-Nck immunoprecipitates were analyzed by 7.5% SDS–PAGE and blotted with an anti-HA antibody (top panel) or an anti-Nck antibody (middle panel). Whole cell lysates were blotted with an anti-HA antibody (bottom panel) showing equivalent expression of HA-PAK1. The experiment shown is representative of two independent experiments. (C) As in (B), but anti-HA immunoprecipitates were tested for kinase activity (n = 4). The increased mean fold activation and large range of PAK1 P13A was due to a single experiment out of four in which PAK1 P13A was uncharacteristically activated 3.5-fold above wild-type PAK1.

Fig. 3. Rac1 activation by the TCR requires ZAP-70 and LAT, but not Slp-76. (A) The cells indicated were stimulated for 2 min with C305 (+) or a buffer control (–) and then lysed. Rac1-GTP was specifically precipitated by incubation with GST–PAK1–CRIB domain for 15 min followed by a rapid wash with lysis buffer. Bound Rac1 was detected by SDS–PAGE and a Rac1-specific antibody (top panel) and compared with total Rac1 (bottom panel). These results are representative of at least three independent experiments. The cells tested were: the ZAP-70-deficient P116 cell, or the ZAP-70 reconstituted P116 C39, the LAT-deficient J.CaM2 cell, a LAT reconstituted J.CaM2 LAT3, the Slp-76-deficient J14 cell and a Slp-76 reconstituted J14 76-11. (B) Jurkat T cells were transfected with either 5 µg of pEF-HA-PAK1 (WT) or 5 µg of pEF-HA-PAK1H83L,H86L (H83,86L) to achieve equal expression. Kinase activity of the HA-tagged PAK1 was measured as in Figure 2C (n = 3).

Fig. 4. PIX binding is required for PAK1 activation. (A) Jurkat T cells were transfected with either 5 µg of pEF-HA-PAK1 (WT) or 3 µg of pEF-HA-PAK1P192G,R193A (P192G, R193A) to achieve equal expression. The cells indicated were stimulated for 2 min with C305 (+) or a buffer control (–) and then lysed. Anti-HA immunoprecipitates were analyzed for PIX content by 7.5% SDS–PAGE and western blotting with an anti-PIX antibody (top panel) or an anti-HA antibody (bottom panel). Results are representative of three independent experiments. (B) PAK1 kinase assays were performed on anti-HA immunoprecipitates prepared as in (A) (n = 3).

Fig. 5. Overexpression of βPIX mutants blocks PAK1 activation. (A) Anti-PIX immunoblot of 1 × 10⁶ cells from parental Jurkat, representative clones overexpressing either wild-type βPIX (WT βPIX), L238R, L239S βPIX (DH* βPIX) or W34P, W44G βPIX (SH3* βPIX). (B) Endogenous PAK1 kinase assays performed as in Figure 1B on representative clones expressing the indicated βPIX allele (n = 3). Results are representative of least three independent clones expressing each PIX allele (data not shown).

Fig. 6. p95PKL–PIX–PAK1 is required for efficient PAK1 activation by the TCR. (A) Either Jurkat cells stably expressing wild-type FLAG-βPIX or untransfected Jurkat cells were stimulated for 2 min with C305 (+) or a buffer control (–) and then lysed. Anti-FLAG immunoprecipitates were analyzed by 7.5% SDS–PAGE and blotting with an anti-p95PKL antibody (top panel) or an anti-PAK1 antibody (middle panel). The blot was then stripped and reprobed with an anti-FLAG antibody (bottom panel). These data are representative of two independent experiments. (B) Jurkat cells were stimulated for 2 min with C305 (+) or a buffer control (–) and then lysed. Endogenous PAK1 was immunoprecipitated from Jurkat cells with a C-terminally directed anti-PAK1 antibody (lanes 1 and 2) or the same antibody pre-incubated with a PAK1-blocking peptide (lanes 3 and 4). The immunoprecipitates were analyzed by 7.5% SDS–PAGE and western blotting with an anti-p95PKL antibody (top panel) and anti-PAK1 antibody (bottom panel). The top panel was stripped and reprobed with an anti-PIX antibody (middle panel), which explains the relatively weaker PIX signal. (C) Jurkat T cells were co-transfected with 15 µg of the GFP parental vector and 5 µg of pEF-HA-PAK1 or with 15 µg of LD4–GFP and 20 µg of pEF-HA-PAK1 to equalize expression of PAK1. Six hours after transfection, the cells were stimulated for 2 min with C305 or a buffer control at 37°C and PAK1 kinase activity was measured (left panel, n = 3). Equivalent immunoprecipitation of HA-PAK1 was confirmed by an anti-HA western blotting (right top panel) and expression of GFP and LD4–GFP was confirmed by an anti-GFP western blot from whole cell lysate (right bottom panel).

Fig. 7. Proposed model for PAK1 and Rac1 activation by the TCR. PAK1 activation requires Lck and ZAP-70, but occurs independently of LAT and Slp-76. Rac1 activation requires ZAP-70 and LAT, but is independent of Slp-76. These contrast the requirement of ZAP-70, LAT and Slp-76 for Ras and phosphatidylinositol pathway activation. Each of these divergent pathways is required to activate the transcriptional responses of T-cell activation. PAK1 activity most likely contributes to cytoskeletal reorganization as well. Undoubtedly, cytoskeletal rearrangements require contributions from many other components besides PAK1, but these have been omitted for clarity.