FIG. 7.

p85 tyrosine phosphorylation is mediated by Jak1, but not by Jak3 or Lck, in an IL-2Rβ-dependent fashion. (A) Jak1 and IL-2Rβ are required for tyrosine phosphorylation of p85. 293 T⁺ cells were cotransfected with plasmids expressing p85, Jak1, DNJ1, and either wild-type (βWT) or mutant forms of IL-2Rβ. Cells were harvested 36 to 48 h posttransfection, washed in phosphate-buffered saline, and lysed in lysis buffer. Supernatants were boiled in SDS reducing sample buffer and immunoblotted to evaluate expression of transfected cDNAs; alternatively, lysates were immunoprecipitated (IP) with antiserum to p85 and immunoblotted with 4G10. Expression of transfected constructs (Jak1, p85, and IL-2Rβ constructs) was evaluated by Western blotting (bottom). (B) Neither Jak3 nor Lck can phosphorylate p85. 293 T⁺ cells were cotransfected with PI 3-K (all lanes), FLAG-tagged Jak1 (lanes 2 and 6), FLAG-tagged Jak3 (lanes 3 and 7), or FLAG-tagged Lck (lanes 4 and 8). In lanes 5 to 8, cells were additionally transfected with IL-2Rβ; cells in lanes 1 to 4 were not. Immunoprecipitations and Western blotting were performed as for panel A. Expression of p85 and FLAG-tagged kinases is shown at the bottom. The expression controls are all from one blot; the cut marks reflect the fact that the loading order for the controls was different from that used in the upper blot; the lanes were repositioned to correspond to the upper blot.