Tyrosine residues of IL-2Rβ are not required for its association with p85. (A) Constitutive association of IL-2Rβ and p85. Freshly isolated PBL (lanes 1 and 2), PHA blasts (lanes 3 and 4), YT cells (lanes 5 and 6), or MT-2 cells (lanes 7 and 8) were starved for ≥4 h in RPMI 1640 medium containing 1% FBS and were not stimulated (lanes 1, 3, 5, and 7) or were stimulated with 2 nM IL-2 (lanes 2, 4, 6, and 8) for 10 min; cell lysates were immunoprecipitated with hMikβ1 and immunoblotted with anti-p85. (B) Coprecipitation of p85 with IL-2Rβ in 32D cells expressing wild-type IL-2Rβ (lanes 3 and 4) or a mutated form of IL-2Rβ in which all six tyrosines were mutated to phenylalanines (lanes 5 and 6). Cells were starved of growth factor for 4 h, not stimulated or stimulated with 2 nM IL-2 for 10 min, washed, and lysed prior to immunoprecipitation and Western blotting. (C) Coprecipitation of p85 with IL-2Rβ constructs. 293 T⁺ cells were cotransfected with plasmids expressing p85, Jak1, and either the empty vector (pME18S; lane 1) or pME18S driving expression of wild-type IL-2Rβ (βYYYYYY; lane 2), IL-2Rβ in which all tyrosines are mutated (βFFFFFF; lane 3), or an IL-2Rβ construct lacking residues 380 to 525 (ΔH mutant; lane 4). Cells were harvested 36 to 48 h posttransfection. Note that the decrease in apparent p85 coprecipitation (lane 4, top) corresponded to the decreased expression of IL-2Rβ ΔH (lane 4, bottom). Immunoprecipitation and Western blotting for panels B and C were performed as for panel A. The lower portions of panels B and C represent Western blots with goat antiserum to human IL-2Rβ (R & D Systems), as controls for expression.

Importance of the S and A regions of IL-2Rβ for Akt IL-2-induced activation. (A) 32D cells expressing either wild-type (βWT) or mutant forms of human IL-2Rβ were rested overnight and then stimulated with IL-2 for 15 min. In vitro kinase (IVK) assays were performed on Akt immunocomplexes, using histone H2B peptide as an exogenous substrate. (B) Loading control for the in vitro kinase assay. Immunoprecipitation (IP) with anti-Akt was followed by Western blotting with anti-Akt antibody. (C) Receptor expression controls are also shown for each cell line.

Both of the SH2 domains of p85 are required for its efficient association with Jak1. (A) 293 T⁺ cells were transfected with FLAG-tagged Jak1 and with the indicated HA-tagged forms of p85. Immunoprecipitations (IP) with anti-FLAG M2 MAb were followed by Western blotting with anti-HA MAb. The p85 constructs (described in reference 10) are as follows: N-SH2, residues 329 to 439, spanning the more N-terminal of the two SH2 domains in p85; C-SH2, residues 563 to 724, spanning the more C-terminal SH2 domain; NC, residues 329 to 439 plus residues 563 to 724, containing both SH2 domains but lacking the p110 binding (inter-SH2) region; NiC, residues 329 to 724, like NC except that it includes the inter-SH2 region; ΔiSH2, full-length p85 that lacks the 439–563 inter-SH2 region; iSH2, residues 425 to 616, a construct that contains only the inter-SH2 region; PBP, residues 79 to 328, containing the Pro-Bcr-Pro region located between the SH3 and N-terminal SH2 domains of p85; and SH3, residues 1 to 78, spanning the SH3 domain. All constructs have N-terminally positioned HA tags. WT, wild type. (B) Expression control for the HA-tagged forms of p85. (C) Expression controls for Jak1.

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.

Jak1 is required for IL-2-induced phosphorylation of PI 3-K p85. (A) Transfection of Jak1 is required for IL-2-induced tyrosine phosphorylation of PI 3-K p85 in A49 cells. A49 cells were transfected with IL-2Rβ, γ_c, and Jak3, with or without wild-type (WT) Jak1 or DNJ1 (DNJak1). Cells were washed and starved overnight in DMEM–1% FBS and then not stimulated or stimulated for 10 min with 2 nM IL-2. The arrowhead points to a band that we believe to be Jak1. IP, immunoprecipitation; αPY, antiphosphotyrosine. (B) The lack of phosphorylation of p85 in lane 6 of panel A was not due to lack of association of DNJ1 with p85. Note that the decreased association of DNJ1 with p85 in the absence of IL-2 was not expected; however, the key point is that the degree of association in lane 6 was similar to that seen with wild-type Jak1 (lane 4). (C) Controls for expression of Jak1, Jak3, p85, IL-2Rβ, and γ_c in the transfected A49 cells.

Importance of the S and A regions of IL-2Rβ for the association of PI 3-K p85 and Jak1. (A) The S and A regions are required for optimal association of IL-2Rβ and p85. 293 T⁺ cells were cotransfected with plasmids expressing Jak1, p85, and either wild-type (βWT) or mutant forms of IL-2Rβ, immunoprecipitated (IP) with hMikβ1, and then Western blotted with anti-p85. (B) The S and A regions are required for optimal association of IL-2Rβ and Jak1. 293 T⁺ cells were cotransfected with plasmids as for panel A, lysed, and immunoprecipitated with hMikβ1 followed by Western blotting with MAb to Jak1. (C) Lysates of the transfected 293T⁺ cells used for panels A and B were blotted with anti-Jak1 (Transduction Laboratories), anti-p85 (UBI), or anti-IL-2Rβ (Santa Cruz).

Jak1 and IL-2Rβ cooperate for efficient recruitment of PI 3-K p85. (A) Wild-type Jak1 (J1) and DNJ1 associate with p85. 293 T⁺ cells were cotransfected with a vector control or a plasmid expressing wild-type IL-2Rβ (βWT), Jak1, or DNJ1. Cellular lysates were immunoprecipitated (IP) with anti-p85 and then Western blotted with anti-Jak1. (B) Constitutive association of Jak1 and p85. PHA blasts and CTLL-2, YT, 32D, 32Dβ, and 32DβFFFFFF cells were starved for 4 h, stimulated with 2 nM IL-2 for 10 min, washed, and lysed. Immunoprecipitations and Western blotting were performed as for panel A. (C) The interaction of IL-2Rβ and p85 is augmented by the presence of Jak1. Murine fibroblasts lacking Jak1 (A49 cells) were transfected with pME18S, wild-type IL-2Rβ, or IL-2Rβ plus Jak1. Cell lysates were immunoprecipitated with hMikβ1 and then blotted with antiserum to Jak1 (top) or p85 (bottom). (D) Lysates of the cells described for panel C were Western blotted to confirm expression of transfected cDNAs. Note that there is somewhat more IL-2Rβ expressed in lane 2 than in lane 3. This makes it even more clear that the increased coprecipitation in panel C, lane 3, is due to the presence of Jak1.

The JH1 and JH2 regions of Jak1 are required for interaction with p85. Jak2 and Jak3 do not interact with p85. (A) 293 T⁺ cells were transfected with p85 and with the indicated forms of FLAG-tagged Jak1 C-terminal deletions or FLAG-tagged Jak2 or Jak3. Immunoprecipitations (IP) with anti-p85 antibody were followed by Western blotting with anti-FLAG M2 MAb. WT, wild type. (B) Expression control for the FLAG-tagged forms of Jak1, Jak2, and Jak3. (C) Expression controls for p85.

IL-2-induced phosphorylation of PI 3-K. (A) IL-2 induces phosphorylation of p85 in normal PHA blasts. (B) IL-2 induces phosphorylation of p85 in Molt4β cells (lane 4 versus lane 3) but not in Molt4 cells (lanes 1 and 2). Immunoprecipitations (IP) and Western blotting were performed as for Fig. 7.