Characterization of anti-pY(904)Jak3 and anti-pY(939)Jak3 antibodies. (A) Phospho-specific Jak3 antibodies to pY904 and pY939 are selective targets. Increasing amounts of Y904 and pY904 peptides (left panel, top), as well as Y939 and pY939 peptides (left panel, bottom), were spotted onto PVDF membranes and tested for recognition by rabbit anti-pY(904)Jak3 and anti-pY(939)Jak3 antibodies by Western blotting (immunoblotting [IB]). Additionally, increasing amounts of pY904 and pY993 peptides were spotted onto PVDF membranes and tested for recognition by rabbit anti-pY(904)Jak3 (right panel, top), anti-pY(939)Jak3 (right panel, middle), and anti-pY (right panel, bottom) antibodies by Western blotting. (B) Anti-pY(904)Jak3 antibody recognizes activated WT Jak3. HEK293 cells were transfected with plasmids for WT Jak3 or Y904F or kinase-inactive K855A mutant Jak3. Jak3 proteins were immunoprecipitated (IP) with anti-Jak3 antibody, followed by an in vitro kinase assay in the presence of 100 μM ATP for 30 min as described in Materials and Methods. Phosphorylation of Y904 was detected by blotting with anti-pY(904)Jak3 (upper panel). The Jak3 expression level was monitored by reblotting the membrane with anti-Jak3 antibody (lower panel). Shown are representative data from three independent experiments. (C) Anti-pY(939)Jak3 antibody preferentially recognizes activated WT Jak3. HEK293 cells were transfected with plasmids for WT or Y939F or K855A mutant Jak3. Jak3 proteins were immunoprecipitated, and tyrosine phosphorylation was monitored following an in vitro kinase assay as described for panel B. The phosphorylation of Jak3 on Y939 was detected by blotting with anti-pY(939)Jak3 antibody (upper panel). The blot was reprobed with anti-Jak3 antibody (lower panel). Shown are representative data from three independent experiments.

Jak3 Y904F and Y939F variants have decreased autophosphorylation and kinase activity toward the exogenous substrate GST-γc. (A) HEK293 cells were transfected with plasmids for WT Jak3 or the Y939F, Y939F, or K855A variant. Jak3 was immunoprecipitated (IP) from cell lysates and tested for kinase activity in the presence of the Jak3 substrate GST-γc. The pY levels of Jak3 and GST-γc were assessed by quantitative anti-pY antibody Western blotting (immunoblotting [IB]) normalized to Jak3 and GST-γc. (B) Phosphorylation of GST-γc was quantified, and data from two representative independent experiments were plotted. (C) Jak3 autophosphorylation was quantified, and data from two representative independent experiments were plotted.

Y904 and Y939 are located within distinct regions of the Jak3 catalytic domain and are conserved among Jak family members. (A) Y904 localizes to the ATP binding pocket of the N lobe of the kinase domain. (B) Y939 is harbored within α-helix E of the C lobe of the kinase domain. Models were made with the DeepView Swiss-pdbviewer 3.7 program, based on the crystal structure of the Jak3 kinase domain solved in complex with the staurosporine analogue ANF941 (4) (accession number 1YVJ). Residues E903, Y904, L905, and Y939 and ANF941 are indicated in color beneath the Jak3 structure. (C) The amino acid sequences surrounding the novel phosphotyrosine sites in human Jak3 were aligned with human Jak1, Jak2, and Tyk2.

Association of Stat5 with phosphorylated Y939 peptides. Peptides containing nonphosphorylated or phosphorylated Y939 were coupled to CNBr-activated beads and used to probe for interacting proteins from YT-cell lysates. YT cells (3 × 10⁷/sample) were left untreated or stimulated with 100 nM IL-2 for 10 min. Soluble cell lysates were incubated with the indicated peptides for 1 h at 4°C. After washing, bound proteins were eluted from the beads with SDS sample buffer. Binding of Stat5, pY Stat5, and other tyrosine-phosphorylated proteins was assessed by Western blotting (immunoblotting [IB]) with anti-Stat5, anti-pY Stat5, and anti-pY antibodies. Molecular mass markers are indicated on the left. One representative set of data from three independent experiments is shown.

Identification of novel phosphorylated tyrosine residues in human Jak3. (A) Jak3 is phosphorylated on novel tyrosine sites in HEK293 cells. HEK293 cells were transfected with empty vector or plasmids for WT Jak3 or Jak3 mutant forms Y980F, Y980F/Y981F, Y981F, Y785F, Y980F/Y981F/Y785F (YYY/FFF), and K855A, as indicated. Cells were harvested at 30 h posttransfection and lysed, and Jak3 proteins were immunoprecipitated (IP) from soluble lysates with anti-Jak3 antibody. The immunoprecipitates were then Western blotted (immunoblotted [IB]) with pY or anti-Jak3 antibodies as indicated. Shown are representative data from three independent experiments. (B) Summary of peptides detected as phosphorylated at Y904 and Y939. (C) Tandem mass spectra of monophosphorylated peptides showing site localization to either Y904 (left panel) or Y939 (right panel), as indicated by asterisks. Methionine oxidation is denoted (#). (D) Domain architecture of human Jak3 with known and newly identified (asterisks) tyrosine phosphorylation sites. Numbers indicate amino acid residues of human Jak3.

Phosphorylation of Jak3 Y904 and Y939 in YT, Kit225, and primary human T cells is mediated by γc-containing cytokines. (A) YT cells were stimulated with IL-2 for the indicated times. Endogenous Jak3 was then immunoprecipitated (IP) with anti-Jak3 antibody and Western blotted (immunoblotted [IB]) with anti-pY(904)Jak3 (left panels) or anti-pY(939)Jak3 (right panels) antibody. Blots were then reprobed with anti-pY and anti-Jak3 antibodies. Shown are representative data from three independent experiments. (B) Kit225 cells were made quiescent in IL-2-free medium overnight and then stimulated with IL-2 or IL-9 for the indicated times. Phosphorylation of Jak3 at Y904 and Y939 was monitored as described for panel A. Shown are representative data from two independent experiments. (C) Purified human T lymphocytes were activated with PHA for 72 h, subsequently made quiescent for 24 h, and then stimulated with IL-2 for the indicated times. Phosphorylation of Jak3 on Y904 and Y939 was determined by Western blotting with the respective phospho-specific antibodies. Representative data from three independent experiments are shown.

Jak3 Y939 and Y904 are required for optimal Stat5 activity in vivo. (A) HEK293 cells were cotransfected with plasmids encoding the Stat5a and Jak3 proteins as indicated. At 30 h posttransfection, cells were harvested and cell lysates were immunoprecipitated (IP) with anti-Stat5a antibody. Stat5a activation was then assessed by Western blotting (immunoblotting [IB]) with anti-pY Stat5 antibody. Total Stat5a levels were determined by reprobing the membrane with anti-Stat5a antibody, as shown in the panel below. (B) Cell lysates were also immunoprecipitated with antibodies to Jak3 and Western blotted for tyrosine phosphorylation. Total Jak3 levels were monitored by reprobing with anti-Jak3 antibodies as shown in the panel below. Shown are representative data from four independent experiments. (C) In six-well dishes, subconfluent cells were transfected in triplicate with the Stat5-activated β-casein-luciferase reporter plasmid, the TK-Renilla luciferase vector, Stat5a, and WT Jak3 or Jak3 mutant forms, as indicated. Control (CTRL) wells were transfected with identical amounts of luciferase and empty vectors. Cells were lysed, and the luciferase activities were determined at 30 h posttransfection. Representative data from three independent experiments are shown. Cell lysates were also immunoblotted with anti-pY Stat5, anti-Stat5a, and anti-Jak3 antibodies to verify equivalent expression of Jak3 and Stat5a (lower panels).

The Jak3 Y904F mutant form shows impaired ATP binding activity. (A) HEK293 cells were transfected with plasmids for WT Jak3 or the Y904F or Y939F mutant form. Jak3 immunoprecipitation (IP) kinase assays were performed with increasing concentrations of ATP in the presence of 2 μg GST-γc for 20 min as indicated. pY levels of GST-γc were assessed by quantitative Western blotting (immunoblotting [IB]) with anti-pY and anti-GST antibodies. Jak3 expression levels were determined by Western blotting with anti-Jak3 antibody. (B) Quantification of the K_mATP and V_max for WT Jak3 and the Y904F and Y939F variants were performed with GraphPad Prism 4 software and Michaelis-Menten kinetics. Each datum point represents at least three independent experiments. Error bars represent the standard error of the mean.