Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity - PubMed (original) (raw)

Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity

Y J Zhou et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, gammac and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.

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Figures

Figure 1

Figure 1

Tryptic phosphopeptide mapping of autophosphorylated JAK3. WT JAK3, mutant Y980F, mutant Y981F, and mutant YY980/981FF were expressed in COS-7 cells and immunoprecipitated using anti-JAK3 C-terminal antiserum, and in vitro kinase assays were performed for 10 min at room temperature. The phosphoproteins were subjected to SDS/PAGE, transferred to nitrocellulose, and exposed to x-ray film. The portions of the membrane containing WT and mutant JAK3 proteins were excised and digested in situ with trypsin. Eluted tryptic phosphopeptides from WT and indicated mutants (A) or eluted tryptic peptides from WT JAK3 mixed with a synthetic phosphopeptide DYpYpVVR (B) were analyzed by two-dimensional peptide mapping. The 32P-labeled phosphopeptides were visualized by autoradiography (B Left), and the position of synthetic phosphopeptide was determined by ninhydrin staining as indicated (B Right). The orientation of the positive and negative electrodes during electrophoresis is indicated along with the direction of chromatography; the origin is indicated by the letter O. The most prominent spots were designated a–d. Because of the effect of the mutations on kinase activity, the exposure time was varied to achieve similar intensities for the map of each mutant (exposure time: Y980F, 96 hr; YY980/981FF, 48 hr; WT JAK3, 16 hr; and Y981F, 6 hr).

Figure 2

Figure 2

Effects of Y980 and Y981 mutation on JAK3 in vitro kinase activity. (A) JAK3 autophosphorylation. (B) γc phosphorylation. (C and D) The quantitation of phosphorylation over time. COS-7 cells were transfected with WT JAK3 (lane 1) or K855A (lane 2), Y980F (lane 3), Y981F (lane 4), and YY980/981FF (lane 5) mutant forms of JAK3. Lysates were immunoprecipitated with a JAK3 antiserum, and kinase assays were performed with GSTγc fusion protein as an exogenous substrate. (A and B) Data are from a 5-min reaction at 0°C. Immunoblotting with anti-JAK3 antiserum (A Lower) and anti-GST antiserum (B Lower) is also shown.

Figure 3

Figure 3

(A) Opposing effects of mutation of Y980 and Y981 on JAK3 phosphorylation in COS-7 cells. COS-7 cells were transfected with cDNAs (5 μg) encoding: WT JAK3 (lane 1), K855A (lane 2), Y980F (lane 3), Y981F (lane 4), and YY980/981FF (lane 5). Two days after transfection, cell lysates were precipitated with a JAK3 C-terminal antiserum. Samples were analyzed by SDS/PAGE, transferred to nitrocellulose, and subjected to immunoblotting with antiphosphotyrosine mAb (4G10, Upper) or anti-JAK3 antiserum (Lower). (B) JAK3 Y980 is required for efficient γc phosphorylation. COS-7 cells were cotransfected with 5 μg Tac-γc cDNA and 5 μg cDNAs encoding: K855A (lane 1), JAK3 (lane 2), Y980F (lane 3), Y981F (lane 4), and YY980/981FF (lane 5), or transfected with 5 μg Tac-γc cDNA only (lane 6), and immunoprecipitated with a mAb anti-Tac (7G7). In vitro kinase assays were performed on the immunoprecipitates at room temperature for 5 min. Samples were analyzed by SDS/PAGE and transferred to nitrocellulose, subjected to autoradiography (Top) and immunoblotting with mAb anti-γc (Middle), and anti-JAK3 antiserum (Bottom).

Figure 4

Figure 4

Autophosphorylation of Y980 is necessary for STAT5A phosphorylation. COS-7 cells were cotransfected with STAT5A cDNA (5 μg) and the cDNAs (5 μg) encoding: WT JAK3 (lane 1), K855A (lane 2), Y980F (lane 3), and Y981F (lane 4), or transfected with 5 μg STAT5A cDNA only (lane 5). Two days later, cells were lysed, immunoprecipitated with anti-STAT5A antiserum, and immunoblotted with mAb 4G10 (Upper) and anti-STAT5A antiserum (Lower).

Figure 5

Figure 5

Regulation of STAT5A DNA binding by mutation of JAK3 residues Y980 and Y981. COS-7 cells were transfected with 5 μg STAT5A cDNA only (lanes 1, 8, 13, and 17) or cotransfected with 5 μg STAT5A and 5 μg cDNAs encoding: WT JAK3 (lanes 2, 9, 14, and 18), K855A (lane 3), Y980F (lanes 4, 10, 15, and 19), Y981F (lanes 5, 11, 16, and 20), or YY980/981FF (lanes 6 and 12), or mock transfected (lane 7). Two days later, cells were harvested and nuclear extracts were prepared. Protein determination was measured using BCA protein assay reagent (Bio-Rad). EMSA was performed using 5 μg protein and a labeled probe from the GAS-like element in the CD23 promoter (lanes 1–7). Competition with unlabeled probe is shown in lanes 8–12. Supershift analysis with anti-STAT5A antiserum (lanes 13–16) compared with a control antibody (lanes 17–20) is also shown.

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