DISTINCT TYROSINE PHOSPHORYLATION SITES IN JAK3 KINASE DOMAIN POSITIVELY AND NEGATIVELY REGULATE ITS ENZYMATIC-ACTIVITY

Cytokines are critically important for the growth and development of a variety of cells, Janus kinases (JAKs) associate with cytokine recept ors and are essential for transmitting downstream cytokine signals. Ho wever, the regulation of the enzymatic activity of the JAKs is not wel l understood. Here, we investigated the role of tyrosine pbosphorylati on of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase d omain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutate d to phenylalanine individually or doubly. We found that JAK3 is autop hosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated marked ly decreased kinase activity, and optimal phosphorylation of JAK3 on o ther sites was dependent on Y980 phosphorylation. The mutant Y980F als o exhibited reduced phosphorylation of its substrates, gamma c and STA T5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/9S1FF, had intermediate activity. The se results indicate that Y980 positively regulates JAK3 kinase activit y whereas Y981 negatively regulates JAK3 kinase activity. These observ ations in JAK3 are similar to the findings in the kinase that is close ly related to the JAK family, ZAP-70; mutations of tyrosine residues w ithin the putative activation loop of ZAP-70 also have opposing action s, Thus, it will be important to determine whether this feature of reg ulation is unique to JAK3 or if it is also a feature of other JAKs. Gi ven the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of thes e and other tyrosine residues in the control of kinase activity and he nce cytokine signaling.