Complex effects of naturally occurring mutations in the JAK3 pseudokinase domain: Evidence for interactions between the kinase and pseudokinase domains

The structure of Janus kinases (JAKs) is unique among protein tyrosine kina ses in having tandem, nonidentical kinase and pseudokinase domains. Despite its conservation in evolution, however, the function of the pseudokinase d omain remains poorly understood. Lack of JAK3 expression results in severe combined immunodeficiency (SCID). In this study, we analyze two SCID patien ts with mutations in the JAK3 pseudokinase domain, which allows for protein expression but disrupts the regulation of the kinase activity. Specificall y, these mutant forms of JAK3 had undetectable kinase activity in vitro but were hyperphosphorylated both in patients' Epstein-Barr virus-transformed B cells and when overexpressed in COS7 cells. Moreover, reconstitution of c ells with these mutants demonstrated that, although they were constitutivel y phosphorylated basally, they were unable to transmit cytokine-dependent s ignals. Further analysis showed that the isolated catalytic domain of JAK3 was functional whereas either the addition of the pseudokinase domain or it s deletion from the full-length molecule reduced catalytic activity. Throug h coimmunoprecipitation of the isolated pseudokinase domain with the isolat ed catalytic domain, we provide the first evidence that these two domains i nteract. Furthermore, whereas the wild-type pseudokinase domain modestly in hibited kinase domain-mediated STAT5 phosphorylation, the patient-derived m utants markedly inhibited this phosphorylation. We thus conclude that the J AK3 pseudokinase domain is essential for JAK3 function by regulating its ca talytic activity and autophosphorylation. We propose a model in which this occurs via intramolecular interaction with the kinase domain and that incre ased inhibition of kinase activity by the pseudokinase domain likely contri butes to the disease pathogenesis in these two patients.