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Molecular and Cellular Biology, February 2000, p. 947-956, Vol. 20, No. 3
Lymphocyte Cell Biology Section, Arthritis
and Rheumatism Branch, National Institute of Arthritis, Musculoskeletal
and Skin Diseases,1 Howard Hughes
Medical Institute
Received 5 October 1999/Accepted 5 November 1999
The structure of Janus kinases (JAKs) is unique among protein
tyrosine kinases in having tandem, nonidentical kinase and pseudokinase domains. Despite its conservation in evolution, however, the function of the pseudokinase domain remains poorly understood. Lack of JAK3
expression results in severe combined immunodeficiency (SCID). In this
study, we analyze two SCID patients with mutations in the JAK3
pseudokinase domain, which allows for protein expression but disrupts
the regulation of the kinase activity. Specifically, 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
cells with these mutants demonstrated that, although they were
constitutively phosphorylated basally, they were unable to transmit
cytokine-dependent signals. Further analysis showed that the isolated
catalytic domain of JAK3 was functional whereas either the addition of
the pseudokinase domain or its deletion from the full-length molecule
reduced catalytic activity. Through coimmunoprecipitation of the
isolated pseudokinase domain with the isolated catalytic domain, we
provide the first evidence that these two domains interact.
Furthermore, whereas the wild-type pseudokinase domain modestly
inhibited kinase domain-mediated STAT5 phosphorylation, the
patient-derived mutants markedly inhibited this phosphorylation. We
thus conclude that the JAK3 pseudokinase domain is essential for JAK3
function by regulating its catalytic activity and autophosphorylation.
We propose a model in which this occurs via intramolecular interaction
with the kinase domain and that increased inhibition of kinase activity
by the pseudokinase domain likely contributes to the disease
pathogenesis in these two patients.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Complex Effects of Naturally Occurring Mutations in
the JAK3 Pseudokinase Domain: Evidence for Interactions between the
Kinase and Pseudokinase Domains
National Institutes of Health Research Scholars
Program,2 Clinical Gene Therapy Branch,
National Human Genome Research Institute,3
National Institutes of Health, Bethesda, Maryland 20892;
Department of Pediatrics, University of Goteborg, SE-41685
Goteborg, Sweden4; and Department of
Pediatrics, University of Brescia, I-25123 Brescia,
Italy5
*
Corresponding author. Mailing address:
LCBS/ARB/NIAMS/NIH, 10/9N262, 10 Center Dr., MSC-1820, Bethesda,
MD 20892. Phone: (301) 496-2541. Fax: (301) 402-0012. E-mail:
chenm{at}arb.niams.nih.gov.
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