JAK3 Protein Mutation And Preventing The Effect Of Mutation

JAK3 Protein Mutation And Preventing The Effect Of Mutation

Introduction

Cytokines are critical regulators of cellular growth and differentiation, a subset of which bind to members of the type I cytokine receptor superfamily and initiate their actions by ligand- induced receptor oligomerization [20]. Although cytokine receptors lack intrinsic kinase activity, they associate with and activate cytoplasmic protein tyrosine kinases [PTKs], which then phosphorylate downstream signaling molecules such as the signal transducers and activators of transcription [STATs]. Activated STATs, in turn, translocate to the nucleus and regulate gene expression [7]. For TYK2, deletion of the JH2 domain abrogated the in vitro catalytic activity and the ability to transmit alpha/beta interferon-dependent signals, suggesting that the JH2 domain was required for catalytic function of TYK2 [42]. In contrast, however, a similar mutant of JAK2 was able to transmit growth hormone-dependent signals [10]. Indeed, in the context of CD16-JAK2 chimeras, a construct lacking JH2 had increased catalytic activity compared to the one containing the JH2 domain. This finding was interpreted as showing that the JH2 domain might serve to tonically inhibit kinase activity. In addition, it was reported that a mutation within the JH2 domain of the Drosophila JAK, Hopscotch, produced a presumably hyperactive JAK that caused leukemia in flies. The equivalent mutation in JAK2 also resulted in a kinase with an increased ability to phosphorylate STAT5 in an overexpression system [6]. However, the catalytic activity of these mutants was not specifically assessed. Thus, the data to this point did not indicate a simple negative or positive regulatory function of the JH2 domain.

Given these discrepancies and the potential differences among the JAKs, it was not clear what would be the consequence of mutations of the JAK3 JH2 domain. Furthermore, none of these studies provided an in-depth biochemical and functional analysis of JAKs with mutations in the JH2 domain, nor did they offer a mechanism by which the JH2 domain might regulate catalytic function. The study of naturally occurring JAK3 variants with mutations in the JH2 domain offers an advantage in elucidating the functional characteristics of this region. Since the initial description of JAK3-SCID patients, who were devoid of JAK3 expression [8], we recently identified two new patients with mutations involving the JH2 domain that allowed for expression of JAK3 protein but who nonetheless had presented with a SCID phenotype [4].

Materials and Methods

Human IL-2 was obtained from C. Reynolds [National Cancer Institute, Frederick, Md.]. Rabbit polyclonal antisera against JAK3 and STAT5a and 7G7 monoclonal antibody [MAb] [anti-Tac] were described previously [5]. The anti-JAK3 N and C terminus antibodies work comparably well in both immunoprecipitation and Western blotting [data not shown]. Rabbit antiserum against STAT3 was obtained from Andrew Larner [Cleveland Clinic Foundation Research Institute, Cleveland, Ohio].

The 4G10 antiphosphotyrosine MAb and anti-JAK2 polyclonal antisera were from Upstate Biotechnology [Lake Placid, N.Y.]. Rabbit polyclonal anti-glutathione S-transferase [anti- GST], anti-IL2Rgc, anti-extracellular signal-regulated kinase [anti-ERK], and anti-Flag MAb [M2] were purchased from Pharmacia [Piscataway, ...