Interferon alpha (IFN-alpha) modulates the proliferation of different human tumour cell lines. It has been shown that IFN-alpha induces the growth inhibition of T-cell acute lymphoblastic leukaemia (T-ALL). However, its intracellular signalling mechanisms remain unknown. This study found that IFN-alpha inhibited the cell proliferation of human T-ALL cell line Jurkat in a dose- and time-dependent manner. A p38 inhibitor (SB203580), but not an extracellular signal-regulated kinase 1/2 inhibitor (PD98059) or c-Jun N-terminal kinase inhibitor (SP600125), eliminated IFN-alpha inhibition of Jurkat cell proliferation, indicating that p38 pathway is crucial for IFN-alpha-mediated growth inhibition. SB203580 targeted two p38 isoforms, p38alpha and p38beta. The expression of p38alpha and p38beta mRNA in Jurkat cells was examined by reverse transcriptase-polymerase chain reaction. The kinase activity of p38alpha and p38beta was activated by IFN-alpha in Jurkat cells. To investigate the role of p38alpha and p38beta isoforms in IFN-alpha-mediated growth inhibition, we generated stable clones that overexpressed the dominant-negative p38 isoform, p38alpha(AF) or p38beta(AF), in Jurkat cells. Overexpression of p38alpha(AF) or p38beta(AF) inhibited IFN-alpha-mediated p38 kinase activity and growth inhibition in Jurkat cells. Similarly, down-regulation of either p38alpha or p38beta by isoform-specific small interference RNAs also reduced IFN-alpha-mediated growth inhibition. These results demonstrate that IFN-alpha can regulate growth inhibition of Jurkat cells through p38alpha and p38beta.

The human interleukin-3 receptor (hIL-3R) consists of a unique alpha subunit (hIL-3Ralpha) and a common beta subunit (betac). Binding of IL-3 to IL-3R activates Janus kinases JAK1 and JAK2. Our previously study showed that JAK2 and JAK1 were constitutively associated with the hIL-3Ralpha and betac subunits, respectively. In this study, we further demonstrate that JAK2 binds to the intracellular domain of hIL-3Ralpha and JAK1 binds to the Box 1 and Box 2 motifs of betac using GST-hIL-3R fusion proteins in pull-down assays. JAK1 mutational analysis revealed that its JH7-3 domains bound directly to the Box 1 and Box 2 motifs of betac. We further examined the role of JAK1 JH7-3 domains in JAK1 and JAK2-mediated signaling using the CDJAKs fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1), JAK2 (CDJAK2), or JAK1-JH7-3 domains (CDJAK1-JH7-3) as intracellular domains. Anti-CD16 antibody crosslinking of wild type fusion proteins CDJAK1 with CDJAK2 could mimic IL-3 signaling, however, the crosslinking of fusion proteins CDJAK1-JH7-3 with CDJAK2 failed to activate downstream proteins. These results suggest that the JAK1-JH7-3 domains are required for betac interaction and abolish wild type JAK1 and JAK2-mediated signaling.