Nuclear localization of the tyrosine kinase Itk and interaction of its SH3 domain with karyopherin α (Rch1α)

We report a physical and functional association between the Tec-family tyrosine kinase Itk (Emt/Tsk) and the nuclear import chaperone karyopherin alpha (Rch1 alpha) in human T cells. The Itk-SH3 domain and the Rch1 alpha proline-rich (PR) motif were crucial for the Itk/Rch1 alpha constitutive interaction as demonstrated by directed mutagenesis of the Rch1 alpha PR motif (proline 242 to alanine, P242A). TCR-CD3 stimulation of Jurkat T cells resulted in increased Itk/Rch1 alpha complex formation, recruitment of karyopherin beta to the protein complex and Rch1 alpha a tyrosine phosphorylation. Analysis of in vitro kinase reactions with a panel of recombinant glutathione-S-transferase (GST) fusion tyrosine kinases (Itk, Lck, ZAP-70 and Jak3) revealed that only GST-Itk efficiently phosphorylated a recombinant GST-Rch1 alpha fusion. We observed constitutive nuclear localization of Itk that was up-regulated following either TCR-CD3 stimulation or over-expression of wild-type Rch1 alpha in T cells. Further, nuclear localization of Itk and TCR-CD3-mediated IL-2 production were significantly down-regulated following expression of the Rch1 alpha -P242A mutant, implicating a role for Rch1 alpha in the nuclear translocation of Itk.