title: Nuclear localization of the tyrosine kinase Itk and interaction of its SH3 domain with karyopherin alpha (Rch1alpha).
abstract: We report a physical and functional association between the Tec-family tyrosine kinase Itk (Emt/Tsk) and the nuclear import chaperone karyopherin alpha (Rch1alpha) in human T cells. The Itk-SH3 domain and the Rch1alpha proline-rich (PR) motif were crucial for the Itk/Rch1alpha constitutive interaction as demonstrated by directed mutagenesis of the Rch1alpha PR motif (proline 242 to alanine, P242A). TCR-CD3 stimulation of Jurkat T cells resulted in increased Itk/Rch1alpha complex formation, recruitment of karyopherin beta to the protein complex and Rch1alpha 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-Rch1alpha fusion. We observed constitutive nuclear localization of Itk that was up-regulated following either TCR-CD3 stimulation or over-expression of wild-type Rch1alpha in T cells. Further, nuclear localization of Itk and TCR-CD3-mediated IL-2 production were significantly down-regulated following expression of the Rch1alpha-P242A mutant, implicating a role for Rch1alpha in the nuclear translocation of Itk.
authors.affiliation: Immunology, Inflammation, Pulmonology and Dermatology Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Perez-Villar
authors.forename: K
authors.initials: K
authors.lastname: O'Day
authors.forename: D H
authors.initials: DH
authors.lastname: Hewgill
authors.forename: S G
authors.initials: SG
authors.lastname: Nadler
authors.forename: S B
authors.initials: SB
authors.lastname: Kanner
mesh_terms: D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D017252:CD3 Complex; D002451:Cell Compartmentation; D015536:Down-Regulation; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell; D013601:T-Lymphocytes / Q000276:immunology*; D020798:Two-Hybrid System Techniques; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D017252:CD3 Complex; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D028901:alpha Karyopherins; C087978:karyopherin alpha 2; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase