RESUMEN
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.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/inmunología , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Complejo CD3 , Compartimento Celular , Regulación hacia Abajo , Humanos , Interleucina-2/biosíntesis , Células Jurkat , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Unión Proteica , Receptores de Antígenos de Linfocitos T , Técnicas del Sistema de Dos Híbridos , alfa Carioferinas/genética , Dominios Homologos srcRESUMEN
The negative regulation of T- or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca2+ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3zeta, ZAP-70, Syk, and phospholipase Cgammal but not the Src family tyrosine kinase p56(lck). By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.