The Zinc binding site in the BH domain of Itk is required for interaction with Gα13 (50.10)

Abstract

Abstract The Tec family kinase, Itk has been shown to be critical in T cell activation and development downstream of the T cell receptor (TCR). Migratory responses to chemokines such as CCL11/Eotaxin and CXCL12/SDF-1α require Itk. However, the mechanism by which chemokine receptor signals connect to Itk is not clear. We speculate Gα13 may serve to couple GPCRs, Itk and downstream signals. Using a Bimolecular Fluorescence Complementation system, we show that Itk and Gα13 are in close physical proximity, less than 80Å. This interaction requires the membrane localization of Itk, as the membrane targeting deficient mutants (S15P, R29C), lost their association with Gα13. Moreover, we found that mutations in the Zn2+ binding Bruton’s tyrosine kinase homology (BH) motif (C132GC133G), disrupted the interaction, even though this Itk mutant was still able to get recruited to membrane associated clusters. However, this interaction is Itk kinase activity independent. We also show that both GTP bound and GDP bound forms of Gα13 can interact with Itk. Surprisingly, co-expression of Itk and Gα13 resulted in tyrosine phosphorylation of Gα13. Taken together, we propose that interactions between Gα13 and Itk may connect chemokine/chemokine receptor stimulation and downstream responses regulated by Itk. Itk may also modulate the function of Gα13 by tyrosine phosphorylation. These data provide evidence for better understanding of the mechanism by which chemokines regulate immune cell responses.