IDENTIFICATION AND CHARACTERIZATION OF INTERACTIONS BETWEEN ITK AND Gα13
Open Access
- Author:
- Huang, Weishan
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- None
- Committee Members:
- Avery August, Thesis Advisor/Co-Advisor
Avery August, Thesis Advisor/Co-Advisor - Keywords:
- protein-protein interaction
Gα13
signal transduction
T cell receptor
Itk
G protein coupled receptor - 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). Other than TCR ligands, migratory responses to chemokines such as CCL11/Eotaxin and CXCL12/SDF-1α that act through G protein coupled receptors (GPCRs), require Itk. However, the mechanism by which chemokine receptor signals connect to Itk is not clear. We hypothesize that the G-protein 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. We extended the investigation to test the properties of Itk and Gα13 that are important for this interaction. The interaction requires the membrane localization of both parties, as a membrane targeting deficient mutant of Itk (R29C), lost its association with WT Gα13, and a membrane targeting deficient mutant of Gα13 (C14S, C18S) lost its interaction with WT Itk. However, this interaction is Itk kinase activity independent (shown by Itk K391M mutant). We also show that both GTP bound and GDP bound forms of Gα13 can interact with Itk. Interestingly, we found that mutations in the Zn2+ binding Bruton’s tyrosine kinase (Btk) homology (BH) motif (C132GC133G, CCGG for further reference), disrupted the interaction between Itk and Gα13, even though this Itk mutant was still able to get recruited by WT Itk and form membrane associated clusters. The striking finding is that co-expression of Itk and Gα13 resulted in tyrosine phosphorylation of Gα13. Moreover, we found that the interaction between Itk and Gα13 is transient upon stimulation against TCR signaling axis. 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.