Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Amy H. Andreotti
Itk is a tyrosine kinase that is implicated in the T cell signal transduction and cytoskeleton reorganization after TCR engagement. It is well established that Itk needs to be recruited to the plasma membrane and transphosphorylated by Lck to be activated. However the detailed mechanisms that regulate Itk kinase activity are still elusive. Itk consists of a PH domain, proline rich region, and a SH3, SH2 and kinase domain. Our group previously reported that the Itk SH3 domain can interact with Itk SH2 domain leading to self-association of the large fragment that includes both the SH3 and SH2 domains. In this thesis, I have extended this observation to the full-length Itk molecule. A full-length Itk-SH3 mutant that exhibits decreased self-association was constructed by replacing the Itk SH3 domain with the Btk SH3 domain. In vitro kinase assays indicate that the wild type full-length Itk kinase activity decreases at higher protein concentration while kinase activity of the Itk-SH3 mutant increased linearly with increasing enzyme concentration. When coexpressed with PLCg1 in insect cells, the full-length Itk-SH3 mutant phosphorylates PLCg1 to a greater extent than does wild type full-length Itk. Furthermore, expression of the full-length Itk-SH3 mutant in primary T cells leads to higher ERK phosphorylation following TCR stimulation than wild-type Itk. Our data suggest that the tyrosine kinase Itk can be down regulated by intermolecular self-association.
PLCg1 is the substrate for Itk in T cells. After the TCR engagement, Itk is activated and phosphorylates its substrate PLCg1. Activated PLCg1 hydrolyzes PIP2 into IP3 and DAG, which mobilize Ca2+ release and activate PKC respectively. Phosphorylation is required for PLCg1 activation. However, the mechanism by which PLCg1 is specifically phosphorylated by Itk is not clear. In this thesis, I discover that a docking interaction is necessary for efficient phosphorylation of PLCg1 by Itk. Interestingly, a similar docking interaction is required for autophosphorylation in the Itk SH3 domain. Further studies indicate that the docking interaction is mediated by the Itk kinase domain and a portion of the substrate SH2 domain. This docking interaction is different from typical SH2 domain mediated interactions as it does not involve phosphor-tyrosine binding pockets of SH2 domain and nor does it involve phosphorylation of the Itk kinase domain. The docking interaction enhances Itk mediated phosphorylation by ~7 fold by increasing the affinity between the Itk kinase domain and its substrates. Furthermore, the Tec family kinases are conserved in exploiting this docking interaction to phosphorylate their substrates.
The docking interaction provides a possibility to selectively target the enzyme-substrate interaction surface instead of the enzyme catalytic site to modulate enzyme activity. The structural basis for the docking interaction is not yet completely understood. Site-directed-mutagenesis was used to map out the docking site on the PLCg1 SH2C. We found that the docking site on the PLCg1 SH2C is composed of a largely basic surface from the BG loop and CD loop of the SH2C. Consistent with the location of the docking site on the SH2C, the presence of phosphor-tyrosine ligand does not interfere with the interaction between the Itk kinase domain and the PLCg1 SH2C. Furthermore the docking site that is mapped out in the SH2C domain fragment can be extended to full-length PLCg1. The presence of excess wild type PLCg1 SH2C domain in an in vitro kinase assay, but not the SH2C docking mutants, can decrease the phoshporylation of full-length PLCg1. Mutation of the residues that are involved in the docking interaction in the full-length PLCg1 substrate diminishes the phosphorylation by Itk. Our data indicate that the docking interaction is required for the full-length PLCg1 phosphorylation by Itk.
We also found an intramolecular interaction between the PLCg1 SH2C and the adjacent linker that can modulate the docking interaction between SH2C and Itk kinase domain. Biochemical and NMR spectroscopic analyses indicate that the intramolecular interaction is different from the canonical SH2/phosphor-tyrosine interaction. Residues in the SH2C domain that mediate linker binding form a hydrophobic groove that overlaps with the canonical pY+3 binding pocket. Our data suggest that the intramolecular interaction might be one of the mechanisms that keep PLCg1 in its inactive conformation before its phosphorylation and activation. Overall this thesis provides significant molecular level insight into mechanisms of the substrate recognition by Itk and regulation of TCR signaling.
Min, Lie, "Interleukin-2-tyrosine kinase substrate docking and its regulation by an intramolecular interaction in phospholipase Cg1" (2008). Graduate Theses and Dissertations. 10590.