The C2 domain of Tollip, a Toll-like receptor signalling regulator, exhibits broad preference for phosphoinositides

Abstract

TLRs (Toll-like receptors) provide a mechanism for host defence immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn promote the activation of IRAKs (interleukin-1 receptor-associated kinases). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Tollip (Toll-interacting protein) modulates IRAK function in the TLR signalling pathway. Tollip is multimodular, with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), in a Ca2+-independent manner. However, NMR analysis demonstrates that the Tollip C2 domain binds Ca2+, which may be required to target the membrane interface. NMR and lipid-protein overlay analyses suggest that PtdIns3P and PtdIns(4,5)P2 share interacting residues in the protein. Kinetic studies reveal that the C2 domain reversibly binds PtdIns3P and PtdIns(4,5)P2, with affinity values in the low micromolar range. Mutational analysis identifies key PtdIns3P- and PtdIns(4,5)P2-binding conserved basic residues in the protein. Our findings suggest that basic residues of the C2 domain mediate membrane targeting of Tollip by interaction with phosphoinositides, which contribute to the observed partition of the protein in different subcellular compartments.

Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides

“…In the absence of polyubiquitinated cargo, Tollip could be partitioned in membrane-bound and membrane-free states, which depend on both the presence of PtdIns(3)P-rich domains and ubiquitin. The affinity of Tollip for PtdIns(3)P is 46 nM, which is ϳ240-fold higher than its isolated C2 domain (18). This increment is not due to additional interactions of the protein via either TBD or CUE domains with PtdIns(3)P (data not shown); thus, oligomerization of Tollip provides high affinity and sensitivity to PtdIns(3)P levels at endosomal membranes.…”

“…The remaining bound protein was washed away by the injection of 30 l of 10 mM HEPES (pH 8.3), 150 mM NaCl, 0.005% Tween 20, 350 mM EDTA, and 1 mM NaN 3 . Tollip-PtdIns(3)P interactions were followed using PtdIns(3)P-free and -enriched liposomes as ligands and GST-Tollip as an analyte, similar to what we previously described (18). In all cases, sensorgrams were obtained from eight different concentrations of each of the tested proteins.…”

“…Cloning, Expression, and Purification of Tollip and Ubiquitin Constructs-The cloning, expression, and purification of human Tollip and the isolated C2 (residues 54 -182) and CUE (residues 223-278) domains as GST fusions and untagged proteins were carried out as previously reported (18,21). Purified His-tagged rat PKC␣ C2 domain and a rat cDNA that codifies for PKC␤ II C2 domain, cloned into the pGEX4T1 vector, were kindly provided by Dr. Robert Stahelin (University of Notre Dame).…”

“…Tollip localizes to endosomal membranes and the subcellular localization of the protein is likely associated with its ability to preferentially bind PtdIns(3)P through its central C2 domain (18). Because Tollip binds ubiquitin through its CUE domain (19), we then asked whether ubiquitin regulates phosphoinositide recognition.…”

“…Tollip TBD associates with the Tom1 GAT domain to facilitate membrane trafficking (10). We have recently demonstrated that the C2 domain of Tollip preferentially binds phosphoinositides, particularly PtdIns(3)P and PtdIns(4,5)P 2 (18). Thus, Tollip likely acts in the recruitment of several proteins on the endosomes via PtdIns(3)P (for membrane trafficking) and the plasma membrane via PtdIns(4,5)P 2 (e.g.…”