Wiskott-Aldrich syndrome protein and its key regulator WIP: NMR investigation of phosphoregulation

Wiskott-Aldrich Syndrome protein (WASP) is a 502-residue polypeptide responsible for cytoskeleton rearrangement and predominantly expressed in hematopoietic cells. WASP-Interacting Protein (WIP) is a 503-residue regulator of WASp activity and degradation, whose C-terminal domain is known to interact via several short epitopes with the EVH1 domain at the WASp N-terminus. Mutations in WASp interfering with this chaperone function of WIP cause immunodeficiencies Wiskott-Aldrich syndrome and X-linked thrombocytopenia, and hematopoietic malignancies result from insufficient WIP expression, highlighting the importance of the WIP/WASp complex. Our investigation focuses on the structural basis for phosphoregulation of this key complex, using the ¹H,¹⁵N-HSQC NMR spectrum as our main tool for viewing the state of this complex.

Contrary to the accepted paradigm that WIP and WASp dissociate due to PKCtheta phosphorylation of residue S488, the HSQC of phosphomimicking mutant WIP(S488E) did not show changes reflecting a dissociation event. However, the HSQC of two other mutants, WIP(Y468E) and WIP(Y475E), exhibits a global change that is consistent with partial dissociation of WIP from WASp. In fact, triple mutant WIP(Y455,468,475E) representing phosphorylation of three tyrosine phosphosites loses much of the affinity to WASp, yet remains bound. We have characterized the phosphorylation-induced structural change occurring in the complex, showing that WIP loses its tight bound structure upon phosphorylation and becomes mostly unstructured, presumably exposing WASp to interactions with other cellular factors. These findings are discussed in the context of our NMR-derived three-dimensional structure of the WIP/WASp complex.