Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex

Abstract

Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeletal machinery. Binding of WASp-interacting protein (WIP) to WASp modulates WASp activity and protects it from degradation. Formation of the WIP-WASp complex is crucial for the adaptive immune response. We found that WIP and WASp interacted in cells through two distinct molecular interfaces. One interaction occurred between the WASp-homology-1 (WH1) domain of WASp and the carboxyl-terminal domain of WIP that depended on the phosphorylation status of WIP, which is phosphorylated by protein kinase C θ (PKCθ) in response to T cell receptor activation. The other interaction occurred between the verprolin homology, central hydrophobic region, and acidic region (VCA) domain of WASp and the amino-terminal domain of WIP. This latter interaction required actin, because it was inhibited by latrunculin A, which sequesters actin monomers. With triple-color fluorescence resonance energy transfer (3FRET) technology, we demonstrated that the WASp activation mechanism involved dissociation of the first interaction, while leaving the second interaction intact. This conformation exposed the ubiquitylation site on WASp, leading to degradation of WASp. Together, these data suggest that the activation and degradation of WASp are delicately balanced and depend on the phosphorylation state of WIP. Our molecular analysis of the WIP-WASp interaction provides insight into the regulation of actin-dependent processes.

A conformational change within the WAVE2 complex regulates its degradation following cellular activation

“…In a manner similar to WASp regulation, WAVE2 also requires the release from its autoinhibitory conformation by dissociating the VCA domain. Furthermore, we previously discovered that upon TCR stimulation, the WASp-WIP molecular complex is partially dissociated, leading to the ubiquitylation of activated WASp on lysine residues 76 and 81 and to its degradation3437. Indeed, we show here the essential role of the non-inhibited open conformation of WAVE2 for its ubiquitylation.…”

“…Förster resonance energy transfer (FRET) technology enables the detection of intra- as well as inter- molecular interactions on a nanometer scale3738. We utilized this technique to monitor possible conformational changes within WAVE2 by constructing a FRET-based probe consisting of WAVE2 tagged with YFP at its N-terminus and with CFP at its C-terminus (YFP-WAVE2-CFP, Fig.…”

“…Furthermore, in these cells, WASp strongly associates with WIP via amino acids 56–102 located within the WASp N-terminal WH1 domain344950. Deletion of the WASp VCA domain disrupts its interaction with WIP, thereby exposing WASp to ubiquitylation and, subsequently, to degradation37. In a manner similar to WASp regulation, WAVE2 also requires the release from its autoinhibitory conformation by dissociating the VCA domain.…”

“…Similar to WAVE2, WASp is intrinsically inactive in naïve T cells and exists in an autoinhibited state in which its C-terminal VCA domain intramolecularly interacts with its GTPase-binding domain (GBD)48 and, intermolecularly, with WASp-interacting protein (WIP)37. Furthermore, in these cells, WASp strongly associates with WIP via amino acids 56–102 located within the WASp N-terminal WH1 domain344950.…”

Nesfatin-1 Suppresses Cardiac L-type Ca2+ Channels Through Melanocortin Type 4 Receptor and the Novel Protein Kinase C Theta Isoform Pathway

“…In brief, cells were Assay for PKC activity PKC acitivity was measured as described previously [25,[27][28][29]. The cells were pretreated with either vehicle or chelerythrine chloride for 30 min, followed by treatment with NF-1 for 15 min.…”