title: 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.
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
authors: Sophia
authors: S
authors: Fried
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
authors: Barak
authors: B
authors: Reicher
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
authors: Maor H
authors: MH
authors: Pauker
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
authors: Shani
authors: S
authors: Eliyahu
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
authors: Omri
authors: O
authors: Matalon
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
authors: Elad
authors: E
authors: Noy
authors: Department of Chemistry, Faculty of Exact Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
authors: Jordan
authors: J
authors: Chill
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel. mira.barda-saad@biu.ac.il.
authors: Mira
authors: M
authors: Barda-Saad
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D000074121:Protein Kinase C-theta; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D013601:T-Lymphocytes / Q000378:metabolism / Q000473:pathology; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D011948:Receptors, Antigen, T-Cell; C495767:WAS protein, human; C109903:WIPF1 protein, human; D051316:Wiskott-Aldrich Syndrome Protein; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta