Triple-color FRET analysis reveals conformational changes in the WIP-WASp actin-regulating complex

Sci Signal. 2014 Jun 24;7(331):ra60. doi: 10.1126/scisignal.2005198.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / chemistry*
  • Actins / metabolism
  • Binding Sites / genetics
  • Blotting, Western
  • Cytoskeletal Proteins / chemistry*
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism
  • Fluorescence Resonance Energy Transfer / methods*
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Isoenzymes / metabolism
  • Jurkat Cells
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Confocal
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism
  • Mutation
  • Phosphorylation
  • Protein Conformation*
  • Protein Kinase C / metabolism
  • Protein Kinase C-theta
  • Receptors, Antigen, T-Cell / metabolism
  • Signal Transduction / genetics
  • T-Lymphocytes / metabolism
  • T-Lymphocytes / pathology
  • Wiskott-Aldrich Syndrome Protein / chemistry*
  • Wiskott-Aldrich Syndrome Protein / genetics
  • Wiskott-Aldrich Syndrome Protein / metabolism

Substances

  • Actins
  • Cytoskeletal Proteins
  • Intracellular Signaling Peptides and Proteins
  • Isoenzymes
  • Luminescent Proteins
  • Multiprotein Complexes
  • Receptors, Antigen, T-Cell
  • WAS protein, human
  • WIPF1 protein, human
  • Wiskott-Aldrich Syndrome Protein
  • PRKCQ protein, human
  • Protein Kinase C
  • Protein Kinase C-theta