Abstract
WASp family Verprolin-homologous protein-2 (WAVE2), a member of the Wiskott-Aldrich syndrome protein (WASp) family of actin nucleation promoting factors, is a central regulator of actin cytoskeleton polymerization and dynamics. Multiple signaling pathways operate via WAVE2 to promote the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. WAVE2 exists as a part of a pentameric protein complex known as the WAVE regulatory complex (WRC), which is unstable in the absence of its individual proteins. While the involvement of WAVE2 in actin polymerization has been well documented, its negative regulation mechanism is poorly characterized to date. Here, we demonstrate that WAVE2 undergoes ubiquitylation in a T-cell activation dependent manner, followed by proteasomal degradation. The WAVE2 ubiquitylation site was mapped to lysine 45, located at the N-terminus where WAVE2 binds to the WRC. Using Förster resonance energy transfer (FRET), we reveal that the autoinhibitory conformation of the WRC maintains the stability of WAVE2 in resting cells; the release of autoinhibition following T-cell activation facilitates the exposure of WAVE2 to ubiquitylation, leading to its degradation. The dynamic conformational structures of WAVE2 during cellular activation dictate its degradation.
Highlights
WASp family Verprolin-homologous (WAVE) proteins contain a WAVE/SCAR homology domain (WHD/SHD) at their N-terminus, immediately followed by a basic region (B)[17,18,19]
We recently demonstrated that WAVE2 is recruited to the T cell receptor (TCR) site and co-localizes with the nucleation promoting factor (NPF), WASp, during the initiation of T-cell activation[15]
These results were confirmed by a reciprocal immunoprecipitation, demonstrating a ladder of bands of ubiquitylated endogenous WAVE2 above the molecular weight (MW) of WAVE2 (75 kDa) with a strong precipitate of ubiquitylated WAVE2 at around 83 kDa, which is increased in stimulated T cells (Fig. 1b)
Summary
WAVE proteins contain a WAVE/SCAR homology domain (WHD/SHD) at their N-terminus, immediately followed by a basic region (B)[17,18,19]. (b) Jurkat T cells were left unstimulated (−), or were co-stimulated with anti-CD3 and anti-CD28 antibodies (+). The factors regulating the stability of the WRC members are not clear. It was shown by Nolz et al that stability of the WRC components is inter-dependent; elimination of any component results in decreased amounts of WAVE as well as of the other constituents of the complex[12]. It was shown in Dictyostelium that cells expressing a mutant SCAR (a WAVE homolog), lacking a WRC binding site, produce a stable protein in both wild type cells and in cells missing various members of the complex[30].
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