T-cell receptor ligation causes Wiskott-Aldrich syndrome protein degradation and F-actin assembly downregulation.

Wiskott-Aldrich syndrome protein (WASP) links T-cell receptor (TCR) signaling to the actin cytoskeleton. WASP is normally protected from degradation by the Ca(++)-dependent protease calpain and by the proteasome because of its interaction with the WASP-interacting protein. We investigated whether WASP is degraded after TCR ligation and whether its degradation downregulates F-actin assembly caused by TCR ligation. Primary T cells, Jurkat T cells, and transfected 293T cells were used in immunoprecipitation experiments. Intracellular F-actin content was measured in splenic T cells from wild-type, WASP-deficient, and c-Casitas B-lineage lymphoma (Cbl)-b-deficient mice by using flow cytometry. Calpeptin and MG-132 were used to inhibit calpain and the proteasome, respectively. A fraction of WASP in T cells was degraded by calpain and by the ubiquitin-proteasome pathway after TCR ligation. The Cbl-b and c-Cbl E3 ubiquitin ligases associated with WASP after TCR signaling and caused its ubiquitination. Inhibition of calpain and lack of Cbl-b resulted in a significantly more sustained increase in F-actin content after TCR ligation in wild-type T cells but not in WASP-deficient T cells. TCR ligation causes WASP to be degraded by calpain and to be ubiquitinated by Cbl family E3 ligases, which targets it for destruction by the proteasome. WASP degradation might provide a mechanism for regulating WASP-dependent TCR-driven assembly of F-actin.

Watanabe Y ，Sasahara Y ，Ramesh N ，Massaad MJ ，Yeng Looi C ，Kumaki S ，Kure S ，Geha RS ，Tsuchiya S ... -  《-》

Ubiquitylation-dependent negative regulation of WASp is essential for actin cytoskeleton dynamics.

Reicher B ，Joseph N ，David A ，Pauker MH ，Perl O ，Barda-Saad M ... -  《-》

WIP is a chaperone for Wiskott-Aldrich syndrome protein (WASP).

de la Fuente MA ，Sasahara Y ，Calamito M ，Antón IM ，Elkhal A ，Gallego MD ，Suresh K ，Siminovitch K ，Ochs HD ，Anderson KC ，Rosen FS ，Geha RS ，Ramesh N ... -  《-》

Mechanism of recruitment of WASP to the immunological synapse and of its activation following TCR ligation.

Sasahara Y ，Rachid R ，Byrne MJ ，de la Fuente MA ，Abraham RT ，Ramesh N ，Geha RS ... -  《MOLECULAR CELL》

A peptide derived from the Wiskott-Aldrich syndrome (WAS) protein-interacting protein (WIP) restores WAS protein level and actin cytoskeleton reorganization in lymphocytes from patients with WAS mutations that disrupt WIP binding.

The Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are caused by mutations in WAS, which encodes for WAS protein (WASP). The WASP-interacting protein (WIP) stabilizes WASP, as evidenced by severely decreased WASP levels in T cells from WIP-deficient mice. The majority of missense mutations in patients with WAS/XLT are located in the WIP-binding domain of WASP and might result in dissociation of the WASP-WIP complex and WASP degradation. To restore WASP levels and correct T-cell function in WAS/XLT patients with mutations in the WIP-binding domain of WASP. WIP, and a WIP-derived 41-amino acid-long peptide, which interacts with WASP and was designated nanoWIP (nWIP), were fused to enhanced green fluorescent protein and introduced by electroporation into EBV-transformed B cells, and by retroviral transduction into purified blood T cells from patients with WAS. WASP levels were measured by intracellular fluorescence-activated cell sorting staining. The actin cytoskeleton was visualized by intracellular phalloidin staining. Introduction of WIP and nWIP restored WASP levels to normal in EBV-transformed B-cell lines from XLT patients with missense mutations in the WIP-binding domain of WASP and residual WASP levels, and corrected the defective spreading and pseudopodia formation of their T cells in response to immobilized anti-CD3. A WASP-binding WIP-derived peptide stabilizes WASP in cells from XLT patients with missense mutations that disrupt WIP binding, and corrects their T-cell actin cytoskeleton defect. This may provide a novel therapeutic strategy for these patients.

Massaad MJ ，Ramesh N ，Le Bras S ，Giliani S ，Notarangelo LD ，Al-Herz W ，Notarangelo LD ，Geha RS ... -  《-》