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

Abstract

Wiskott-Aldrich syndrome protein (WASP) is in a complex with WASP-interacting protein (WIP). WASP levels, but not mRNA levels, were severely diminished in T cells from WIP(-/-) mice and were increased by introduction of WIP in these cells. The WASP binding domain of WIP was shown to protect WASP from degradation by calpain in vitro. Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP levels in T cells from WIP(-/-) mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupt WIP binding. The calpain inhibitor calpeptin increased WASP levels in activated T and B cells from the WASP patients, but not in primary T cells from the patients or from WIP(-/-) mice. Despite its ability to increase WASP levels proteasome inhibition did not correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient. These results demonstrate that WIP stabilizes WASP and suggest that it may also be important for its function.

Fig. 1.

WASP is unstable in WIP^−/− T cells. (A) WASP expression in T cells from WIP^−/− and WT control mice. Vav and actin protein expression levels are shown as controls. (B) FACS analysis of intracellular WASP expression in WT, WIP^−/−, and WASP^−/− splenic T cells. Cells were stained with anti-WASP mAb 5A5 and isotype-matched mouse IgG2a as control. (C) Northern blot analysis of WASP mRNA in splenic T cells from WIP^−/− mice and WT controls. (D) WASP expression in B cells from WIP^−/− and WT controls. (E) N-WASP expression in fibroblasts from WIP^−/− mice and WT controls. (F) WIP expression in T cells from WASP^−/− mice and WT controls.

Fig. 2.

Overexpression of WIP in cells results in increased levels of WASP, but not N-WASP. (A) WASP binding to Myc-tagged N-WIP (1–400) and C-WIP (401–503) in Jurkat cells transfected with Myc-tagged C-WIP and N-WIP under the control of a DOX-inducible promoter. The presence of WASP in Myc immunoprecipitates was examined before and after DOX treatment. To achieve optimal resolution of Myc-N-WIP (≈50 kDa) and to avoid Myc-C-WIP (≈11 kDa) running off the gel, the gels were run for different time periods. (B) WASP expression in the same Jurkat cells. Representative results from one of four independent clones for each construct are shown. The asterisk denotes nonspecific bands. (C) Forced expression of WIP in T293 cells does not alter N-WASP levels.

Fig. 3.

Forced expression of WIP in WIP^−/− T cells increases WASP levels. (A) Expression of GFP proteins in mouse transfected T cells. The asterisks denote degradation products. (B) WASP binding to GFP proteins in transfected thymocytes. (C) FACS analysis of intracellular WASP expression in WIP^−/− T cells expressing WIP–GFP, WIPΔWASP–GFP, and GFP alone.

Fig. 4.

Effect of immunodepletion of WIP on WASP levels (A) and of WASP on WIP levels (B) in T cells. T cell lysates were immunoprecipitated sequentially five times with anti-WASP or anti-WIP mAbs, and the lysates and immunoprecipitates were run on gels and Western blotted and probed for WASP or WIP. The numbers 0–5 refer to the number of times sequential immunoprecipitation was performed. The numbers below the Western blot of the lysates refer to the percent residual WASP or WIP with 100 being the value in the lysates before immunodepletion.

Fig. 5.

Inhibitors of calpain and the proteasome increase WASP levels in T cells from WIP^−/− mice. T cells from WT and WIP^−/− mice were incubated with medium containing DMSO vehicle (Med), MG132 (M), calpeptin (C), or both (M+C) for 6 h, then analyzed for WASP by flow cytometry by using mAb B-9 conjugated to phycoerythrin (A) or by Western blot using B-9 mAb (B). Expression of Vav1 was used as control. The numbers in B represent the ratio of WAS:Vav1 in inhibitor-treated cells versus untreated control cultured with medium alone.

Fig. 6.

Effect of calpain and proteasome inhibitors on WASP levels in lymphocytes from WAS patients. Effect of inhibitors on WASP levels in purified peripheral blood T cells and PHA/IL-2 propagated T cells from a WAS patient with R86H mutation (A and B) and in EBV-transformed B cells from a WAS patient with T45M mutation (C).