Characterization of an Aldolase-binding Site in the Wiskott-Aldrich Syndrome Protein

Carlos A Buscaglia,Deepak Penesetti,Mingyuan Tao,Victor Nussenzweig

doi:10.1074/jbc.m506346200

Abstract

The thrombospondin-related anonymous protein (TRAP) is an essential transmembrane molecule in Plasmodium sporozoites. TRAP displays adhesive motifs on the extracellular portion, whereas its cytoplasmic tail connects to actin via aldolase, thus driving parasite motility and host cell invasion. The minimal requirements for the TRAP binding to aldolase were scanned here and found to be shared by different human proteins, including the Wiskott-Aldrich syndrome protein (WASp) family members. In vitro and in vivo binding of WASp members to aldolase was characterized by biochemical, deletion mapping, mutagenesis, and co-immunoprecipitation studies. As in the case of TRAP, the binding of WASp to aldolase is competitively inhibited by the enzyme substrate/products. Furthermore, TRAP and WASp, but not other unrelated aldolase binders, compete for the binding to the enzyme in vitro. Together, our results define a conserved aldolase binding motif in the WASp family members and suggest that aldolase modulates the motility and actin dynamics of mammalian cells. These findings along with the presence of similar aldolase binding motifs in additional human proteins, some of which indeed interact with aldolase in pull-down assays, suggest supplementary, non-glycolytic roles for this enzyme.

Highlights

In addition to cytoskeletal proteins, aldolase binds to the erythrocyte anion exchanger Band 3 [7, 8], different subunits of the vacuolar-type Hϩ-ATPase [9, 10], glucose transporter GLUT4 [11], Sorting-Nexin 9 [12], phospholipase D2 [13], Mycobacterium superoxide dismutase [14], fructose 1,6-bisphosphatase [15], calcium-signaling protein S100A12 [16], calcium/calmodulin-dependent protein kinase phosphatase [17], and the light chain 8 of dynein [18]
The parasite actin-myosin motor localized underneath the plasma membrane promotes the antero-posterior redistribution of thrombospondin-related anonymous protein (TRAP) molecules, which is critical for parasite motility and host cell invasion
Mapping of the Aldolase-binding Site on TRAP—In a previous paper we showed that the last ϳ25 residues of the cytoplasmic tail of TRAP family proteins are sufficient to promote its binding to aldolase [21]

Summary

Introduction

In addition to cytoskeletal proteins, aldolase binds to the erythrocyte anion exchanger Band 3 [7, 8], different subunits of the vacuolar-type Hϩ-ATPase [9, 10], glucose transporter GLUT4 [11], Sorting-Nexin 9 [12], phospholipase D2 [13], Mycobacterium superoxide dismutase [14], fructose 1,6-bisphosphatase [15], calcium-signaling protein S100A12 [16], calcium/calmodulin-dependent protein kinase phosphatase [17], and the light chain 8 of dynein [18]. The binding of TRAP molecules to aldolase is mediated by a short acidic sequence containing a sub-terminal tryptophan (Trp) conserved across the protein family and is competitively inhibited by aldolase substrate/products [21, 22].

Results

Conclusion