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The PX domains of p47phox and p40phox bind to lipid products of PI(3)K

Abstract

PX domains are found in a variety of proteins that associate with cell membranes, but their molecular function has remained obscure. We show here that the PX domains in p47phox and p40phox subunits of the phagocyte NADPH oxidase bind to phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-3-phosphate (PtdIns(3)P), respectively. We also show that an Arg-to-Gln mutation in the PX domain of p47phox, which is found in patients with chronic granulomatous disease, eliminates phosphoinositide binding, as does the analogous mutation in the PX domain of p40phox. The PX domain of p40phox localizes specifically to PtdIns(3)P-enriched early endosomes, and this localization is disrupted by inhibition of phosphoinositide-3-OH kinase (PI(3)K) or by the Arg-to-Gln point mutation. These findings provide a molecular foundation to understand the role of PI(3)K in regulating neutrophil function and inflammation, and to identify PX domains as specific phosphoinositide-binding modules involved in signal transduction events in eukaryotic cells.

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Figure 1: Sequence alignment of PX domains.
Figure 2: Phosphoinositide-binding properties of PX domains.
Figure 3: PX domains bind to lipid vesicles that contain phosphoinositides.
Figure 4: In vivo localization of p40PX–EGFP.

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Acknowledgements

We thank J. Miyagi for technical assistance, S.-Y. Pai for suggesting the R42Q mutation, and members of M.B.Y.'s laboratory for discussions. F.K. was supported by fellowships from the Cell Science Research Foundation and Sankyo Foundation of Life Science. S.J.F. was supported by a Howard Hughes Medical Institute Postdoctoral Fellowship. This work was funded by NIH grants to M.B.Y. and L.C.C, and a Burroughs-Wellcome Career Development Award to M.B.Y.

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Correspondence to Michael B. Yaffe.

Supplementary information

Movie S1

Loss of endosomal localization of p40PX–EGFP after inhibition of PI(3)K with Wortmannin. (MOV 1067 kb)

Figure S1

Expression of GST–fusion proteins of the PX domains from p40phox and p47phox. (PDF 145 kb)

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Kanai, F., Liu, H., Field, S. et al. The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat Cell Biol 3, 675–678 (2001). https://doi.org/10.1038/35083070

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