Proteomic identification of phosphatidylinositol (3,4,5) triphosphate-binding proteins in Dictyostelium discoideum

Abstract

Phosphatidylinositol (3,4,5)-triphosphate (PtdInsP(3)) mediates intracellular signaling for directional sensing and pseudopod extension at the leading edge of migrating cells during chemotaxis. How this PtdInsP(3) signal is translated into remodeling of the actin cytoskeleton is poorly understood. Here, using a proteomics approach, we identified multiple PtdInsP(3)-binding proteins in Dictyostelium discoideum, including five pleckstrin homology (PH) domain-containing proteins. Two of these, the serine/threonine kinase Akt/protein kinase B and the PH domain-containing protein PhdA, were previously characterized as PtdInsP(3)-binding proteins. In addition, PhdB, PhdG, and PhdI were identified as previously undescribed PH domain-containing proteins. Specific PtdInsP(3) interactions with PhdB, PhdG, and PhdI were confirmed using an in vitro lipid-binding assay. In cells, PhdI associated with the plasma membrane in a manner dependent on both the PH domain and PtdInsP(3). Consistent with this finding, PhdI located to the leading edge in migrating cells. In contrast, PhdG was found in the cytosol in WT cells. However, when PtdInsP(3) was overproduced in pten(-) cells, PhdG located to the plasma membrane, suggesting its weak affinity for PtdInsP(3). PhdB was found to bind to the plasma membrane via both PtdInsP(3)-dependent and -independent mechanisms. The PtdInsP(3)-independent interaction was mediated by the middle domain, independent of the PH domain. In migrating cells, the majority of PhdB was found at the lagging edge. Finally, we deleted the genes encoding PhdB and PhdG and demonstrated that both proteins are required for efficient chemotaxis. Thus, this study advances our understanding of the PtdInsP(3)-mediated signaling mechanisms that control directed cell migration in chemotaxis.

Fig. 1.

Identification of PtdIns(3,4,5)P₃-binding proteins. (A) High-speed supernatants were prepared from WT cells and cells expressing PHcrac-GFP (PHcrac-GFP/WT). Proteins eluted from PtdInsP₃-, PtdIns(4,5)P₂-, or lipid-free beads were resolved by SDS/PAGE before silver staining. The asterisk indicates PHcrac-GFP. (B) Nine identified proteins. Molecular weight (MW), gene identification number (GI), expected values calculated by the MASCOT program (E-value), numbers of peptide matches (PMs), and coverage of proteins by identified peptides (Cov) are shown. (C) Domain structures of PhdB, PhdG, and PhdI were analyzed using Prosite (http://ca.expasy.org/prosite/). (D) Lipid dot blot assays.

Fig. 2.

Localization of PhdB, PhdG, and PhdI in undifferentiated growing cells. Localization of PHcrac-GFP, PhdB-GFP, PhdG-GFP, PhdI-GFP, and GFP in undifferentiated WT and pten⁻ cells was analyzed by fluorescence microscopy in the presence or absence of 100 μM LY294002 (+LY) and 5 μM latrunculin A (+Lat A). Arrows indicate macropinocytic cups. (Scale bar: 10 μm.)

Fig. 3.

cAMP regulates the localization of PhdB and PhdI in differentiated cells. Cells expressing PHcrac-, PhdB-, PhdG-, or PhdI-GFP were differentiated for 5 h. Cells were uniformly stimulated with 1 μM cAMP and examined by time-lapse fluorescence microscopy in the absence (−LY) (A) or presence (+LY) of 100 μM LY294002 (B) and 5 μM latrunculin A (Lat A) (D). (C) Quantitation of membrane association of PHcrac-GFP and PhdB-GFP. Cells were stimulated with 1 μM cAMP in the presence or absence of 100 μM LY294002 and lysed at the indicated time points. A membrane fraction was collected by centrifugation (8, 11) and analyzed by Western blot analysis using anti-GFP antibodies. Values are mean ± SEM (n = 4). (E) Localization of PHcrac-, PhdB-, PhdG-, and PhdI-GFP in migrating cells. Cells were moving upward in the images. (Scale bars: A–C, E; 10 μm.)

Fig. 4.

Mutational analysis of PhdB, PhdG, and PhdI. Point and deletion mutations were introduced into PhdB (A and B), PhdG (A and C), and PhdI (A and D). Asterisks indicate mutations introduced in PH domains. Lipid binding (A) and subcellular localization of mutant proteins in undifferentiated WT and pten⁻ cells (B–D) were examined by dot blot and fluorescence microscopy, respectively.

Fig. 5.

Analysis of phdB⁻ and phdG⁻ cells. Cell proliferation in shaking culture (A) and on plastic dishes (B). (C and D) Cells were placed on nonnutrient agar and observed at the indicated time points. (Scale bars: 1 mm.) (E and F) Small-drop chemotaxis assay. All values are mean ± SEM (n = 3). Results were statistically analyzed using t test (*P < 0.05; **P < 0.01).