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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.

SUBMITTER: Zhang P

PROVIDER: S-EPMC2900710 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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

Zhang Pingbo P Wang Yu Y Sesaki Hiromi H Iijima Miho M

Proceedings of the National Academy of Sciences of the United States of America 20100614 26

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 ...[more]

PMID: 20547830

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Project description:Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P₃ that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P₃ headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P₃ headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P₃-binding proteins.

Dataset Information

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

Publications

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

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