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

Research output: Contribution to journalArticle

Abstract

Phosphatidylinositol (3,4,5)-triphosphate (PtdInsP3) mediates intracellular signaling for directional sensing and pseudopod extension at the leading edge of migrating cells during chemotaxis. How this PtdInsP3 signal is translated into remodeling of the actin cytoskeleton is poorly understood. Here, using a proteomics approach, we identified multiple PtdInsP3-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 PtdInsP3-binding proteins. In addition, PhdB, PhdG, and PhdI were identified as previously undescribed PHdomain-containingproteins. Specific PtdInsP3 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 PtdInsP3. 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 PtdInsP3 was over-produced in pten- cells, PhdG located to the plasma membrane, suggesting its weak affinity for PtdInsP3. PhdBwas found to bind to the plasma membrane via both PtdInsP3-dependent and -independent mechanisms. The PtdInsP3-independent interaction was mediated by the middle domain, independent of the PH domain. In migrating cells, themajority 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.

Original languageEnglish (US)
Pages (from-to)11829-11834
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number26
DOIs
StatePublished - Jun 29 2010

Keywords

  • Chemotaxis
  • Dictyostelium
  • PI3 kinase
  • Phosphatase and tensin homolog
  • Pleckstrin homology domain

ASJC Scopus subject areas

  • General

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