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Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium.

ABSTRACT: We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3' kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain-containing proteins. We demonstrate that in response to chemoattractants three PH domain-containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.

SUBMITTER: Funamoto S

PROVIDER: S-EPMC2192389 | biostudies-literature | 2001 May

REPOSITORIES: biostudies-literature

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Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium.

Funamoto S S Milan K K Meili R R Firtel R A RA

The Journal of cell biology 20010501 4

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3' kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain-containing proteins. We demonstrate ...[more]

PMID: 11352940

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Project description:Background:Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P2 through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P2/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways. Methods:Surface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used. Results:In this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P2/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC50 ranges from 53 to 159 nM for PI(4,5)P2/PLC?1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], PI(4,5)P2 specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C ?1 (PLC?1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLC?1-PH domain from plasma membrane to cytosol in A549 cells. Conclusions:Overall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment. General significance:These results suggest specific but differential binding of these compounds to the PLC?1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.

Dataset Information

Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium.

Publications

Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium.

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