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Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

ABSTRACT: Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.

SUBMITTER: Isakoff SJ

PROVIDER: S-EPMC1170863 | biostudies-other | 1998 Sep

REPOSITORIES: biostudies-other

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Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Isakoff S J SJ Cardozo T T Andreev J J Li Z Z Ferguson K M KM Abagyan R R Lemmon M A MA Aronheim A A Skolnik E Y EY

The EMBO journal 19980901 18

Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI ...[more]

PMID: 9736615

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Project description:BackgroundFor the success of the malaria control and eradication programme it is essential to reduce parasite transmission by mosquito vectors. In the midguts of mosquitoes fed with parasite-infected blood, sexual-stage parasites fertilize to develop into motile ookinetes that traverse midgut epithelial cells and reside adjacent the basal lamina. Therefore, the ookinete is a promising target of transmission-blocking vaccines to break the parasite lifecycle in mosquito vectors. However, the molecular mechanisms of ookinete formation and invasion of epithelial cells have not been fully elucidated. A unique structure called the crystalloid body has been identified in the ookinete cytoplasm by electron microscopy, but its biological functions remain unclear.MethodsA recombinant protein of a novel molecule, designated as crystalloid body specific PH domain-containing protein of Plasmodium yoelii (PyCryPH), was synthesized using a wheat germ cell-free system. Specific rabbit antibodies against PyCryPH were obtained to characterize the expression and localization of PyCryPH during sexual-stage parasite development. In addition, PyCryPH knockout parasites were generated by targeted gene disruption to examine PyCryPH function in mosquito-stage parasite development.ResultsWestern blot and immunofluorescence assays using specific antibodies showed that PyCryPH is specifically expressed in zygotes and ookinetes. By immunoelectron microscopy it was demonstrated that PyCryPH is localized within crystalloid bodies. Parasites with a disrupted PyCryPH gene developed normally into ookinetes and formed oocysts on the basal lamina of midguts. In addition, the number of sporozoites residing in salivary glands was comparable to that of wild-type parasites.ConclusionsCryPH, containing a signal peptide and PH domain, is predominantly expressed in zygotes and ookinetes and is localized to crystalloid bodies in P. yoelii. CryPH accumulates in vesicle-like structures prior to the appearance of typical crystalloid bodies. Unlike other known crystalloid body localized proteins, CryPH does not appear to have a multiple domain architecture characteristic of the LAP/CCp family proteins. Although CryPH is highly conserved among Plasmodium, Babesia, Theileria, and Cryptosporidium, PyCryPH is dispensable for the development of invasive ookinetes and sporozoites in mosquito bodies.

Dataset Information

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Publications

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

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