Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.
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.
Isakoff
S J
SJ
The Skirball Institute for Biomolecular Medicine and Department of Pharmacology, New York University Medical Center, New York, NY 10016, USA.
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
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000235:genetics; D002462:Cell Membrane; D016384:Consensus Sequence; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D015290:Second Messenger Systems / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D018631:ras Proteins / Q000502:physiology
D001798:Blood Proteins; D005656:Fungal Proteins; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D018631:ras Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; C039077:platelet protein P47