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Identification and analysis of PH domain‐containing targets of phosphatidylinositol 3‐kinase using a novel in vivo assay in yeast
Author(s) -
Isakoff Steven J.,
Cardozo Tim,
Andreev Julian,
Li Zhai,
Ferguson Kathryn M.,
Abagyan Ruben,
Lemmon Mark A.,
Aronheim Ami,
Skolnik Edward Y.
Publication year - 1998
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/17.18.5374
Subject(s) - biology , phosphatidylinositol , yeast , in vivo , pleckstrin homology domain , kinase , identification (biology) , saccharomyces cerevisiae , domain (mathematical analysis) , biochemistry , protein kinase domain , microbiology and biotechnology , computational biology , genetics , gene , botany , mutant , mathematical analysis , mathematics
Phosphatidylinositol 3‐kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 . 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)P 2 and PtdIns(3,4,5)P 3 in vivo which we have called TOPIS ( T argets o f P I3K I dentification S ystem). 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)P 2 (AKT) or PtdIns(3,4,5)P 3 (BTK) rescues yeast growth at the non‐permissive temperature of 37°C. Using this assay, we have identified several amino acids in the β1–β2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P 2 and/or PtdIns(3,4,5)P 3 , 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)P 2 and/or PtdIns(3,4,5)P 3 , 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.