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 re sponses by generating PtdIns(3,4)P-2 and PtdIns(3,4,5)P-3, These 3-pho sphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) do mains in these signaling molecules, We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind P tdIns(3,4)P-2 and PtdIns(3,4,5)P-3 in vivo which we have called TOPIS ((T) under bar argets (o) under bar f (P) under bar I3K (I) under bar dentification (S) under bar ystem), The assay uses a plasma membrane-t argeted Pas to complement a temperature-sensitive CDC25 Ras exchange f actor in yeast. Coexpression of PI3K and a fusion protein of activated Pas joined to a PH domain known to bind PtdIns(3,4)P-2 (AKT) or PtdIn s(3,4,5)P-3 (BTK) rescues yeast growth at the non-permissive temperatu re of 37 degrees C. Using this assay, we have identified several amino acids in the beta l-beta 2 region of PH domains that are critical for high affinity binding to PtdIns(3,3)P-2 and/or PtdIns(3,4,5)P-3, and we have proposed a structural model for how these PH domains might bin d PI3K products with high affinity. From these data, we derived a cons ensus sequence which predicts high-affinity binding to PtdIns(3,4)P2 a nd/or PtdIns(3,4,5)P-3, and we have identified several new PH domain-c ontaining proteins that bind PI3K products, including Gab1, Dos, myosi nX, and Sbf1, Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful app roach for uncovering additional targets of PI3K.