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)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 (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)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.

Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities

“…However, recent work indicates that six conserved residues that lie in the N-terminal region of the PH domain in a Lys-Xaa-SmaXaa ' -"" -Arg\Lys-Xaa-Arg-Hyd-Hyd motif (where ' Xaa ' is any amino acid, ' Sma ' is a small amino acid and ' Hyd ' is a hydrophobic amino acid) appear to correlate with high-affinity binding of PtdIns(3,4,5)P $ [11]. To date, all of the specific PtdIns(3,4,5)P $ -binding proteins identified possess this putative PtdIns(3,4,5)P $ binding motif (PPBM) (see Table 1).…”

Insulin-independent and wortmannin-resistant targeting of IRS-3 to the plasma membrane via its pleckstrin homology domain mediates a different interaction with the insulin receptor from that of IRS-1

“…Since only high-affinity binding (Kd of < 1 mmol/l) to phosphoinositides could be detected in their system [48], these data suggest that high-affinity binding of the PH domain of Gab1 to PI(3,4,5)P 3 enables tyrosine phosphorylation of Gab1 even in the absence of the domains that directly associate with the activated receptor. In contrast, as the interaction between the PH domain of IRS-1 and the phosphoinositides or other ligands in the plasma membrane seems to be weak [46,48], the cooperation of the PTB domain interacting with the insulin receptor could be required for efficient tyrosine phosphorylation of IRS-1 as well as other members of the IRS family. From our data we could deduce that the extent of the interaction between the IRSs and the insulin receptor, which is required for transducing signals from the insulin receptor, could be influenced by the affinity of the PH domain for phosphoinositides or other ligands in the plasma membrane as follows (Fig.…”

“…Among the endogenous substrates of the insulin receptor tyrosine kinase, Gab1 contains the PH domain but not the PTB domain or any other domain in its N-terminus which interacts with the insulin receptor [5,47]. Using an in vivo assay in yeast, the association of the PH domain of Gab1, but not that of IRS-1, with the PI 3-kinase products was detected [48]. Since only high-affinity binding (Kd of < 1 mmol/l) to phosphoinositides could be detected in their system [48], these data suggest that high-affinity binding of the PH domain of Gab1 to PI(3,4,5)P 3 enables tyrosine phosphorylation of Gab1 even in the absence of the domains that directly associate with the activated receptor.…”

“…Using an in vivo assay in yeast, the association of the PH domain of Gab1, but not that of IRS-1, with the PI 3-kinase products was detected [48]. Since only high-affinity binding (Kd of < 1 mmol/l) to phosphoinositides could be detected in their system [48], these data suggest that high-affinity binding of the PH domain of Gab1 to PI(3,4,5)P 3 enables tyrosine phosphorylation of Gab1 even in the absence of the domains that directly associate with the activated receptor. In contrast, as the interaction between the PH domain of IRS-1 and the phosphoinositides or other ligands in the plasma membrane seems to be weak [46,48], the cooperation of the PTB domain interacting with the insulin receptor could be required for efficient tyrosine phosphorylation of IRS-1 as well as other members of the IRS family.…”

GRB2-mediated recruitment of GAB2, but not GAB1, to SF-STK supports the expansion of Friend virus-infected erythroid progenitor cells

“…In addition to the Grb2-binding site, all Gab proteins contain an N-terminal PH domain with sequence similarity to the Btk PH domain which recognizes primarily PIP3, the lipid products of PI3K (Isakoff et al, 1998). Therefore, the requirement for PI3K signaling in the transformation of cells by FV could also reflect a role for this signaling pathway in the recruitment of Gab2.…”