Summary of the results obtained for the different protein–protein interactions tested in the RTK/FRAP network with the DHFR survival-selection assay in CHO DUKX-B11 (DHFR⁻) cells. On the x axis are the fusions to DHFR[1,2] fragment, and on the y axis the fusions to DHFR[3] fragment. The orientations of the fusions (N-terminal or C-terminal) also are indicated. Positive interactions, green (+); absence of interaction, red (−); not tested, gray squares.

Fluorometric and microscopic analysis of the rapamycin-sensitive components of the network (C, calyculin A; see legend to Fig. 3 for all other abbreviations). The fusion protein pairs used in these experiments were: PP2A-F[1,2]/PKB-F[3], PP2A-F[1,2]/p70S6K-F[3], F[1,2]-FRAP/FKBP-F[3], F[1,2]-Cdc42/P70S6K-F[3], F[1,2]-Rac1/p70S6K-F[3], F[1,2]-p70S6K/S6-F[3], and F[1,2]-p70S6K/4EBP1-F[3].

(A) Schematic representation of the strategy for generating a functional validation profile of a biochemical network by using the DHFR PCA. Positive clones are detected with the DHFR survival-selection assay. They correspond to interacting component proteins of two convergent signal transduction pathways (Path 1 and Path 2). An interaction matrix (upper left) represents all positive (green) and negative (red) interacting pairs observed in the survival-selection assay. Positive clones from survival selection are propagated and subjected to two functional analyses. First, with use of the DHFR fluorescence assay, interactions are probed with pathway-specific stimulators (1 and 2) and inhibitors (A and B). Pharmacological profiles are established on the basis of the pattern of response of individual interactions to stimulators and inhibitors, represented in the histograms. (Ordinate axes represent fluorescence intensity.) For example, stimulation of pathway 1 will augment all of the interactions composing that pathway. Inhibitor A will inhibit protein interactions downstream, but not upstream of its site of action in pathway 1. Second, cellular locations of the interactions are determined by fluorescence microscopy, also by use of the DHFR fluorescence assay. (B) Well established connections within RTK (growth factor-activated)- and FRAP-mediated pathways that control initiation of translation and sites of action of inhibitors of these pathways. Broken line indicates indirect action. PI3K, phosphatidylinositol-3-kinase.

Fluorometric and microscopic analysis of the wortmannin-sensitive/rapamycin-resistant components of the network. The grid represents all positive (green) and negative (red) interactions observed in survival selection. The pharmacological profiles are represented by the histograms (right). Cells were treated with stimulants and inhibitors as described in Materials and Methods (x axis; NT, no treatment; I, insulin; S, serum; R, rapamycin; W, wortmannin). Fluorescence intensity is given in relative fluorescence units (y axis). The background fluorescence intensity corresponding to nontransfected cells was subtracted from the fluorescence intensities of all of the samples. Error bars represent standard errors for the mean calculated from at least three independent experiments. Microscopy revealing patterns of locations also is presented. The dimerization of GCN4 leucine zipper (GCN4/GCN4) is used as a control. The fusion protein pairs used in these experiments were: PDK1-F[1,2]/PKB-F[3], F[1,2]-p70S6K/PKB-F[3], F[1,2]-FRAP/PKB-F[3], F[1,2]-p70S6K/PDK1-F[3], PDK1-F[1,2]/F[3]-FRAP, F[1,2]-FRAP/F[3]-FRAP, F[1,2]-FRAP/4EBP1-F[3], and F[1,2]-GCN4/GCN4-F[3].

Summary of the protein–protein interactions involved in the RTK/FRAP network and their responses to inhibitors. This summary is based on the results obtained from survival-selection and pharmacological profile experiments. The effects of each drug are indicated (C, calyculin A; R, rapamycin; W, wortmannin). The blue arrows indicate protein–protein interactions that have not been previously observed. Single-head arrows indicate kinase- or phosphatase-substrate catalytic events. Double-head arrows represent equilibrium noncatalytic interactions. Broken arrows indicate that the nature of the interactions is unknown.