An experimentally derived database of candidate Ras-interacting proteins

Abstract

We used a TAP-tag approach to identify candidate binding proteins for the related Ras family GTPases: H-Ras, R-Ras, and Rap1A. Protein complexes were isolated from mouse fibroblasts, and component proteins were identified by a combination of nanoflow HPLC and tandem mass spectrometry. H-Ras was found to associate with numerous cytoskeletal proteins including talin-1. R-Ras and Rap1A each associated with various signaling molecules, many of which are membrane-associated. Thus, we have established the first database of potential Ras interactors in mammalian cells.

Figure 1

(A) Domain structure of TAP-Ras constructs (R-Ras is shown). CBD, caldmodulin-binding domain; PP, proline-rich sequence; SW1/2, switch1/2; TEV, TEV protease cleavage site; *, palmitoylation site; ¥, methylation and geranylgeranylation site; canonical CAAX sequence shown in red. (B) Regulation of integrin activation by TAP-Ras fusion proteins. Integrin activation was measured in CHO cells expressing constitutively active chimeric integrins. TAP-tagged H-Ras(G12V) suppressed integrin activation in GFP-positive cells (relative to the activation state in control GFP-transfected cells, set to 0). Activated R-Ras with or without a TAP tag fusion or PEA-15 (control) rescued H-Ras-mediated suppression. Data are shown +SEM for two independent experiments.

Figure 2

(A) Ras purification strategy. TAP-Ras-transfected cells were lysed in NP-40 buffer. TAP-Ras protein complexes were enriched on Ni⁺⁺-sepharose beads, followed by elution with imidazole, recapture on calmodulin-sepharose beads, and final elution with EGTA. Purified protein complexes were digested with trypsin and subjected to LC–MS/MS analysis and protein identification using a combination of manual interpretation of MS/MS spectra and the search algorithm, SEQUEST. (B) Silver stain (top) and immunoblot with antibodies to the TAP tag (bottom). The purified fractions contained the TAP-R-Ras protein, and endogenous proteins which did not co-purify with the isolated TAP tag. These proteins were assigned as putative R-Ras interactors. M, molecular weight marker; S, sample.

Figure 3

Interaction of Abi1 and activated R-Ras. FLAG-tagged Abi1 was coexpressed with TAP-tagged R-Ras(G38V), R-Ras-(T43N), H-Ras(G12V) or Rap1A(G12V). TAP-Ras proteins were precipitated from cell lysates with IgG-coupled sepharose beads, and bead-bound proteins were visualized by Western blotting with anti-FLAG and anti-TAP antibodies.

Figure 4

Interaction of activated H-Ras and talin-F23. GST-talin-F23 domain fragment protein (GST-F23) or GST was coupled to GSH-sepharose and incubated with lysates of cells expressing TAP-tagged H-Ras, R-Ras, or Rap1A bearing the indicated mutations. Bead-bound Ras proteins were visualized by immuno-blotting with TAP tag antibodies (TAP); GST-F23 was visualized with GST antibodies.