The WW domain of the scaffolding protein IQGAP1 is neither necessary nor sufficient for binding to the MAPKs ERK1 and ERK2

Abstract

Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (K_d ) for this interaction of 8 μm These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding.

Keywords: IQGAP1; cancer; cell signaling; extracellular-signal-regulated kinase (ERK); mitogen-activated protein kinase (MAPK); protein complex; protein kinase; protein-protein interaction; scaffold protein.

Figure 1.

The IQGAP1 scaffold protein. A, schematic depicting full-length human IQGAP1 protein, and the domains it contains. CHD, calponin homology domain; IR, internal repeated sequence/coiled-coil domain; WW, WW domain; IQ, IQ domain; GRD, GTPase-activating protein-related domain; RGCT, RasGAP C-terminal domain. B, schematic interpretation of proposed model of the function of IQGAP1 as a scaffold protein for the MAPK pathway, based on a similar figure in Ref. . According to the model, the IQ domain of IQGAP1 binds to RAF and MEK, and the nearby WW domain binds to ERK. These interactions are thought to facilitate RAF phosphorylation of MEK, and MEK phosphorylation of ERK. C, proposed mechanistic model for the anti-tumor efficacy of the isolated IQGAP WW domain studied by Jameson et al. (21). In this model, the WW domain binds to ERK and blocks the ability of ERK to productively interact with IQGAP1 (8, 21). The WW domain fragment studied by Jameson et al. (21) consisted of IQGAP1 residues 680–711, plus N-terminal myc and polyarginine tags.

Figure 2.

The IQ domain of IQGAP1 is necessary for binding to ERK2; the WW domain is not sufficient. A, rat ERK2, fused to GST, was tested for binding to full-length human IQGAP1, or to truncated derivatives of IQGAP1. Qualitative results of these experiments are shown on the right: +++ indicates strong binding; — indicates minimal binding. B, as shown in A, ³⁵S radiolabeled full-length human IQGAP1 protein and truncated derivatives were prepared by in vitro translation and partially purified by ammonium sulfate precipitation, and portions (10% of the amount added in the binding reactions) were resolved on a 10% SDS-polyacrylamide gel (Input). Samples (∼1 pmol) of the same proteins were incubated with 25 μg of GST or GST-ERK2 bound to glutathione-Sepharose beads, and the resulting bead-bound protein complexes were isolated by sedimentation and resolved by 10% SDS-PAGE on the same gel. The gel was analyzed by staining with GelCode Blue (Thermo Scientific) for visualization of the bound GST fusion protein (a representative example is shown in the lowest panel) and by X-ray film exposure for visualization of the bound radiolabeled protein (upper four panels). C, quantification of the binding of IQGAP1 derivatives to GST or GST-ERK2, normalized to the percent binding of full-length IQGAP1 to GST-ERK2. The results shown are the average of at least 5 independent repetitions of the binding assay shown in A and B, with duplicate points (i.e. technical replicates) in each repetition. S.E. bars are shown (n = 5 to 7). The scatter of the individual normalized data points is also shown for the binding of ERK2 to IQGAP1(1–863). The means for ERK2-IQGAP1 and ERK2-IQGAP1(1–863) binding were significantly different from all other the means shown (p < 0.01), but were not significantly different from each other (p = 0.98, thus the null hypothesis that the population means are the same cannot be rejected with confidence). The minimal binding of ERK2 to IQGAP1(1–719) was not significantly different from that of ERK2 to IQGAP1(1–678) (p = 0.91), nor was it significantly different from the minimal binding of GST alone to IQGAP1(1–719) (p = 0.41).

Figure 3.

The IQ domain of IQGAP1 is sufficient for binding to ERK2; the WW domain is not necessary. A, rat or human ERK2, fused to GST, were tested for binding to full-length human IQGAP1, or to fragments of IQGAP1. Qualitative results of these experiments are shown on the right: +++ indicates strong binding; — indicates minimal binding. B, autoradiograms of representative experiments of binding assays are described in A. Each binding assay shown was repeated three separate times (i.e. three independent experiments), with duplicate points (i.e. technical replicates) in each experiment. Other details are as described in the legend to Fig. 2.

Figure 4.

Further characterization of the ERK2-IQGAP1 interaction. A, human ERK2, or mutant derivatives thereof, fused to GST, were tested for binding to full-length human IQGAP1, or to IQGAP1(1–863). The ERK2 alleles tested were the wild-type allele (ERK2), catalytically inactive (K54A mutation, K54A), unphosphorylatable and unactivatable (T185A Y185F mutations, AF), and docking groove mutated (L115A, Q119A, D318A, D321A mutations, ”DGM“). Small circles on the schematics indicate the wild-type residues and the alterations thereof. B, autoradiograms of representative experiments of binding assays described in A. Each binding assay shown was repeated three separate times (i.e. three independent experiments), with duplicate points (i.e. technical replicates) in each experiment. Other details are as described in the legend to Fig. 2.

Figure 5.

The IQ domain of IQGAP1 is sufficient for binding to ERK1, MEK1, and MEK2. A, human ERK1, MEK1, or MEK2, fused to GST, was tested for binding to full-length human IQGAP1, or to fragments of IQGAP1. Qualitative results of these experiments are shown on the right: +++ indicates strong binding; — indicates minimal binding. B, autoradiograms of representative experiments of binding assays described in A. Each binding assay shown was repeated three separate times (i.e. three independent experiments), with duplicate points (i.e. technical replicates) in each experiment. Other details are as described in the legend to Fig. 2.

Figure 6.

The WW domain does not contribute to the ERK-IQGAP1 interaction. A, the top three lines show an alignment of the amino acid sequences of the first WW domain from human WWOX1 (accession number NP_057457, residues shown are 22–47), and the single WW domains in human PIN1 (NP_006212, residues 11–37) and human IQGAP1 (NP_003861, residues 685–710). Residues identical in all three domains are boxed; these include the two tryptophan residues (positions 685 and 707 in IQGAP1) that give the WW domain its name. Residues that were the site of inactivating mutations in other studies (75, 76) are shaded orange. The bottom line shows the sequence of the quintuplely-mutated WW domain in the derivative IQGAP1(1–863)wwmut; residues mutated to alanine are shown in red and underlined. B and C, human ERK1, human ERK2, and rat ERK2, fused to GST, were tested for binding to human IQGAP1(1–863) or IQGAP1(1–863)wwmut. Other details are as described in the legend to Fig. 2. D, quantification of the binding of IQGAP1(1–863) or IQGAP1(1–863)wwmut to GST-hERK2. The results shown are the average of 4 independent repetitions of the binding assay shown in B and C, with duplicate points (i.e. technical replicates) in each repetition. S.E. bars are shown (n = 4). The scatter of the individual data points is also shown. The ERK2-IQGAP1(1–863) and ERK2-IQGAP1(1–863)wwmut interactions were not significantly different from each other (p = 0.57, thus the null hypothesis that the population means are the same cannot be rejected with confidence).