MAPK scaffold IQGAP1 binds the EGF receptor and modulates its activation

Abstract

Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca(2+) attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser(1443), a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKCα isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation.

FIGURE 1.

EGFR co-immunoprecipitates with IQGAP1 in a Ca²⁺-sensitive manner. A, COS-7 cells were transiently transfected with empty pcDNA3 vector (V) or full-length wild-type Myc-IQGAP1 (IQGAP1). After lysis, equal amounts of protein were immunoprecipitated with anti-Myc antibody. Both unprocessed lysates (lysate) and immunoprecipitates (IP) were resolved by SDS-PAGE and transferred to PVDF membranes. Identical blots were probed with either anti-EGFR or anti-Myc antibodies. B, A431 cells were starved overnight and incubated with vehicle (−) or 100 ng/ml EGF (+) for 10 min. Equal amounts of protein lysates were immunoprecipitated with anti-IQGAP1 (IQGAP1) or anti-TIM23 (ctrl) antibodies and probed with anti-EGFR antibody (top). 10% of the immunoprecipitate was processed in parallel and probed with anti-IQGAP1 antibody (bottom). Aliquots of lysate from each sample not subjected to pull-down were also processed by Western blotting. C, A431 cells were incubated with vehicle (−) or 100 ng/ml EGF (+) and processed as described in A, except that cells were lysed in buffer containing 1 mm EGTA or 1 mm CaCl₂. Equal amounts of protein lysate were immunoprecipitated with anti-IQGAP1 antibody. Immunoprecipitates (IQGAP1) and unprocessed lysates were probed with anti-EGFR antibody (top) and anti-IQGAP1 antibody (bottom). D, identical aliquots were resolved separately by SDS-PAGE, transferred to PVDF, and probed with anti-phosphotyrosine antibody (pTyr). All data shown above are representative of three independent experiments.

FIGURE 2.

EGFR colocalizes with IQGAP1 in cells. A431 cells were plated on poly-d-lysine coverslips, serum-starved, and treated with (+) or without (−) 100 ng/ml EGF for 5 min and then fixed and processed as described under “Experimental Procedures.” Cells were double-stained with rabbit anti-IQGAP1 antibody/Alexa Fluor 594-conjugated secondary antibody and mouse anti-EGFR/Alexa Fluor 488-conjugated secondary antibody (A and B), rabbit anti-IQGAP1 antibody/Alexa Fluor 594-conjugated secondary antibody and mouse anti-E-cadherin/Alexa Fluor 488-conjugated secondary antibody (C), or rabbit anti-IQGAP1 antibody/Alexa Fluor 594-conjugated secondary antibody and mouse anti-phospho-EGFR (Tyr¹⁰⁶⁸) (pY) antibody/Alexa Fluor 488-conjugated secondary antibody (D). Slides were analyzed by Zeiss LSM510 confocal laser microscopy. A, serum-starved A431 cells stained for EGFR (green) and IQGAP1 (red) after incubation with vehicle (top) or EGF (bottom). B, x-z plane comparison of serum-starved A431 cells stained for EGFR (green) and IQGAP1 (red) after incubation with vehicle (left) or EGF (right). C, serum-starved A431 cells stained for E-cadherin (green) and IQGAP1 (red) after incubation with vehicle (top) or EGF (bottom). D, serum-starved A431 cells stained for phospho-Tyr¹⁰⁶⁸ (green) and IQGAP1 (red) after incubation with vehicle (top) or EGF (bottom). The arrows indicate examples of areas where IQGAP1 and EGFR (A) or phospho-EGFR (B) strongly colocalize. Merge represents the composite of both channels, with yellow regions indicative of colocalization. Colocalization of EGFR and phospho-EGFR with IQGAP1 was analyzed with Zeiss LSM software (colocalization; right). Negative controls showed no evidence of nonspecific staining or bleed-through. The images are representative of three independent experiments, with at least six fields examined in each assay. Scale bar, 20 μm.

FIGURE 3.

Calmodulin disrupts the binding of IQGAP1 to EGFR in vitro. A, GST-IQGAP1 (IQGAP1) or GST alone bound to glutathione-Sepharose was incubated with equal amounts of purified EGFR-KD in the presence of Ca²⁺ or EGTA. The complexes were isolated as described under “Experimental Procedures” and probed with antibody to EGFR amino acids 985–996. Input is purified EGFR. B, GST-IQGAP1 or GST alone bound to glutathione-Sepharose was incubated with equal amounts of purified EGFR-KD in the presence of Ca²⁺/calmodulin (CaM) or BSA. The complexes were isolated and washed. Input, EGFR-KD not subjected to pull-down. After proteins were resolved by SDS-PAGE, the gel was cut into two pieces. The piece containing EGFR-KD was transferred to PVDF and probed with anti-EGFR kinase domain antibody (top). The piece containing IQGAP1 was stained with Coomassie Blue (bottom). C, the experiment described in B was carried out in the presence of EGTA. All of the data in this figure are representative of three independent experiments.

FIGURE 4.

Regions of IQGAP1 necessary for EGFR binding. A, schematic representation of IQGAP1 constructs. Full-length IQGAP1 and deletion mutants of IQGAP1 are depicted. The identified protein interaction motifs and the specific amino acid residues in each construct are indicated. IQGAP1ΔIQ lacks amino acids 746–860. CHD, calponin homology domain; WW, polyproline binding domain; IQ, four tandem calmodulin binding motifs; GRD, Ras-GAP-related domain; IQGAP1-N, N-terminal half of IQGAP1; IQGAP1-C, C-terminal half of IQGAP1. [³⁵S]Methionine-labeled IQGAP1, IQGAP1-N, and IQGAP1-C (B) and IQGAP1ΔIQ and IQGAP1-IQ (C) produced with the TNT Quick Coupled Transcription/Translation system were incubated with purified EGFR intracellular domain. Complexes were isolated by immunoprecipitation with either anti-EGFR cytoplasmic domain antibody or anti-glycogen phosphorylase control (ctrl) antibody (pulldown). Complexes were resolved by SDS-PAGE. Gels were dried and processed by autoradiography. An aliquot of [³⁵S]methionine-labeled TNT product (equivalent to 10% of the amount that was subjected to pull-down) was resolved by SDS-PAGE, dried, and processed by autoradiography (input). Data are representative of three independent experiments.

FIGURE 5.

Mass spectrometry-based IQGAP1 Ser¹⁴⁴³ phosphoassay. A431 cells were serum-starved overnight and stimulated with 1 μm PMA for 20 min. Cells were lysed and immunoprecipitated with rabbit anti-IQGAP1 polyclonal antibody. Following SDS-PAGE, immunopurified IQGAP1 was excised and digested with trypsin, producing the four Ser¹⁴⁴³ epitope peptides shown. Two phosphorylated and two unmodified forms were produced as a result of alternate lysine cleavage at the C terminus. The phosphorylated Ser¹⁴⁴³ peptide sequence differs from the unmodified sequence at the N terminus as a result of a hindered tryptic cleavage site flanking the phosphorylated residue. Continuous LC-MS/MS was performed on the triply charged ion (3+) observed for each of the four peptides, as described under “Experimental Procedures.” Extracted ion chromatograms for the y₅ or y₆ fragment ion produced in each reaction are shown.

FIGURE 6.

IQGAP1 is phosphorylated at Ser¹⁴⁴³ in response to EGFR activation. Serum-starved HeLa cells were treated with either vehicle or EGF at the indicated concentrations and times. IQGAP1 was immunoprecipitated and isolated by SDS-PAGE, digested with trypsin, and analyzed by LC-MS/MS using the assay described above. Ser¹⁴⁴³ phosphorylation data are expressed as apparent stoichiometry (described under “Experimental Procedures”) and represent the means ± S.E. (n = 3 independent experiments). *, a significant difference of p < 0.01 compared with vehicle. A, effect of increasing EGF concentration on Ser¹⁴⁴³ phosphorylation. Responses were quantified by fitting a four-parameter Hill equation to individual experiments and calculating an EC₅₀ value. B, dynamics of EGF-induced IQGAP1 Ser¹⁴⁴³ phosphorylation. Serum-starved HeLa cells were treated with either vehicle (0 min) or 100 ng/ml EGF for 2, 5, 10, 20, or 60 min. C, reconstructed ion chromatograms from one channel of the LC-MS/MS assay showing the dynamics of EGF-dependent phosphorylation of Ser¹⁴⁴³. Each trace shows the production of the y₅ ion produced by fragmentation of the Ser¹⁴⁴³ phosphopeptide SKpSVKEDSNLTLQEK (M³⁺ ion, m/z 618.3). D, dynamics of EGF-induced activation of EGFR. Additional aliquots of lysate from each sample described in B were probed with anti-EGFR and anti-phosphotyrosine (pY) as a measure of activated EGFR and anti-IQGAP1 antibodies. The data are representative of three independent experiments, each performed in triplicate.

FIGURE 7.

Specificity of IQGAP1 Ser¹⁴⁴³ phosphorylation to EGFR pathway activation. Serum-starved HeLa cells were pretreated with either vehicle, 1 μm bisindolylmaleimide (Bis), or 1 μm 4557W for 60 min, followed by activation with 100 ng/ml EGF for 5 min (A) or 1 μm PMA for 20 min (B) where indicated. The data are expressed as apparent stoichiometry (described under “Experimental Procedures”) and represent the means ± S.E. (n = 3 independent experiments). *, a significant difference of p < 0.01 compared with either EGF- or PMA-treated samples.

FIGURE 8.

siRNA knockdown of PKC alpha abrogates EGFR-mediated IQGAP1 Ser¹⁴⁴³ phosphorylation. HeLa cells were transfected for 48 h with non-targeting control siRNA (NT) or siRNAs targeting the α, δ, and ϵ isoforms of PKC individually (PKCa, PKCd, and PKCe, respectively) or α and ϵ together (PKCa-e). After overnight serum starvation, cells were treated with either vehicle (−) or 100 ng/ml EGF (+) for 5 min. A, equal aliquots of protein lysate were blotted and probed to confirm PKC isoform-specific protein knockdown (PKCα, -δ, and -ϵ), EGFR activation (EGFR and pTyr), and protein loading (GAPDH). B, IQGAP1 was immunoprecipitated and isolated by SDS-PAGE, digested with trypsin, and analyzed by LC-MS/MS. Ser¹⁴⁴³ phosphorylation data are expressed as apparent stoichiometry (described under “Experimental Procedures”) and represent the means ± S.E. (n = 3 independent experiments). *, a significant difference of p < 0.01 compared with non-targeting control siRNA.

FIGURE 9.

IQGAP1 enhances EGFR activation. A, IQGAP1^−/− MEFs were transfected with empty pcDNA vector (V) or Myc-tagged IQGAP1 constructs and starved of serum. Cells were treated with either vehicle (−EGF) or 100 ng/ml EGF (+EGF) for 5 min. Equal aliquots of protein lysate were used to measure the expression and activation of the receptor by probing the blots for EGFR and phosphotyrosine (pTyr). Blots were also probed for IQGAP1 to evaluate expression of the constructs and for GAPDH to evaluate protein loading. B, tyrosine phosphorylation of EGFR was quantified by densitometry, and the data are expressed as the -fold increase in EGFR phosphotyrosine following EGF relative to vehicle for each condition. Data represent the means ± S.E. (n = 3 independent experiments). *, significant difference of p < 0.005 compared with vector.