Hierarchical scaffolding of an ERK1/2 activation pathway

Abstract

Background: Scaffold proteins modulate cellular signaling by facilitating assembly of specific signaling pathways. However, there is at present little information if and how scaffold proteins functionally interact with each other.

Results: Here, we show that two scaffold proteins, caveolin-1 and IQGAP1, are required for phosphorylation of the actin associated pool of extracellular signal regulated kinase 1 and 2 (ERK1/2) in response to protein kinase C activation. We show by immunofluorescence and proximity ligation assays, that IQGAP1 tethers ERK1/2 to actin filaments. Moreover, siRNA experiments demonstrate that IQGAP1 is required for activation of actin-bound ERK1/2. Caveolin-1 is also necessary for phosphorylation of actin-bound ERK1/2 in response to protein kinase C, but is dispensible for ERK1/2 association with actin. Simultaneous knock down of caveolin-1 and IQGAP1 decreases total phorbol ester-induced ERK1/2 phosphorylation to the same degree as single knock down of either caveolin-1 or IQGAP1, indicating that caveolin-1 and IQGAP1 operate in the same ERK activation pathway. We further show that caveolin-1 knock down, but not IQGAP1 knock down, reduces C-Raf phosphorylation in response to phorbol ester stimulation.

Conclusions: Based on our data, we suggest that caveolin-1 and IQGAP1 assemble distinct signaling modules, which are then linked in a hierarchical arrangement to generate a functional ERK1/2 activation pathway.

Figure 1

ERK1/2 subcellular localization does not show stimulus-specific differences. A7r5 cells were stimulated for 5 minutes with either a phorbol ester (DPBA) or with serum (FCS), or left unstimulated before subcellular fractionation by differential ultracentrifugation. Samples were analyzed by western blotting. (A) Average ERK1/2 subcellular distribution from four independent experiments is shown along with a typical western blot. (B) Control staining with marker proteins for the cytoskeletal fraction (caldesmon), the membrane fraction (integrin) and the cytosolic fraction (GAPDH). Please note that the integrin band appears as a doublet. Significance (*compared to unstimulated samples) and p-values are indicated on chart. n.s., not significant; error bars represent standard errors.

Figure 2

IQGAP1 and KSR1 interact with ERK1/2 in a stimulus-specific manner. (A) Lysates of unstimulated, DPBA-stimulated or FCS-stimulated cells were subjected to immunoprecipitation (IP) experiments. Co-immunoprecipitated proteins were detected by western blotting and analyzed by densitometry. (A) The graph shows average band intensities of ERK1/2 co-immunoprecipitated with an anti-IQGAP1 antibody. (B) Representative western blot for an anti-IQGAP1 IP experiment. (C) Average band intensities of ERK1/2 co-immunoprecipitated with an anti-KSR1 antibody. (D) Representative western blot for an anti-KSR1 IP experiment. (E) Average band intensities of IQGAP1 co-immunoprecipitated with an anti-ERK1/2 antibody. (F) Average band intensities of KSR1 co-immunoprecipitated with an anti-ERK1/2 antibody. (G) Representative western blot for an anti-ERK1/2 IP experiment. Please note that a non-specific band of 38 kDa (*) was seen in ERK1/2 IPs. (H) Analysis of pERK to total ERK ratios in input samples as well as in immunoprecipitates. Significance (*compared to unstimulated samples; # compared to ) and p-values are indicated on chart; error bars represent standard errors. IP, immunoprecipitation. Please note that ERK2 appears as a doublet after stimulation, with the upper band representing phosphorylated ERK2.

Figure 3

ERK1/2 scaffold IQGAP1 mediates stimulus-specific ERK1/2 activation. Cells transfected with siRNA directed against IQGAP1 or KSR1, or with undirected control siRNA, were stimulated with DPBA or FCS, or left unstimulated. (A and B) Western blot analysis of cell lysates from (A) IQGAP1 knock down and (B) KSR1 knock down experiments. (C) Densitometric analysis of ERK1/2 activation after siRNA knock down. Significance (*) and p-values are indicated on chart; error bars represent standard errors.

Figure 4

IQGAP1 is required for activation of cytoskeletal ERK1/2. (A) IQGAP1 knock down interferes with filamentous localization of phospho-ERK1/2. A7r5 cells on coverslips transfected with siRNA directed against IQGAP1 were stimulated with DPBA or FCS. After fixing, cells were co-stained for IQGAP1 (red channel) and phospho-ERK1/2 (green channel); filamentous actin was stained with Alexa350-phalloidin (blue channel). The panels show representative images from one out of three independent experiments. (B) The same images as in (A) shown in black and white for enhanced contrast. Scale bar, 20 μm.

Figure 5

IQGAP1 mediates actin association of ERK1/2. Cells were transfected with siRNA as indicated. Before fixing, cells were stimulated with either FCS or DPBA, or left unstimulated. Proximity between ERK1/2 and actin was analyzed by proximity ligation assays (PLA). (A) Immunofluorescence and statistical analysis of PLA with a negative control (anti-actin and anti-tubulin) and positive control (anti-ERK1/2 and anti-phospho-ERK1/2). (B) Immunofluorescence images of the ERK1/2-actin PLA pair show that IQGAP1 siRNA reduces actin association of ERK1/2. (C) Statistical analysis of 60 cells for each test from three independent experiments (*compared to unstimulated samples; # compared to corresponding control samples; + between DPBA and FCS; error bars represent standard errors). Scale bar, 20 μm.

Figure 6

IQGAP1 and caveolin-1 are required for activation of cytoskeletal ERK1/2. Cells were transfected with siRNA as indicated. Before fixing, cells were stimulated with either FCS or DPBA, or left unstimulated. Proximity between phospho-ERK1/2 and actin was analyzed by proximity ligation assays (PLA). (A) Immunofluorescence images of the phospho-ERK1/2-actin pair show that both, caveolin-1 and IQGAP1, are required for phosphorylation of actin-associated ERK1/2. (B) Statistical analysis of 60 cells for each test from three independent experiments (*compared to unstimulated samples; # compared to corresponding control samples; + between DPBA and FCS; error bars represent standard errors). Scale bar, 20 μm.

Figure 7

IQGAP1 and caveolin-1 are upstream and downstream scaffolds in the same ERK1/2 activation pathway. (A) Double knock down of IQGAP1 and caveolin-1 shows the same decrease in DPBA-induced ERK1/2 activation as single knock down. Cells were transfected with siRNA as indicated, then stimulated with DPBA for 5 minutes. Lysates were subjected to western blotting for analysis of ERK1/2 phosphorylation. (B) C-Raf activation is reduced after knock down of caveolin-1, but not after knock down of IQGAP1. Cells were transfected as indicated, then stimulated with DPBA for 5 minutes. The ratio of phospho-C-Raf (S338) to total C-Raf was analyzed on duplicate membranes after normalization to GAPDH. (C-E) To enrich the cytoskeletal fraction, DPBA stimulated A7r5 cells were subjected to Triton X-extraction before preparation of soluble and insoluble cell extracts. Insoluble and soluble lysates were analyzed for ERK1/2 phosphorylation as well as target protein expression by western blotting and densitometry. Data represent five independent experiments. (C) Western blots show siRNA knock down of caveolin-1, IQGAP1, B-Raf and C-Raf. Insoluble samples are shown for caveolin-1 and IQGAP1 expression, soluble samples are shown for B-Raf, C-Raf and GAPDH. (D) The graph shows average ERK1/2 phosphorylation in the TX-insoluble fraction, along with a representative western blot. (E) The western blot shows phosphorylated ERK1/2 and total ERK1/2 in TX-soluble samples. Significance compared to control (*) and compared to IQGAP1 siRNA (#), as well as p values are indicated on graphs; error bars represent standard errors.

Figure 8

B-Raf and C-Raf interactions in A7r5 smooth muscle cells. (A) Interactions between Raf isoforms, IQGAP1 and caveolin-1 were assessed by immunoprecipitation experiments. Lysates were immunoprecipitated with an anti-GFP antibody as control, or with an anti-B-Raf or anti-C-Raf antibody. Co-immunoprecipitated B-Raf, C-Raf, caveolin-1 and IQGAP1 were detected by western blotting. (B) Statistical analysis of co-immunoprecipitated C-Raf, caveolin-1 and IQGAP1 from 7 independent IPs with the anti-B-Raf antibody (GFP shown as control). (C) Statistical analysis of co-immunoprecipitated B-Raf, caveolin-1 and IQGAP1 from 7 independent IPs with the anti-C-Raf antibody (GFP shown as control). Significance compared to control (*) and p values are indicated on graphs; error bars represent standard errors. n. s., not significant.

Figure 9

Model for hierarchical scaffolding by caveolin-1 and IQGAP1. (A) We suggest a model in which an upstream signaling module, associated with caveolin-1 and consisting of PKC, Ras, and C-Raf, is linked via Raf heterodimerization to a downstream signaling module, scaffolded by IQGAP1 and consisting of B-Raf, MEK and ERK. (B) Knock down of caveolin-1 prevents activation of actin-associated ERK1/2 by PKC, but does not interfere with actin association of ERK1/2. (C) Knock down of IQGAP1 disconnects ERK1/2 from actin.