IQGAP1 enhances EGFR signaling by Promoting Oligomerization and Preventing Degradation

doi:10.21203/rs.3.rs-3620514/v1

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IQGAP1 enhances EGFR signaling by Promoting Oligomerization and Preventing Degradation

https://doi.org/10.21203/rs.3.rs-3620514/v1

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IQGAP1 is a large, multi-domain scaffold that connects and modulates different signaling networks including the one initiated by epidermal growth factor (EGF)by localizing and concentrating pathway components to promote activation. In this study, we have used live cell fluorescence imaging methods along with other biochemical techniques to follow the mechanisms used by IQGAP1 to enhance EGF signaling. We show that IQGAP1 enhances EGF signaling by promoting the oligomerization of its receptor, EGFR upon EGF addition along with concurrent IQGAP oligomerization. Using cellular markers, we find that IQGAP1 promotes the plasma membrane localization of EGFR and promotes association to one of its phosphoinositide lipid pathway ligands, PI(3, 4, 5)P₃. Additionally, we find that binding of EGFR to IQGAP1 protects EGFR from lysosomal degradation. Taken together, our results show that IQGAP1 enhances and prolongs EGF-mediated pathway progression.

IQGAP1 (IQ-domain containing Ras GTPase Activating Protein 1) is a multi-domain protein involved in mediating multiple cell activities. IQGAP1 lacks catalytic activity (1), but serves as a scaffold for components of several signaling pathways (2) such as the PI3K/Akt pathway, where IQGAP1 binds PI(4, 5)P₂ and PI(3, 4, 5)P₃, the phosphorylation kinases involved in their generation(i.e. PIPKI and PI3K), and signaling molecules downstream of PI3K such as Akt and PDK1(3–5). IQGAP1 is also thought to modulate the epidermal growth factor (EGR) signaling pathway that mediates growth (6). IQGAP1 is known to directly bind to EGFR and has been thought to modulate EGFR’s activation (7) although this idea has not been directly shown. It has been shown that EGFR binding to IQGAP1 specifically modulates the PI3K-Akt pathway (8).

EGFR is a member of the tyrosine kinase family (9) where the binding of ligands induces receptor dimerization (10) leading to phosphorylation and subsequent activation of downstream signaling that include the PI3K/Akt and Ras/Erk signaling pathways (6, 11). The signal from activated EGFR is terminated through endocytosis of ligated EGFR oligomers in early endosomes (12). Two major destinations exist for EGFR trafficking: return to the cell surface or lysosomal degradation. These pathways balance the enhanced signaling from recycled EGFR at the cell surface against signal attenuation via the degradative pathway. Dysregulation of EGFR signaling results in gene amplification and overexpression, and activating mutations of EGFR have been identified in a variety of cancers (13). These mutations include ones that cause trafficking defects of EGFR (14). Because of its dysregulation in many cancers, EGFR has been used as a target with mixed success (15, 16) pointing to the need for alternative approaches.

Increased IQGAP1 expression has also been found in a variety of cancers (17–23). IQGAP1 plays a role in receptor trafficking through its interactions with several endosomal and lysosomal proteins, including the recycling endosomes-associated Rab11 (24). IQGAP1 is also involved in the trafficking and recycling of the chemokine receptor CXR4 (25), and forms complexes with PIPKIγ by way of endosomal transportation possibly by physically scaffolding endosomes and microtubules. IQGAP1 is also known to impact EGFR signaling. A mutant that blocks IQGAP1’s binding to phosphoinositide kinases was shown to suppress EGF-mediated activation of the PI3K-Akt but not the Ras-ERK pathway. This mutant did not result in changes to colocalization between IQGAP1 and internalized EGFR (8), suggesting that there may be multiple sites for IQGAP1 to interact with EGFR.

Here, we have studied the role of IQGAP1 in EGFR signaling, including changes in EGFR localization and oligomerization by fluorescence imaging. We find that IQGAP1 scaffolds EGFR to the pools of PI(3, 4, 5)P₃, and protects EGFR against lysosomal degradation. Our studies show that IQGAP1 offers an additional level of EGFR regulation.

Materials. HeLa cells were obtained from American Type Culture Collection (Manassas, VA, USA). Untagged eGFP and mCherry plasmids were obtained from Clontech (Mountain View, CA, USA). All other plasmids were obtained through AddGene (Boston, MA, USA) from the following investigators: eGFP-IQGAP1 (Dr. David Sacks), mCherry-PH-Akt1(Dr. Moritoshi Sato). On-Target Plus SmartPool IQGAP1-siRNA (a pool of 4 different siRNAs) (GE Dharmacon, Lafayette, CO, USA) was used to knockdown IQGAP1 while the scrambled siRNA was obtained from Ambion (ThermoFisher Scientific, Waltham, MA, USA). Lysosomal (Lysotracker) and membrane (CellMask Deep Red Plasma Membrane) markers were also obtained from ThermoFisher.

Cell Culture. HeLa cells were incubated in high-glucose DMEM (ThermoFisher Scientific) supplemented with 5% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA, USA) at 37°C with 5% CO₂. Transfection of plasmids and small interfering RNA (siRNA) was performed using Lipofectamine 3000 (ThermoFisher Scientific) in antibiotic-free medium as per the manufacturer’s instructions. For treatment with agonist, cells were cultured in a serum-free high-glucose DMEM for 2 hours, following which they were supplemented with EGF (1000 ng/ml or 100 ng/ml) and incubated for at least 1 hour. Experiments were performed on different days using completely different cell cultures as replicates.

Imaging. Cells plated on MatTek chambers (MatTek, Ashland, MA, USA) were fixed using 3.7% formaldehyde and permeabilized with 0.2% nonyl phenoxypolyethoxylethanol (NP40) in phosphate buffered saline (PBS) for 5 min and then blocked in PBS containing 1% BSA for 1 h. If appropriate, cells were then incubated with the primary antibody diluted to 1:1000 for 1.5 hours at 37°C, followed by incubation with Alexa-labeled secondary antibody for 0.5 hours at the same temperature. Cells were washed with Tris buffered saline (TBS) solution after the incubations. Images of the cells were obtained using a Zeiss LSM 510 confocal microscope. Live cell images were taken on the same microscope while the cells were incubated in a custom-built chamber at 37°C and 5% CO₂. The fluorescence images were obtained with a Nikon (Minato, Tokyo, Japan) Eclipse Ti inverted microscope with a TIRF Illuminator and a Nikon 60 X CFI Apo TIRF oil objective. For excitation sources, coherent 488 nm, 561 nm, and 647 nm sapphire lasers were used. Images were captured with an Andor3iXon CCD camera (Belfast, UK). The images were analyzed using Zeiss software and/or ImageJ (27) (National Institutes of Health, Bethesda, MD,USA).

Number and Brightness (N&B) analysis. The Number and Brightness (N&B) analysis is a powerful tool that has been used previously to quantify graphically the aggregation state of diffusing proteins in living cells (28–31). N&B analysis can determine the number (N) of diffusing particles within a given focal area and the intrinsic brightness (B) of each particle represented by pixels in an image and provides a map of brightness for every pixel. While we used the B-value as a measure of aggregation and oligomerization, it does not increase linearly with increased oligomerization. The mathematical equations that describe the apparent brightness B for every pixel are:

$$\varvec{B}= \frac{{\varvec{\sigma }}^{2}-{\varvec{\sigma }}_{0}^{2}}{<\varvec{I}>-\varvec{o}\varvec{f}\varvec{f}\varvec{s}\varvec{e}\varvec{t}}$$

where I denotes the intensity of the signal, σ² is the variance of the signal, σ₀² is the readout noise variance of the detection electronics and offset is the detector offset. Based on the control measurements, we chose to use the percentage of pixels with B values above 1.5 to quantify the oligomeric aggregates in the cells. The analysis has been described in more detail (29, 30, 32).

N&B studies were performed, by acquiring images of live cells plated on MatTek chambers (MatTek, MA, USA) using a 2-photon MaiTai laser (Spectra-Physics), a Nikon inverted confocal microscope in an ISS Alba System. The Images were analyzed using ISS VistaVision and SimFCS 4 (Irvine, CA, USA) software packages.

Western Blotting. HeLa cells were lysed with 500 ul of buffer containing 150 mM NaCl, 20 mM HEPES, 2 mM MgCl₂, 5 mM 2-mercaptoethanol, 1% NP40, 1 mM phenylmethylsulfonyl fluoride and cOmplete™ protease inhibitor cocktail tablet (Roche/Millipore Sigma, St. Louis, MA, USA). The lysed cells were boiled in sample buffer at 95°C for 3 minutes and were analyzed using SDS-PAGE and Western blotting.

Fluorescence lifetime imaging microscopy (FLIM). FLIM is a method that allows to measure physical interactions as quantified by Förster resonance energy transfer (FRET) between labeled proteins in cells. FRET reduces the fluorescence lifetime of a donor molecule due to transfer of energy to an acceptor. Because of its steep distance dependence in the low nanometer range, FRET transfer requires a direct physical interaction between the proteins to which the probes are attached. The methodology has been discussed previously(32).

In our experiments, we used green fluorescent protein donors (such as eGFP, enhanced Green Fluorescent Protein) with red fluorescent proteins such as mCherry and dsRed acting as the FRET acceptors. FLIM was performed by acquiring images of live cells plated on MatTek chambers using a 2-photon MaiTai laser (Spectra-Physics, Santa Clara, CA, USA) (excitation 850 nm at 80 MHz) and a Nikon inverted confocal microscope in an ISS Alba System (Champaign, IL, USA). The Images were analyzed using ISS VistaVision and ImageJ software packages. Atto 425 fluorescent dye (t = 3.61 ns) was used to calibrate the sample lifetimes.

Statistics. All data were analyzed using Sigmaplot (SysStat, San Jose, CA, USA) using Student’s t-test substituted by Mann-Whitney’s rank sum test if the data was not normally distributed. A difference between two groups was considered statistically significant only with a p-value below 0.05.

IQGAP1 oligomerizes upon cell stimulation with EGF. IQGAP1 binds a broad array of proteins (33). In HeLa cells, endogenous IQGAP1 is detected both at the plasma membrane and in the cytosol (Fig. 1A). Upon stimulation with EGF (100 ng/ml), we observe a statistically significant increase in IQGAP1 intensity localized to the plasma membrane, including cell-cell junctions (Fig. 1C). To better understand these dynamics, we used eGFP-IQGAP1 whose fluorescent tag does not interfere with its functionality (34, 35) and localization (see Supplemental Fig. 1).

IQGAP1 is known to oligomerize (34, 36) and we wondered whether the shift in localization with EGF signaling seen in Fig. 1A is accompanied by changes in oligomerization. We assessed changes in oligomerization by a quantitative spectroscopy-based method that analyzed the number & brightness (N&B, see Methods). This method follows changes in the intensity and movement of pixels in a time series of images to determine whether the movement corresponds to a monomer, as calibrated using free eGFP, or a higher order species. Here, the fluorescence intensity of each pixel in an image represents a molecule of eGFP-IQGAP1 giving us both the quantification and visualization of its oligomerization in the cell image. Pixel oligomerization is calculated as the percentage of pixels that correspond to a brightness (B) value above 1.5 as a after background correction and calibration to a monomer value of 1.0 using free eGFP (for further details, please see (31)) (Fig. 1D, E). We see a significant increase of oligomeric species (i.e., those with B value above 1.5) from 6% to about 12% upon treatment with EGF (Fig. 1F), whereas no changes are seen for free eGFP (Fig. 1F). This increase in B values indicates that EGF stimulation increases both plasma-membrane localization and oligomerization of IQGAP1(Fig. 1D, E).

We used fluorescence correlation spectroscopy (FCS) to observe the changes in the mobility of eGFP-IQGAP1 as it responds to EGF stimulation. FCS quantifies the diffusion coefficient of the fluorescent protein which will decrease if the protein aggregates to a size that is at least 5-fold larger. We see that there is a significant decrease in the diffusion coefficient in the plasma membrane population of IQGAP1 that accompanies the clustering observed by the N&B analysis (Fig. 1G). However, there is no change in the mobility of the cytosolic population. These studies show that EGF signaling promotes the formation of domains enriched in IQGAP1 on or close to the plasma membrane.

IQGAP1 enhances EGFR Expression, Oligomerization and Phosphorylation. Overstimulation of EGFR by its ligands such as EGF is known to cause its internalization through endocytosis to terminate the signal (12). When we stimulate HeLa cells expressing eGFP-EGFR with EGF, we see punctate cytosolic populations of EGFR after 30 minutes as the receptor is internalized, and this population survives if the stimulation is sustained (Fig. 2A). We used N&B to determine whether changes in eGFP-EGFR oligomerization are associated with clustering after long-term EGF stimulation. We find that EGF activation produces EGFR oligomerization both on or close to the plasma membrane and in the cytosol in accord with its activation on the plasma membrane and subsequent internalization in a dose-dependent manner (Representative image Fig. 2B, Fig. 2C, left panel).

IQGAP1 enhances EGFR’s localization to the Plasma Membrane and Lysosomes. We assessed the localization of eGFP-EGFR in unstimulated HeLa cells by visualizing its colocalization to a plasma membrane marker (Fig. 3A). EGF stimulation results in a statistically significant change to its localization (Fig. 3B), but this effect is eliminated when IQGAP1 is downregulated, indicating that IQGAP1 may enhance membrane localization of EGFR.

IQGAP1 is known to localize to various organelles such as endosomes and lysosomes in the cytoplasm (5), endosomes (37), and lysosomes (38) that are involved in the recycling and degradation of the excess receptors as a part of receptor trafficking. We find that the cytoplasmic populations of eGFP-EGFR co-localize with lysotracker (an indicator of lysosomes), indicating that EGFR is being targeted to the lysosomes (Fig. 3C-D), and that the increased cytoplasm-localized population of EGFR after EGF stimulation observed above corresponds to a shift of this EGFR population to lysosomes. Importantly, knockdown of IQGAP1 results in a statistically significant (p < 0.0001) increase in EGFR-lysosome localization and eliminates any EGF-induced effects (Fig. 3E). Overall, these results indicate that IQGAP1 expression protects EGFR from lysosome degradation.

EGFR – phosphoinositide association is enhanced by IQGAP1. IQGAP1’s binding to EGFR is known to be modulated by PI(4, 5)P₂ (39) and PI(3, 4, 5)P₃ levels (40). Here, we quantified the effect of EGF stimulation on the interactions between eGFP-EGFR and the PI(3, 4, 5)P₃ sensor mCherry PH-Akt1 using fluorescent lifetime imaging (FLIM). FLIM results can be observed in the form of a phasor plot (Figs. 4A: unstimulated 4B: EGF stimulated, left panels) which provides a graphical representation of fluorescence lifetime distributions allowing individual lifetime populations within a FLIM image to be easily distinguished, see (41). Choosing a particular pixel cluster on the phasor plot (shown in green, purple, and yellow circles) and overlaying the spatial distribution of these lifetimes on cell images, enables the visualization of the cellular distribution of these lifetimes (Figs. 4A: unstimulated cells 4B: EGF stimulated cells, right panels). In FRET experiments, decreased donor lifetimes are shifted inside the phasor arc (purple and yellow regions) and away from the non-FRET population (green). Although we see FRET in the unstimulated and stimulated case, we observe increased FRET between eGFP-EGFR and mCherry PH-Akt1 upon EGF stimulation. This increased FRET population is distributed across the whole cell including the plasma membrane (Figs. 4A: unstimulated 4B: EGF stimulated). Downregulation of IQGAP1 eliminates this EGF-enhanced increase in the EGFR-PH-Akt1 association (Fig. 4C), suggesting that IQGAP1 enhances an interaction between EGFR and PI(3, 4, 5)P₃.

Scaffold proteins play key roles in enhancing or attenuating cell signals by organizing and localizing pathway proteins to promote their associations in specific cellular localization. Here, we have studied the ability of the ubiquitous scaffolding protein, IQGAP1 to mediate EGF signals in HeLa cells. By imaging live cells expressing fluorescent-tagged proteins, we were able to directly observe the impact of IQGAP1 on EGFR’s localization, aggregation, interactions with ligands and movement to lysozymes. We note that over-expression of fluorescent-tagged proteins may influence the impact of the behavior seen here, but our expression levels are low, as seen by single molecular fluorescence data, relative to the levels of endogenous proteins.

IQGAP1 is characteristically over expressed in various types of cancers (42) including hepatocellular (43, 44), prostate (45), glioma (46), head-and-neck (22) and breast (23). IQGAP1’s cellular localization and expression levels impact various signaling systems, and redistribution of IQGAP1 from the cytoplasm to the membrane correlates to tumor grade and poor disease outcome prognosis (23, 45–47). Here, we find that stimulation of HeLa cells with EGF results in an increased plasma membrane localization of IQGAP1 suggesting that the increase in cellular calcium, PIP₂ hydrolysis, and subsequent lipid and protein phosphorylation help drive IQGAP1 membrane association. It is notable that the increase in the plasma membrane pools of IQGAP1 is accompanied by an increase in IQGAP1 homo-oligomerization correlating with the increased effective concentration on the quasi-two-dimensional membrane surface. The enhancement of IQGAP1 membrane localization and oligomerization on the membrane surface will in turn concentrate EGF pathway components on the membrane surface which may be associated with the increase EGF stimulation seen in cancer (13, 48). We note that our measurements cannot clearly distinguish between proteins localized on the plasma membrane and proteins incorporated in plasma membrane associated vesicles that may appear during receptor internalization. Thus, enhanced membrane localization and oligomerization by IQGAP may be either direct or indirect.

We find that IQGAP1 knockdown results in an increased expression of EGFR. We also see in preliminary studies that knockdown of IQGAP1 results in higher basal EGFR phosphorylation but a weaker EGF-mediated increase of EGFR activity (Fig. S3). EGFR phosphorylation is a result of EGFR homodimerization caused by stimulation by its agonist EGF that culminates in the activation of downstream signaling. When normalized to the increased overall levels of EGFR protein, these results show that downregulation of IQGAP1 results in a decrease in EGF-mediated EGFR phosphorylation despite the overall increase in EGFR expression.

ur studies suggest that the cytosol-localized population of EGFR is linked to endosomes and lysosomes. Interestingly, we show that IQGAP1 expression protects EGFR against lysosomal degradation revealing a new aspect of IQGAP1’s promotion of EGFR signaling. It is possible that the increase in EGFR expression for IQGAP1 knockdown cells occurs in response to the increased lysosomal trafficking of the receptors. We also see that IQGAP1 is localized close to the pools of PI(3, 4, 5)P₃ generated by PI3K activity which is enhanced by EGF.

A summary of our findings is depicted in Fig. 5. Our results, therefore, further establish a model of a multi-pronged promotion of EGFR signaling by IQGAP1, especially in the context of ligand activation. These strategies include promotion of EGFR activation, protecting EGFR against degradation, and the activation of PI3K/Akt and Ras/Erk signaling pathways. Our studies further reveal the many facets of IQGAP1’s role in mediating EGF signaling, hence indicating the potential of targeting IQGAP1 as a target for novel anti-cancer therapies that can supplement the existing anti-EGFR chemotherapy.

Author Contributions: Conceptualization, VSY; AHR; AG; and SS.; methodology, VSY; AHR; AG; and SS.; formal analysis, VSY; AHR; AG; and SS.; investigation, VSY; JLR; RMH; SKL; and KP; resources, AHR; AG; and SS.; writing—original draft preparation, VSY writing—review and editing, VSY; AHR; AG; and SS.; supervision, AHR; AG; and SS.; funding acquisition, AG; and SS.;

Funding: This work has been funded by NSF CHE 1904886 (AG) and Whitcomb Award (SS). In addition, we would like to acknowledge funding through the NSF REU program awards (NSF CHE 1659529 and NSF CHE 1950512)

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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No competing interests reported.

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IQGAP1 enhances EGFR signaling by Promoting Oligomerization and Preventing Degradation

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