Research ArticleCancer

KIF22 coordinates CAR and EGFR dynamics to promote cancer cell proliferation

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Sci. Signal.  30 Jan 2018:
Vol. 11, Issue 515, eaaq1060
DOI: 10.1126/scisignal.aaq1060

The EGFR-cytoskeleton connection

Growth factor signaling stimulates cell proliferation and migration, which requires changes in cell-cell adhesion and the cytoskeleton. Increased activity of the growth factor receptor EGFR is implicated in various cancers. Pike et al. found that EGFR signaling directs changes in cell-cell junctions and the cytoskeleton in lung cancer cells by inducing the phosphorylation of the cell adhesion receptor CAR (see also the Focus by Chiasson-MacKenzie and McClatchey). Phosphorylated CAR interacted with the microtubule motor protein KIF22 to stabilize the peripheral microtubule network, which facilitated cell division and anchorage-independent growth associated with metastasis. It also altered the trafficking of EGFR such that its signaling was prolonged. Thus, CAR or KIF22 might be alternative targets for therapeutically inhibiting EGFR signaling in some cancers.

Abstract

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane receptor that plays a key role in cell-cell adhesion. CAR is found in normal epithelial cells and is increased in abundance in various human tumors, including lung carcinomas. We investigated the potential mechanisms by which CAR contributes to cancer cell growth and found that depletion of CAR in human lung cancer cells reduced anchorage-independent growth, epidermal growth factor (EGF)–dependent proliferation, and tumor growth in vivo. EGF induced the phosphorylation of CAR and its subsequent relocalization to cell junctions through the activation of the kinase PKCδ. EGF promoted the binding of CAR to the chromokinesin KIF22. KIF22-dependent regulation of microtubule dynamics led to delayed EGFR internalization, enhanced EGFR signaling, and coordination of CAR dynamics at cell-cell junctions. These data suggest a role for KIF22 in the coordination of membrane receptors and provide potential new therapeutic strategies to combat lung tumor growth.

INTRODUCTION

Coxsackievirus and adenovirus receptor (CAR) was initially identified as the primary docking receptor for coxsackie B viruses and members of the adenovirus family (1). Further work has since demonstrated that CAR is an important cell adhesion molecule (2, 3) as a member of the junction adhesion molecule family that forms homodimers across cell-cell junctions (4, 5). We have previously shown that CAR is phosphorylated at Thr290 and Ser293 within the cytoplasmic domain by protein kinase C δ (PKCδ) and that it controls E-cadherin stability at adherens junctions (6, 7). Its role in cancer may be tissue-specific; the expression of the gene that encodes CAR is up-regulated in some cancers and down-regulated in others (8). However, in the lungs, CAR abundance is consistently increased in tumor tissue compared to normal tissue, and reducing its expression in lung cancer cells reduces the growth of xenografts in animal models (9). Increased CAR abundance in lung cancer is associated with a more mesenchymal cell phenotype and increased expression of several mesenchymal markers (9). Other studies have shown that CAR promotes cell-cell adhesion and facilitates cell survival (10) and that transforming growth factor–β–induced epithelial-to-mesenchymal transition (EMT) is coupled with the down-regulation of CAR (11), potentially leading to enhanced metastasis in vivo (12). In vitro, CAR depletion reduces the growth of lung cancer cells in soft agar, suggesting an important role in anchorage-independent growth (13). CAR may play a role in lung cancer cell adhesion and invasion (8) and in being a potential marker of cancer stem cells in non–small cell lung cancers (NSCLCs) that are resistant to paclitaxel and radiation treatment (14). Despite this growing evidence that implicates CAR in lung tumor progression, its mechanisms of action in this context are not clear.

Growth factor signaling is an important driver of tumor growth, and mutations in growth factor receptors and downstream signaling molecules are frequently found in lung cancers (15). Gain-of-function mutations in the epidermal growth factor receptor (EGFR) are particularly prominent and well characterized in adenocarcinomas and provide a proliferative advantage (16). EGFR acts a node for a number of complex signaling networks and controls many cellular processes as well as proliferation, including DNA replication, adhesion, and migration (17). In addition to the well-characterized role as a mitogen, EGFR also signals both upstream and downstream of cell-cell adhesion molecules (18). For example, cytokines are able to induce the disassembly of tight junctions in lung epithelial cells by activating EGFR and mitogen-activated protein kinase (MAPK) signaling (19). EGFR is also able to drive the phosphorylation of the polarity protein Par3 at tight junctions to determine the rate of tight junction assembly (20). Similarly, EGFR activity acts to regulate transcription of claudin and, in turn, positively regulates transepithelial resistance (21). E-cadherin promotes the activation of EGFR and MAPK signaling directly, suggesting that adhesion molecules regulate receptor tyrosine kinase signaling (18). The loss of E-cadherin during EMT can also activate MAPK signaling and invasive behavior specifically in NSCLC cells (22). This highlights the importance of cross-talk between EGFR signaling and cell adhesion complexes in the regulation of tumor growth.

The cytoskeleton plays a key role in regulating cell adhesion and proliferation. CAR and EGFR require F-actin and/or microtubule cytoskeletons for membrane localization, signaling, and trafficking (23, 24), and both localize to cell-cell contacts and play a role in controlling epithelial cell junction stability (6, 7, 25). Here, we aimed to determine whether cooperation exists between these two receptors and found that CAR and EGFR act in concert to coordinate and enhance cancer cell proliferation. Our data demonstrate a role for CAR in controlling EGFR signaling through a direct interaction with the chromokinesin KIF22. We show that CAR promotes tumor cell proliferation downstream of EGFR both in vivo and in vitro. We further show that EGFR indirectly phosphorylates CAR that, in turn, provides junctional adhesion maintenance in EGF-stimulated cells through relocation at cell contact sites. Moreover, we show that this relocation relies upon an EGF-induced CAR-KIF22 complex. Our data reveal a new interplay between two key receptors known to be dysregulated in tumors and provide potential new avenues for therapeutic targeting of solid tumor growth.

RESULTS

CAR mediates EGF-dependent lung cancer cell proliferation

A previous study has shown that antisense-mediated depletion of CAR in NSCLC cells (NCI-H1703) resulted in reduced proliferation (13). To determine whether depletion of CAR in other human lung cancer cells alters proliferation, we used short hairpin RNA (shRNA) to stably deplete CAR from A549 and H1975 cells (Fig. 1A and fig. S1A). Proliferation was monitored over 48 hours in the presence or absence of serum as growth stimulants. Resulting analysis demonstrated that depletion of CAR significantly reduced proliferation in both cells lines (Fig. 1B and fig. S1B). Given that normal lung epithelial cells also express CAR, we also determined whether CAR could control proliferation in this context. We depleted or overexpressed CAR in the human lung epithelial line 16HBE using two targeted shRNAs and a green fluorescent protein (GFP)–tagged wild-type CAR construct (7, 26) and analyzed cell proliferation over 48 hours. CAR knockdown significantly reduced, whereas CAR overexpression significantly increased, proliferation in 16HBE cells (fig. S1C), confirming that CAR plays a key role in this process in both normal epithelial cells and epithelial-derived carcinoma cells. Because EGF is known to play a key role in the proliferation of lung cancer cells in vivo, we next tested whether CAR contributed to EGF-specific proliferation in these cells. CAR depletion significantly reduced proliferation in both A549 and H1975 cells treated with EGF (Fig. 1B and fig. S1D). However, knocking down CAR had no effect on cell proliferation induced by hepatocyte growth factor (fig. S1E), suggesting that CAR plays a role in EGF but not in other ligand-dependent signals that promote cell growth.

Fig. 1 CAR regulates lung cancer cell proliferation in vitro and in vivo.

(A) Western blots of coxsackievirus and adenovirus receptor (CAR) knockdown (KD) in A549 cells, either untransfected [wild type (WT)] or transfected with control short hairpin RNA (shRNA) (−ve) or one of two shRNA sequences targeting CAR (shA and shB). (B) A549 cell proliferation over 48 hours in response to serum (left) or epidermal growth factor (EGF) (10 ng/ml; right). Data are normalized to serum-free control samples for both and are from three independent experiments. (C) Representative images (left) and analysis (right) of agar colony growth assays in A549 control or CAR KD cells (shA and shB). Scale bar, 100 μm. Data are the average number of colonies per field in all cells from 10 fields per cell line and are representative of three independent experiments. (D) Representative images of resected tumors from xenograft models using H1975 control or CAR KD cells. Graphs on the right show tumor volume and weight over time in nine (control), five (siCAR-1), or five (siCAR-2) mouse models pooled from two independent experiments. (E) Example images of phospho-histone H3 (p-histone H3; top) and Ki-67 (bottom) staining in xenograft tissues from H1975 cell tumors in (D). Scale bar, 50 μm. (F) Analysis of the p-histone H3 and Ki-67 staining in xenografts represented in (E). n ≥ 12 tumors per condition over two independent experiments. Data in all graphs are means ± SEM. *P < 0.01, **P < 0.005, ***P < 0.001 by two-way analysis of variance (ANOVA).

Soft agar colony assays revealed that CAR knockdown markedly reduced anchorage-independent growth in both A549 and H1975 cells (Fig. 1C and fig. S1F). To further determine whether the in vitro defects in proliferation seen in CAR-depleted cells also translated to in vivo settings, we performed subcutaneous injections of matched control or CAR-depleted H1975 cells into the flanks of immunocompromised mice. Analysis of tumor growth over time revealed a significant defect in tumor growth in CAR-depleted cells, resulting in smaller tumors at the time of sacrifice (Fig. 1D). To confirm that this reduced tumor size was due to reduced cell proliferation, we stained sections of fixed tumors for both phosphorylated histone H3 and Ki-67 (Fig. 1E). Quantification demonstrated a significant reduction in both markers in CAR-depleted tumors (Fig. 1F). Therefore, these data demonstrate that CAR promotes the proliferation of human lung cancer cells both in vitro and in vivo.

CAR promotes postmitotic daughter cell attachment and spreading

To determine the nature of the defect in CAR knockdown cells that leads to reduced proliferation, we performed time-lapse imaging of control or CAR-depleted A549 cells under growth-inducing conditions. Analysis of resultant movies demonstrated that CAR knockdown cells were significantly slower to respread after division (Fig. 2, A and B), and junctions between daughter cells were more transient than in control cells, resulting in a greater proportion of cells undergoing complete separation after division (Fig. 2B). This suggested that CAR plays a role in coordinating and maintaining contacts between adjacent daughter cells and between other neighboring cells within colonies, potentially supporting efficient respreading and growth signaling. To determine localization of CAR during mitosis, we further analyzed movies of A549 cells coexpressing CAR-GFP and histone H2B type 1-K (H2BK)–mCherry to mark the nuclei during division. These movies revealed that CAR was rapidly recruited to newly forming contacts between daughter cells after division, further supporting the notion that CAR may play a key role in stabilizing these adhesions (Fig. 2C).

Fig. 2 CAR promotes postmitotic daughter cell attachment and spreading.

(A) Representative images from time-lapse movies of shControl or CAR KD (shA) A549 cells undergoing division. Green arrows denote dividing cells, and magenta arrows denote initiation of daughter cell separation after division. (B) Quantification of time-lapse movies of control and CAR KD cells (shA and shB) represented in (A), assessing the time taken for cells to respread after division (left) and the percentage of cells that separate completely after division (right). Data are quantified from at least 20 cells over three independent experiments. Data are means ± SEM. *P < 0.05, ***P < 0.005. (C) Representative images from time-lapse movies of A549 cells expressing CAR–green fluorescent protein (GFP) and H2BK-mCherry; n = 4 experiments. Green arrows denote sites of high CAR-GFP at cell-cell contact points. Scale bars, 10 μm.

EGF promotes CAR phosphorylation and relocalization at cell-cell adhesions

Given that our data demonstrated that CAR played a key role in EGF-dependent proliferation, we next sought to further investigate the potential mechanisms governing this response. We first analyzed whether CAR plays a role in regulating EGFR activation or signaling upon ligand binding. There was no difference in the phosphorylation of EGFR or the activation of the key downstream signaling molecule extracellular signal–regulated kinase 1 (ERK1) and ERK2 in control or CAR knockdown A549 cells (fig. S2A). In agreement with this, phospho-EGFR levels were also unchanged in tissue samples of CAR knockdown A549 xenografts compared to controls (fig. S2B). Moreover, neither phospho-ERK levels nor degradation of EGFR over longer time courses upon high concentrations of EGF stimulation (100 ng/ml) was altered by CAR depletion (fig. S2C). We therefore hypothesized that EGFR may be acting upstream of CAR in controlling proliferation. To better define the relationship between CAR and EGFR, we performed live-cell confocal time-lapse imaging of CAR-GFP expressed in A549 cells after stimulation with EGF. Analysis of resultant movies demonstrated that CAR moved from the periphery of junctions between cells toward the center in response to EGF (Fig. 3A). The same CAR redistribution was also seen in images of cells fixed after EGF stimulation and stained for endogenous CAR and EGFR (fig. S2D). Analysis of CAR and EGFR in these images also demonstrated a high degree of colocalization between the two receptors at cell-cell adhesion sites, which was significantly reduced after 15 and 60 min of EGF stimulation and EGFR internalization, further demonstrating that CAR is retained at adhesion sites after EGFR endocytosis (fig. S2D).

Fig. 3 CAR is phosphorylated in response to EGF, leading to CAR movement within cell junctions.

(A) Representative images from time-lapse movies of A549 cells expressing CAR-GFP at time 0 (untreated; top) versus 30 min after EGF addition (10 ng/ml; bottom). Graph shows quantification of CAR intensity at junctions measured at 0- and 30-min time points (example analysis areas shown in zoomed regions in images are shown on the left). Data are pooled from >20 junctions from at least three independent experiments and are means ± SEM. (B) Western blots of lysates of A549 cells treated with EGF (10 ng/ml) for the specified times and probed for the specified proteins. Graph shows quantification of phosphorylated CAR (p-CAR) abundance (normalized to total CAR levels) relative to time 0 from three independent experiments. Data are means ± SEM. *P < 0.05, ***P < 0.001. p-EGFR, phosphorylated EGF receptor; p-ERK, phosphorylated extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Representative confocal images of A549 cells fixed after 0 or 60 min of EGF stimulation (10 ng/ml) and stained for 4′,6-diamidino-2-phenylindole (DAPI) (blue, top), phospho-CAR (white, top), and actin (bottom). Graph shows quantification of p-CAR staining from images at 5, 15 and 60 min after EGF stimulation from 100 cells per condition. Data are means ± SEM. **P < 0.005, ***P < 0.001. (D) Western blots of control or PKCδ KD A549 cells treated with EGF for the specified time periods and probed for specified antibodies. Graph shows quantification of p-CAR levels relative to time 0 from three independent experiments. Data are means ± SEM. ***P < 0.001. RFP, red fluorescent protein; PKCδ, protein kinase C δ. (E) Representative images from time-lapse movies of A549 cells expressing WT CAR-GFP (top) or AA-CAR-GFP (bottom) at 0 and 30 min after EGF stimulation. Graphs show analysis of AA-CAR-GFP movement at junctions as in (A). Data are means ± SEM. All data were analyzed for statistical differences using two-way ANOVA.

Our previous studies have shown that the phosphorylation of the CAR cytoplasmic tail at Thr290/Ser293 can regulate dynamics of CAR at epithelial cell junctions (7, 26). To determine whether EGF may act through the phosphorylation of CAR to elicit junctional movement, we analyzed the levels of Thr290/Ser293 phosphorylated CAR (p-CAR) in EGF-stimulated A549 cells. Western blotting revealed a significant increase in the amount of p-CAR after 15 min of EGF stimulation that was maintained up to 60 min (Fig. 3B), and this increase was also observed by immunostaining for p-CAR in fixed cells (Fig. 3C). We further demonstrated that depletion of PKCδ, the kinase that phosphorylates these sites in CAR (7), inhibited the EGF-induced phosphorylation of CAR, suggesting that EGFR-dependent PKCδ activation may contribute to CAR dynamics (Fig. 3D). To test this concept further, we expressed a nonphosphorylatable mutant of CAR (AA-CAR) (26) in A549 cells and analyzed CAR redistribution after stimulation with EGF in A549 cells by live-cell confocal time-lapse imaging. AA-CAR-GFP did not undergo translocation within cell junctions in response to EGF as compared to wild-type CAR-GFP (Fig. 3, A and E). These data together demonstrate that EGF stimulation can promote the phosphorylation of CAR and that this, in turn, acts to promote translocation of CAR within cell-cell junctions.

CAR binds to KIF22

Our data thus far show that CAR can move laterally within the junction in an EGF- and phosphorylation-dependent manner, but we had yet to determine the molecular mechanisms that may mediate this dynamic repositioning. To identify potential binding partners for CAR involved in this process, we analyzed the proteins that associated with the CAR cytoplasmic tail in pull-downs from A549 cell lysates. Silver staining and mass spectrometry analysis of CAR-bound complexes subsequently revealed that multiple peptides mapping to the chromokinesin KIF22 (also known as KID) pulled down with the wild-type but not AA-mutant CAR cytoplasmic tail (fig. S3A). This interaction was subsequently validated in pull-down assays using glutathione S-transferase (GST)–CAR cytoplasmic domain protein (Fig. 4A) and by immunoprecipitation (IP) of CAR followed by probing with antibodies specific to KIF22 (Fig. 4B). Compared to wild-type CAR, AA-CAR-GFP showed significantly lower binding to KIF22 (Fig. 4B), suggesting that the phosphorylation of CAR promotes the formation of this complex. Moreover, we observed an increase in CAR-KIF22 binding by co-IP in cells treated with EGF, suggesting a potential role for this complex in mediating CAR-dependent responses to EGF (Fig. 4C). Preincubation of these cells with the EGFR tyrosine kinase inhibitor AG1478 before IP led to a significant reduction in both CAR-associated KIF22 and levels of p-CAR (Fig. 4C), further suggesting that EGFR activation is required for the formation of the KIF22 via CAR phosphorylation. To better define the binding region(s) within KIF22 for CAR, we expressed a series of FLAG-tagged truncation mutants of KIF22 (Fig. 4D) (27) in human embryonic kidney (HEK) 293 cells and performed pull-downs from lysates using the GST-CAR cytoplasmic domain. The resulting blots confirmed binding to full-length KIF22 (FL-KIF22) and additionally demonstrated specific binding to mutant constructs (called D1 and D2) that contain N-terminal motor domains but lack the DNA binding domain (DBD) (Fig. 4D). This suggests that the CAR-binding interface within KIF22 lies within its N-terminal region. Moreover, binding to CAR was greater for the KIF22-D1 mutant construct [which also lacks the coiled-coil domain (CCD)] than for either FL-KIF22 or the FL-KIF22-D2 mutant, suggesting that the presence of the CCD may act to limit binding to CAR.

Fig. 4 CAR binds to KIF22.

(A) Representative blot of pull-downs using glutathione S-transferase (GST)–tagged cytoplasmic domain of CAR incubated with lysates from A549 cells and probed for KIF22. GST alone was used a control. Coomassie-stained equivalent gel shows the input (bottom). (B) Representative blots of A549 cells expressing GFP, WT CAR-GFP, or AA-CAR-GFP and subjected to GFP immunoprecipitation (IP). Blots were probed for KIF22 or GFP as indicated. (C) A549 cells expressing CAR-GFP were serum-starved (−) or EGF-stimulated (10 ng/ml for 15 min; +) in the presence or absence of AG1478 and subjected to GFP IP followed by probing for specified proteins. Top two panels show IP complexes, and bottom three panels show input lysates. Quantifications of KIF22 levels in IP complexes below blots are mean values from three independent experiments ± SEM. IgG, immunoglobulin G. (D) Schematic cartoon of FLAG-tagged KIF22 constructs used for binding analysis. Representative levels of KIF22-FLAG binding to GST-CAR cytoplasmic domain after pull-down from transfected human embryonic kidney (HEK) 293 cell lysates are shown (right). Coomassie-stained gel shows protein input (bottom). Quantifications of KIF22 binding are provided beneath and represent mean values from four independent experiments ± SEM. (E) Schematic cartoon of GST-tagged N-terminal (NT) and C-terminal (CT) KIF22 constructs used for pull-downs. Blots show representative results from pull-downs of KIF22-FLAG–expressing HEK293T cell lysates using GST-NT-KIF22 or GST-CT-KIF22 (bottom). Data are representative of five independent experiments. (F) Representative blots from GST-tagged KIF22 CT pull-downs from cell lysates expressing KIF22-FLAG in the presence or absence of coexpressed KIF22-CCD-HALO. GST was used as a control. Coomassie-stained gel shows protein input (bottom). Values beneath show levels of KIF22-FLAG in pull-downs and represent mean values from three independent experiments ± SEM. All data were analyzed for statistical differences using two-way ANOVA.

The CCD region in other kinesin family members, such as kinesin-1 and kinesin-5, has previously been shown to regulate dimerization and cargo binding (28), but the potential for this region to regulate KIF22 self-association remains unclear. To test whether KIF22 may self-associate, we performed in vitro GST pull-down assays using purified GST-tagged N-terminal or C-terminal domain constructs of KIF22 (GST-NT-KIF22 and GST-CT-KIF22, respectively; Fig. 4E) coincubated with a purified FLAG-tagged FL-KIF22 construct (FLAG-FL-KIF22). The C-terminal, but not the N-terminal, domain constructs containing the CCD of KIF22 associated with the full-length construct, supporting the notion that the CCD may mediate self-binding (Fig. 4E). To test this directly, we performed GST pull-downs using GST-CT-KIF22 incubated with FL-KIF22 protein in the presence or absence of recombinant purified CCD-HALO. Data demonstrated a significant reduction in binding between the C-terminal and full-length constructs in the presence of excess CCD (Fig. 4F), suggesting that the CCD of KIF22 acts to maintain self-binding. Moreover, the expression of CCD-HALO in A549 cells led to a relocalization of endogenous KIF22 from the nucleus to the cytoplasm (fig. S3B), suggesting that interfering with the potentially dimeric self-association of KIF22 can promote cytoplasmic localization of this protein. Together, this data demonstrate that KIF22 is able to bind to itself to potentially form dimers via the CCD and that preventing this interaction can induce its cytoplasmic localization.

KIF22 regulates EGFR signaling and internalization

KIF22 has previously been shown to positively regulate proliferation through control of chromosome separation (2931), but its potential roles in the cytoplasm during interphase are not known. Because our data thus far showed that KIF22 forms a complex with CAR and potentially localizes to the cytoplasm of cells during interphase, we explored the potential role of KIF22 in mediating CAR-dependent, EGF-induced proliferation. We first sought to confirm previous studies showing a role for KIF22 in controlling proliferation. To this end, we depleted KIF22 from A549 cells using two specific small interfering RNAs (siRNAs) (fig. S4A) and analyzed proliferation under normal culture conditions using both fixed and live imaging approaches. Both methods demonstrated a significant reduction in the proliferation in KIF22 knockdown cells (fig. S4, B and C). We further analyzed responses of these cells specifically in response to stimulation with EGF. KIF22 knockdown significantly reduced EGF-dependent proliferation (Fig. 5A) and ERK activation in A549 cells (Fig. 5B), suggesting that EGFR responses to ligand are mediated by KIF22.

Fig. 5 KIF22 promotes EGFR retention at the plasma membrane and signaling in response to EGF.

(A) Analysis of the proliferation of A549 cells transfected with control or one of two KIF22-targeted small interfering RNAs (siRNAs) and treated with EGF and quantified relative to serum-starved control cells. Data are means ± SEM. **P < 0.005. (B) Representative blots of lysates from control or KIF22 KD A549 cells treated with EGF over the specified time periods and probed with the specified antibodies. (C) Representative blots of surface biotinylation experiments showing the amount of surface EGFR [basal unstripped (BU)] and internalized EGFR after 0, 15, and 60 min of EGF stimulation (10 ng/ml) in control or KIF22 KD A549 cells. Blots were probed with specified antibodies. Data in the graph (right) were normalized to basal unstrapped (black bars) and are means ± SEM from four independent experiments. *P < 0.01 versus equivalent time point in Ctrl samples. (D) Representative images of control or KIF22 KD A549 cells, either untreated or after stimulation with EGF (10 ng/ml for 15 min), fixed and stained for DAPI (blue) and EGFR (white). Scale bars, 10 μm. Graph shows quantification of junctional EGFR levels after stimulation with EGF (right). Data are means ± SEM from 30 cells across three independent experiments. *P < 0.01, ***P < 0.001. All data were analyzed for statistical differences using two-way ANOVA.

EGFR undergoes dimerization at the plasma membrane in response to ligand binding, and this results in recruitment of adaptor proteins that are essential to initiate downstream signaling. This then leads to EGFR endocytosis resulting in termination of signaling and either receptor recycling or degradation (32, 33). However, because a number of reports have also shown that EGFR internalization can sustain signaling in certain cell types (34, 35), we sought to determine the extent to which EGFR endocytosis contributes to signaling in A549 cells. Cells preincubated with dynasore (to block dynamin-dependent endocytosis) or nocodazole (to depolymerize microtubules and prevent post-endocytic transport) were treated with EGF, and lysates were analyzed by Western blotting. There was no significant change in EGF-dependent p-EGFR or p-ERK levels in either case (fig. S5), suggesting that endocytosis does not substantially contribute to the magnitude of receptor activation or p-ERK signaling in these cells.

To investigate whether KIF22 may regulate EGFR retention at the plasma membrane and thus promote signaling, we performed surface biotinylation assays in control or KIF22 knockdown A549 cells. Biotinylation of all plasma membrane proteins was performed, followed by EGF stimulation at different time points to induce surface EGFR internalization. A mild acid strip was then performed to remove any remaining surface biotin, followed by cell lysis and streptavidin IP to isolate only the internalized pool of biotinylated EGFR. Analysis of internalization kinetics revealed no change in surface EGFR levels under serum-starved conditions but a significant increase in EGFR internalization 15 min after the addition of EGF when KIF22 was knocked down (Fig. 5C). Reprobing these blots revealed that no internalization of CAR occurred over the same time frame (Fig. 5C), in agreement with confocal images of CAR and EGFR in control cells (fig. S2C). We further confirmed this rapid internalization of EGFR in KIF22 knockdown cells treated with EGF by immunostaining for and quantifying junction-associated EGFR over time (Fig. 5D).

Because our data indicated that KIF22 regulates EGFR signaling and that CAR is relocalized upon EGF stimulation, we investigated whether KIF22 also played a role in CAR localization within junctions. Live imaging and analysis demonstrated that CAR-GFP in KIF22-depleted cells was localized across the entire junction and did not undergo relocalization in response to EGF (Fig. 6A), similar to that seen in cells expressing AA-CAR-GFP (Fig. 3E). To further define the KIF22-dependent coordination of EGF-induced CAR and EGFR movement within junctions, we performed live imaging of A549 cells coexpressing CAR-GFP and EGFR-mCherry transfected with control or KIF22 siRNA. Resulting images (Fig. 6B) and subsequent intensity analysis of signal at junctions (Fig. 6C) demonstrated that the abundance of CAR within the central region of the junction increased after ~10 min of EGF stimulation and that this was rapidly followed by a reduction of EGFR at the membrane [Fig. 6, B (left) and C (left)]. Conversely, KIF22-depleted cells showed very little change in CAR abundance within the junction over the 30-min time course, but a rapid induction of EGFR internalization from the membrane (~2 to 4 min) was seen in response to EGF, further supporting the notion that KIF22 can stabilize EGFR at the plasma membrane. Together, these findings demonstrate a role for KIF22 in controlling both movement of CAR within junctions and more controlled, slower internalization kinetics of EGFR from the plasma membrane. This further suggests that by acting upstream of both receptors, KIF22 is playing a key role in controlling the initiation of EGF-dependent proliferative responses in lung cancer cells.

Fig. 6 KIF22 regulates EGF-dependent movement of EGFR and CAR within junctions.

(A) Representative images of CAR-GFP from time-lapse movies of control or KIF22 KD A549 cells expressing CAR-GFP, either untreated or after 30 min of EGF stimulation. CAR-GFP movement into junctions was quantified as described in Fig. 3 (A and E) and is presented as means ± SEM. (B) Representative images of stills taken at specified time points after EGF treatment from time-lapse confocal movies of A549 cells coexpressing CAR-GFP (top), EGFR-mCherry (bottom), and control or KIF22 siRNA. (C) Quantification of movies as in (B) showing the intensity of CAR-GFP (green) and EGFR-mCherry (magenta) at junctions over time after stimulation with EGF. Data are means ± SEM from 60 cells across three independent experiments. All data were analyzed for statistical differences using two-way ANOVA. au, arbitrary units.

KIF22 regulates peripheral microtubule stability in cells in interphase

Because KIF22 is known to bind to microtubules and microtubules are known to be important in receptor traffic, including EGFR (24, 36), we next sought to determine whether KIF22 could regulate EGFR through control of cytoskeletal dynamics in cells in interphase. To analyze the potential effects of KIF22 on the microtubule network architecture, we first stained fixed control or KIF22 knockdown cells for tubulin and F-actin. Images and subsequent quantification revealed that KIF22-depleted cells exhibit a more spread, peripherally extended microtubule network (Fig. 7A). To further define whether this defect might be due to altered microtubule dynamics, we imaged GFP-tubulin expressed in control or KIF22 knockdown A549 cells using live-cell confocal time-lapse microscopy for up to 60 min after the application of EGF. Analysis of resulting movies demonstrated that treatment of control cells with EGF resulted in more stable microtubules that showed a reduction in growth rate coupled with significantly longer time periods in the growth phase and reduced catastrophe events (Fig. 7, B and C). Conversely, KIF22 knockdown cells showed constitutively more stable microtubules under basal conditions compared to control cells, and tubulin dynamics were unchanged in the presence of EGF (Fig. 7, B and C). Moreover, levels of acetylated tubulin, which is associated with a more stable microtubule network, were also increased in KIF22 knockdown cells, further suggesting that KIF22 may enhance microtubule dynamics (Fig. 7D). These findings support the notion that KIF22 can regulate the stability and organization of the microtubule cytoskeleton in cells in interphase and that this, in turn, may affect receptor traffic and movement at the plasma membrane.

Fig. 7 KIF22 regulates microtubule organization and dynamics in cells in interphase.

(A) Representative examples of confocal images of control or KIF22 KD A549 cells, fixed and stained for DAPI (nucleus; blue), β-tubulin (red), or F-actin (phalloidin; green). Graphs show the number of nuclei per field and the area of cells with no tubulin staining present represented as area (in square micrometers per field). Data are means ± SEM pooled from nine fields per condition across three independent experiments. *P < 0.01, ***P < 0.001. (B) Representative example images from time-lapse movies of GFP-tubulin expressed in control or KIF22 KD A549 cells after stimulation with EGF. Time-dependent changes are depicted as color scales from time-projected stacks in which blue represents regions of highly dynamic microtubule (MT) growth and white denotes regions of static or disassembling MTs. Scale bar, 2 μm. (C) Analysis of MT growth rate (in square micrometers per minute), time spent in growth phase (in seconds), and catastrophe events per minute in control or KIF22 KD cells, with and without EGF (10 ng/ml). Data are means ± SEM pooled from 22 cells in total across two independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001. (D) Representative Western blots of acetylated tubulin in control and KIF22 KD cells. Data are means ± SEM from four independent experiments. **P < 0.005. All data were analyzed for statistical differences using two-way ANOVA.

Cytoplasmic KIF22 coordinates EGFR signaling

Thus far, our data have demonstrated that KIF22 can self-associate via the CCD (Fig. 4, E and F) and that this may occlude the KIF22-CAR binding site within the N terminus (Fig. 4D) and regulate subcellular localization of KIF22 (fig. S3B). We therefore hypothesized that the N terminus of KIF22 may be sufficient to regulate EGFR membrane retention and signaling in response to EGF binding. To investigate this possibility, we depleted KIF22 from A549 cells, reexpressed either FLAG-KIF22-D3 (in which the N terminus lacks the CCD and DBD) or FLAG-KIF22-D5 (in which the C terminus lacks the motor domain) (Fig. 8A), and quantified EGFR abundance at the plasma membrane after 15 min of EGF stimulation. Analysis of confocal images demonstrated clear retention of EGFR at the plasma membrane of cells reexpressing KIF22-D3, but not KIF22-D5, compared to the KIF22-depleted cells within the same field of view (Fig. 8, A and B). Moreover, closer inspection of the localization of KIF22-D3 revealed cytoplasmic microtubule–like association of this mutant and a proportion of plasma membrane–associated KIF22 that showed overlap with EGFR at this location (Fig. 8C). Staining of parallel coverslips or Western blotting of lysates with antibodies to p-EGFR further demonstrated that the KIF22-D3–associated, plasma membrane–localized EGFR was phosphorylated (Fig. 8, D and E), suggesting that cytoplasmic KIF22 is important for sustained EGFR signaling in response to ligand. Our previous data suggested that KIF22 acts upstream of both EGFR and CAR in controlling proliferation. To further confirm this relationship, we performed double knockdown of CAR and KIF22 in A549 cells and analyzed EGFR retention at the membrane in cells reexpressing KIF22-D3. Resulting quantification showed that KIF22-D3 was able to restore membrane-associated EGFR in CAR-depleted cells (Fig. 8E), confirming that KIF22 acts on both EGFR and CAR to ultimately promote efficient cell division.

Fig. 8 Cytoplasmic KIF22 sustains EGFR at the plasma membrane.

(A) Western blots (top) assessing the efficiency of KIF22 KD and reexpression and representative images (bottom) of control (siCtrl) or KIF22 siRNA–treated A549 cells coexpressing FLAG-tagged D3 (N terminus) or D5 (C terminus) truncations of KIF22 and treated with EGF (10 ng/ml) for 15 min, followed by fixation and staining for DAPI (blue), FLAG (green), and EGFR (red). EGFR staining is shown as single-channel images (white) below the merged images; red stars denote FLAG-expressing cells. Scale bar, 20 μm. (B) Quantification of junctional EGFR levels after stimulation with EGF from experiments shown in (A). Data are means ± SEM pooled from at least 40 fields of view across two independent experiments. **P < 0.005. (C) Representative images of KIF22-D3 expressed in KIF22 siRNA–treated A549 cells treated with EGF for 15 min followed by fixation and staining for EGFR (magenta) and KIF22-D3-FLAG (green). White arrows denote areas of colocalized EGFR and KIF22-D3 at the plasma membrane. Scale bar, 10 μm. (D) Representative images of p-EGFR staining in KIF22 siRNA–treated cells reexpressing KIF22-D3-FLAG after EGF treatment (10 ng/ml for 15 min). Cells were fixed and stained for p-EGFR (magenta) and FLAG (green). (E) Representative images of control or KIF22 siRNA–treated CAR KD A549 cells coexpressing FLAG-tagged D3 mutants of KIF22 treated with EGF (10 ng/ml) for 15 min followed by fixation and staining for DAPI (blue), FLAG (green), and EGFR (red). Stars denote channels as described in (A). Graph shows quantification of junctional EGFR abundance after stimulation with EGF. Data are means ± SEM. **P < 0.005. All data were analyzed for statistical differences using two-way ANOVA. Scale bar, 20 μm. (F) Proposed model of KIF22 function on EGFR and CAR upon EGF binding: EGF binding to EGFR drives mitogen-activated protein kinase (MAPK) activation, PKCδ activation, and phosphorylation of CAR. EGFR activation also promotes KIF22-CAR binding and decreases KIF22-dependent microtubule dynamics. Resulting stabilized microtubules promote EGFR retention at the plasma membrane and enhance EGFR signaling.

DISCUSSION

The precise coordination of growth factor and adhesion receptors is essential to facilitate cancer cell division in solid tumors. Data presented in this study reveal a novel requirement for cooperation between two key plasma membrane receptors, CAR and EGFR, in controlling proliferation in tumor cells. We additionally demonstrate a novel role for the chromokinesin KIF22 in coordinating these receptors. Our data support a model whereby KIF22 can act in interphase to regulate microtubule stability and promote EGFR signaling that, in turn, regulates positioning of CAR at the plasma membrane to support efficient progression to cell division (Fig. 8F). Potential functions for KIF22 outside of chromosome movement are poorly studied, and our data provide new insight into previously unexplored roles for KIF22 within the cytoplasm in interphase.

A recent study showed that the putative Drosophila homolog of KIF22 (KLP68D) is involved in synaptic development and neuromuscular junctions (37). Moreover, the same family of kinesins has been implicated in transporting junctional proteins such as E-cadherin as cargo to facilitate adherens junction formation in Drosophila photoreceptors (38). A large-scale mass spectrometry screen recently identified the junctional protein MUPP1 as a putative interaction partner of KIF22, although this interaction has yet to be validated (39). A number of other non-nuclear proteins were also identified as potential interaction partners in this screen, including the adhesion scaffold protein PINCH and EPB41L4B, a regulator of RhoA guanosine triphosphatase activity. This supports our findings and further suggests that KIF22 may form additional interactions outside of the nucleus to control adhesion stability and cytoskeletal dynamics.

KIF22 depletion led to the reorganization of CAR at cell-cell adhesion sites to a more stable, tightly defined contact site. Microtubules can target tight and adherens junctions to regulate dynamics (40, 41); therefore, KIF22 may regulate CAR localization by bridging CAR and the microtubule network to allow junctional repositioning. Mechanical forces are known to influence cell-cell adhesion protein dynamics, and it is plausible that the change in CAR localization may also be due to local tension changes at the junction (42). Moreover, submembranous microtubules can disrupt the actomyosin network, which can increase the diffusion of receptors through membrane regions where actin resistance is low (43). Future studies aimed at understanding how cytoskeletal-dependent mechanical forces at cell-cell adhesion sites influence CAR homodimerization, and how this is regulated by KIF22, will be important to provide further insight into this process.

CAR has two potential binding sites within KIF22: one at the N-terminal region of the protein and the other within the region between the primary motor domain and the CCD. Similar regions in KIF22 are also required for interactions with the check point protein with FHA (forkhead-associated domain) and RING domains (CHFR) (27), suggesting that KIF22 may adopt a certain conformation that exposes these specific sites to promote binding. KIF22 has previously been reported to be a monomer and that the putative CCD is shorter than conventional kinesins that dimerize (44). Our data and those from mass spectrometry analysis (39) contradict this notion and rather suggest that KIF22 can self-associate. A number of other kinesins have been reported to adopt a conformation to induce autoinhibition and prevent microtubule binding (45). Binding data between KIF22 and CAR shown here support this theory, because the C-terminal binding site of CAR is obstructed when the CCD is present, and the CCD can compete self-association of KIF22. It has also been suggested that the CCD modulates the polar ejection force generated by KIF22 and that this, in turn, prevents recongression of chromosomes during anaphase (46). This provides further evidence that CCD is a key regulatory domain within KIF22, affecting its function and potential binding partners.

KIF22 binds to the C terminus of CAR, and that CAR phosphorylation at Thr290/Ser293 occurs in an EGF- and PKCδ-dependent manner, increasing the affinity of KIF22 for the cytoplasmic tail of CAR. EGF stimulation may also promote transient translocation of KIF22 to the plasma membrane to facilitate this interaction. However, KIF22 is required in the nucleus during mitosis, suggesting that EGF stimulation may promote KIF22-CAR complex formation to facilitate CAR relocalization in the short term, after which KIF22 is then shuttled to the nucleus where it acts on chromosome movement. KIF22 is also spatially regulated during the cell cycle through phosphorylation by CDK1 (47), and it is plausible that this may also regulate KIF22 localization into and out of the nucleus. Our data further show that depletion of KIF22 from lung cancer cells decreases EGFR phosphorylation and signaling and increases the rate of EGFR internalization, leading to a marked reduction in EGF-dependent proliferation. Other kinesins have been implicated in later stages of EGFR trafficking [for example, KIF16B modulates the recycling and degradation of EGFR through early endosome regulation (48)]. Nothing is currently known about potential roles for specific kinesin family members in very early stages of EGFR endocytosis. EGFR becomes dephosphorylated by protein tyrosine phosphatases when internalized (32). Therefore, an increase in the rate of internalization also increases the rate at which EGFR is dephosphorylated, supported by our demonstration of enhanced membrane-associated EGFR phosphorylation in cells expressing non-nuclear targeted KIF22. EGFR retention at the plasma membrane is important for the assembly of EGFR signaling complexes, including initiation of MAPK and phosphatidylinositol 3-kinase pathways (33). These pathways are key for promoting cell proliferation, and accelerated internalization of EGFR into endosomes reduces signaling through these pathways. EGFR internalization is widely considered to act as a stop signal for EGFR signaling; however, EGFR has also been reported to retain some activity at endosomes in some cell types (49). Our data suggest that endocytosis does not significantly contribute to EGFR activation or downstream signaling to ERK in A549 cells, suggesting that these cells rely on plasma membrane–associated EGFR for initiating and sustaining signaling after EGF binding. Further analysis will be required to determine the precise mechanism by which KIF22 acts on EGFR to retain signaling activity at the plasma membrane and whether phosphorylation downstream of specific extracellular cues acts to control the subcellular distribution of KIF22.

Our data indicate that KIF22 may have a novel role in microtubule polymerization in cells in interphase. Our analysis of microtubules in both fixed and live cells (Fig. 5) demonstrated that the loss of KIF22 leads to a more spread microtubule network and higher growth rates and reduced dynamic instability in live cells. We also demonstrate that KIF22-depleted cells show higher levels of acetylated tubulin, again indicating increased stability of the network. These three distinct sets of data combined strongly support our conclusions that the loss of KIF22 leads to a net increase in microtubule growth resulting in expansion of the microtubule network toward the plasma membrane. We propose that this expanded microtubule network promotes EGFR internalization and that this notion is supported by our data showing that cytoplasmic, but not nuclear, KIF22 can enhance levels of EGFR at the plasma membrane in KIF22-depleted cells (Fig. 8). Other characterized kinesins that function as depolymerases include the kinesin-8 family. This family of kinesins also has two microtubule binding sites, with the second site located at the C terminus of the protein (50, 51). Therefore, the putative second microtubule binding site of KIF22 identified may be aiding depolymerase activity of KIF22 identified in this chapter (44). Increasing evidence suggests the importance of microtubule stability on EGFR internalization. It has been shown that microtubules facilitate the diffusion of EGFR clusters at the plasma membrane (52). Microtubule stabilization by acetylation in EGFR endocytosis is also controlled through the activity of the tubulin deacetylase HDAC6 that negatively regulates EGFR endocytosis through modulation of microtubule stability and transport of the receptor along microtubules (24). Moreover, microtubule-targeting drugs that disrupt the tubulin network decrease the phosphorylation of EGFR and downstream signaling in esophageal cancer (53). It has been proposed that blocking the addition of tubulin subunits can increase catastrophe by enabling guanosine 5′-triphosphate hydrolysis to reach the plus end of the microtubules (54). Within 5 min of EGFR activation, microtubules are stabilized, possibly in preparation for EGFR internalization and trafficking. Longer-term EGFR signaling drives an increase in KIF22 expression, and this may be a feedback loop to rectify the microtubule network through the depolymerase activity of KIF22.

In summary, we have shown that KIF22 is an important, previously unreported regulator of both EGFR and CAR dynamics in human cancer cells. We postulate that these three molecules act in concert to control efficient EGF-dependent proliferation in lung cancer cells that may ultimately promote CAR- and EGFR-dependent tumorigenesis. Future experiments will be aimed at understanding the mechanisms and spatiotemporal events controlling the assembly of the CAR-KIF22 complex with a view to using blockade of this complex as a potential route for new therapeutic intervention in solid lung tumors.

MATERIALS AND METHODS

Antibodies and reagents

Anti-CAR (H300), anti-HSC70, and anti-PKCδ antibodies were from Santa Cruz Biotechnology. p-CAR Thr290/Ser293 polyclonal antibody was previously described (7), developed by Perbio Science (Thermo Fisher Scientific) using the peptide Ac-RTS(pT)AR(pS)YIGSNH-C, and affinity-purified before use. Anti–β-tubulin, anti-acetylated tubulin, and anti-FLAG antibodies were from Sigma-Aldrich. Anti–phospho-EGFR (Y1173), anti–phospho-ERK (T202/Y204), anti-EGFR, and anti-ERK antibodies were from Cell Signaling Technology. Anti-KIF22 antibody was from GeneTex, anti–glyceraldehyde-3-phosphate dehydrogenase was from Chemicon. Anti-GFP antibodies were from Roche (immunoblotting) and MBL (IP). Anti–Ki-67 and anti–phospho-H3 antibodies were from Leica Biosystems. Anti–HaloTag antibody and HaloTag direct ligand were from Promega. Anti-mouse horseradish peroxidase (HRP) and anti-rabbit HRP were from Dako. Anti-mouse 568, anti-rabbit 568, and phalloidin 647 were all obtained from Invitrogen. Recombinant human EGF was acquired from PeproTech, and calyculin A, sodium orthovanadate, and protease inhibitor cocktail 1 were obtained from Calbiochem. Nocodazole and dynasore were from Sigma-Aldrich. KIF22-targeted siRNAs were from OriGene, and CAR and nontargeting siRNA were acquired from Dharmacon. PKCδ-targeting siRNAs were from Ambion.

Plasmids

Full-length and mutant CAR sequences were cloned in-frame into pHR9SIN-SEW lentiviral expression vector, which was a gift from A. Thrasher (Institute of Child Health, University College London), and into pGEX-2T. Phospho-mutant CAR constructs were generated using site-directed mutagenesis and have been described previously (7). Control and CAR knockdown shRNA vectors (shA and shB) were in pLKO.1 backbones purchased from Sigma MISSION collection (clone ID NM_001338). H2BK-GFP and mCherry plasmids and β-tubulin–GFP were gifts from J. Monypenny (King’s College London). FLAG-tagged KIF22 constructs were a gift from M. Subba Reddy [Centre for DNA Fingerprinting and Diagnostics (CDFD) (27)]. The complementary DNAs (cDNAs) encoding the GST-NT- KIF22 and GST-CT-KIF22 domains were provided by A. Wilde (University of Toronto). The CCD of human KIF22 was cloned in pHTC HaloTag CMV-neo Vector (a gift from M. Dodding, King’s College London) between Nhe I and Xho I sites. The following primers were used for polymerase chain reaction: 5′-AAAGCTAGCATGGACCGTCTGCTTGCCTC-3′ (sense) and 5′-CTCGAGTTGATCCAGTATTTTTTGGCGCC-3′ (antisense).

Cell culture

A549 human lung adenocarcinoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells expressing shRNA to target CAR were maintained in DMEM containing 10% FCS and puromycin (1.2 μg/ml). Wild-type H1975 adenocarcinoma cells were grown in RPMI 1640 medium with 10% FCS, puromycin (1.5 μg/ml), and G418 (1 mg/ml). HEK293 packaging cells were used to generate lentiviral particles for viral transduction as previously described (7). 16HBE human bronchial epithelial cells were a gift from D. Gruenert [University of Vermont (55)] and cultured in MEM containing 10% FCS and supplemented with glutamine. All CAR-expressing stable cell lines were produced using lentiviral expression as previously described (7, 26).

Confocal microscopy

Cultured cells were washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) in PBS for 10 min or ice-cold methanol for 2 min, and permeabilized with 0.1% Triton X-100 for 10 min. Cells were incubated with primary antibodies for 2 hours and with appropriate secondary antibodies conjugated to Alexa Fluor 568 or 488 and phalloidin conjugated to Alexa Fluor 647, including Hoechst, for 1 hour. Cells stained using HaloTag TMR Direct Ligand were incubated live with the ligand overnight before fixation. Cells were mounted onto slides using FluorSave (ICN). Confocal microscopy was performed using a Nikon A1R inverted confocal laser scanning microscope with a 60× oil objective and laser excitation wavelengths of 405, 488, 561, and 633 nm.

Analysis of microtubule area

Images were all taken at the same laser settings and objectives using Nikon Imaging Software Elements and the same Nikon A1R confocal microscope. Actin, β-tubulin, and nuclei channels were saved separately as TIFF files. These images were analyzed using ICY software; images were thresholded to remove background, and the nucleus signal was merged with both the actin and β-tubulin signals. Masks were produced of these merged images and overlaid, after which the difference between the two masks was calculated to determine the number of pixels representing the actin signal without a corresponding β-tubulin signal. This value was converted to square micrometers to determine the surface area per field of view with the presence of actin present and absence of β-tubulin.

Live imaging

For live-cell imaging experiments, A549 were plated onto six-well plastic plates (cell division assay) or glass-bottomed dishes (CAR junctional analysis and microtubule tracking) (Ibidi). For cell division assays, Hepes (25 mM) was added to the cells that were then imaged every 5 min for 12 hours using phase-contrast imaging and 488- and 568-nm laser excitation using a 20× objective on an Olympus IX71 inverted fluorescence microscope equipped with a humidified environmental chamber heated to 37°C. All images were saved as avi files. For CAR junctional analysis, cells were imaged every minute using 488-nm laser excitation using a 60× oil objective on a Nikon A1R inverted confocal microscope (Nikon) equipped with a humidified environmental chamber heated to 37°C, with perfect focus system activated. After 5 min, EGF was added to the imaging media at a final concentration of 10 ng/ml, and the imaging was immediately resumed for a further 60 min. All images were saved as nd2 files and analyzed in ImageJ or exported as tif files for presentation. For microtubule tracking assays, time-lapse movies of A549 cells expressing tubulin-GFP were acquired on an inverted Nikon A1R confocal laser scanning microscope using a 100× oil objective (numerical aperture, 1.45) at a rate of one frame per 2 s with or without EGF addition (10 ng/ml). Images were saved as nd2 files and analyzed in ImageJ or exported as tif files for presentation.

Quantification of live imaging

To analyze cell division, the beginning of division was determined by morphological rounding of cells using phase-contrast imaging before DNA condensation determined by the H2BK-GFP or mCherry signal. The number of frames until the end of DNA separation or complete cell division (cell spreading of daughter cells) was quantified and used to determine the overall time of cell division. Cells were considered separated if less than a third of the cell boundaries of the daughter cells were attached to each other; this was analyzed up to 90 min after nuclear division. To analyze CAR localization at junctions, videos were opened in ImageJ and line scans (4.2 μm) were drawn perpendicular to junctions. The intensity profile of CAR-GFP was then calculated; 30 junctions were analyzed per condition, which were then averaged and normalized. To analyze CAR-GFP and EGFR-mCherry dynamics in live cells, identically sized region-of-interest boxes were placed over central (5 μm wide) junctional regions at time 0, and the resulting intensity measurements over time were calculated for both GFP and mCherry channels. These values were then exported into GraphPad Prism and normalized to the starting intensity for each junction analyzed to provide relative intensity changes over time. For analysis of single microtubules at the cell periphery (microtubule tracking assay), acquired movies were subjected to a band-pass filter (20:2 pixels) in ImageJ (background-subtracted using a rolling ball radius of 15 pixels), and a three-dimensional Gaussian blur filter was applied. Resulting movies were overlaid with the originals to avoid image processing–derived artifacts, and single microtubule growth was measured over time from a defined starting point proximal to the cell periphery. Frequency of catastrophe, growth rate, and time spent in growth phase were quantified as in (56). Example representative images of time-dependent changes to microtubule dynamics were generated using the Temporal Color Code plug-in in FIJI (Centre for Molecular and Cellular Imaging, European Molecular Biology Laboratory, Germany).

Western blotting and IP

Cells were lysed in sample buffer containing β-mercaptoethanol at room temperature. Lysates were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and blotted using nitrocellulose membrane. Blots were blocked and probed using 3% milk/PBS–0.2% Tween 20 or 5% bovine serum albumin (BSA)/tris-buffered saline (TBS)–0.1% Tween 20 and quantified using ECL Plus Western blot detection system (GE Healthcare). For IP experiments, GFP, CAR-GFP A549, or wild-type A549 expressing FLAG-tagged constructs were lysed in IP lysis buffer [50 mM tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% NP-40, and protease inhibitor cocktail]. Lysates were incubated with 5 μg of anti-GFP or anti-FLAG antibody prebound to A/G agarose beads overnight before washing the beads with 1 ml of IP lysis buffer three times. Immunocomplexes were separated using SDS-PAGE and immunoblotted for specified proteins. Where relevant for quantification purposes, phospho-proteins levels were normalized to levels of the same total protein before comparing between conditions.

Biotinylation assay

Cells were incubated for 40 min at 4°C with EZ-Link Sulfo-NHS-SS-Biotin solution (0.5 mg/ml in PBS; Thermo Fisher Scientific). Surface biotinylation was then quenched with PBS containing 50 mM glycine. To initiate EGFR internalization, cells were incubated in Opti-MEM containing EGF for the specified time periods at 37°C and then cell surface biotin was stripped using MesNa buffer [50 mM 2-mercaptoethanesulfonic acid sodium salt (Sigma-Aldrich)] for 1 hour at 4°C. One sample of cells was left unstripped as a control for surface labeling efficiency. Cells were then lysed with radioimmunoprecipitation assay buffer containing protease inhibitor cocktail and phosphatase inhibitors (100 μM vanandate and 1 μM calyculin A). Biotinylated proteins were then isolated by pull-down using NeutrAvidin Agarose resin (Thermo Fisher Scientific) overnight at 4°C. Samples were then boiled with equal volumes of SDS-PAGE sample buffer and analyzed by immunoblotting. The level of internalized EGFR at different time points was determined as the percentage of difference between non-MesNa stripped cells and stripped cells and corrected for any variations of the total cell lysate protein of each sample.

Proliferation assays

A549 or A549s expressing H2BK-GFP cells (5 × 103) were plated into 12-well tissue culture plates and incubated for 24 or 48 hours. Wild-type H1975 EGFR cells (8 × 103) were plated into a sterile 12-well tissue culture plate and incubated for 24, 48, or 72 hours. Wild-type 16HBE or 16HBE stable cell lines (8 × 104) expressing CAR-GFP or transfected with CAR-targeted siRNA were plated into a sterile 12-well tissue culture plate and incubated for 24 or 48 hours. Hoechst was added to the medium of the cells for 30 min to stain DNA and then cells were fixed for 15 min in 4% PFA/PBS in the dark. Using a 10× objective on an Olympus IX71 inverted fluorescence microscope, cells were imaged using the same exposure with the 406-nm laser and six images per well were saved as TIFF files. H2BK-GFP A549s were grown for 24 hours, and using an Olympus IX71 inverted fluorescence microscope (10× objective), cells were imaged live using the same exposure with the 488-nm laser, six images per well were saved as TIFF files, and then the cells were incubated for a further 24 hours, after which they were imaged again. TIFF files were opened in ImageJ, and the number of nuclei per image was analyzed by thresholding the nuclei followed by selecting on the basis of size and circularity to determine the number of particles. The number of particles was counted per field, enabling the total number of nuclei to be calculated across multiple fields.

Agar colony assay

Agar plates were prepared by mixing complete growth medium with 2% noble agar to produce a solution of 0.7% agar. Agar solution (1.5 ml) was added to a well of a sterile six-well tissue culture plate and incubated at room temperature for 30 min to set. A549 (4 × 104 cells per well) and wild-type H1975 (1 × 105 cells per well) were mixed with preheated 2% noble agar to make a 0.3% agar solution, and 1.5 ml of the solution was added to each well on top of the bottom agar layer. One milliliter of fresh growth medium was then added over the top of the agar solution and replaced every 2 to 3 days. A549 cells were grown for 2 weeks, and wild-type H1975 cells were grown for 3 weeks, after which colonies were fixed and stained for 30 min in a 0.5% crystal violet/20% methanol/PBS. Colonies were imaged on an Olympus IX71 inverted fluorescence microscope (4× objective) and analyzed using ImageJ. Colony number and size were calculated in ImageJ.

Recombinant protein production and pull-downs

The cDNAs encoding the GST-NT-KIF22 and GST-CT-KIF22 domains were provided by A. Wilde (University of Toronto). BL21 cells containing DNA of interest were grown in 400 ml of LB broth at 37°C and then protein production was induced with isopropyl β-d-1-thiogalactopyranoside (100 μM) and incubated for a further 4 hours at 30°C. The bacterial culture was pelleted and frozen overnight at −80°C and then lysed in 50 μl of ice-cold PBS per milliliter of original culture volume containing protease inhibitors (Calbiochem). Bacterial pellets were disrupted by sonication for 2 min using 10-s pulses at 10 A on ice. The solution was centrifuged, and the supernatant was collected and filtered using a 0.45-μm filter. Prewashed glutathione Sepharose 4B beads (GE Healthcare) were added to the supernatant to 1 μl of beads/1 ml of original culture and left to mix overnight at 4°C. The beads were washed twice with PBS containing 0.4 M NaCl and 1% Brij and resuspended in 1 ml of buffer with protease inhibitors. SDS-PAGE and Coomassie blue analysis were run to confirm protein purification.

A549 or HEK293T cells were washed and scraped into cold lysis buffer [50 mM tris (pH 7.2), 150 mM NaCl, 20 mM EDTA, 1% NP-40, and 1% Triton X-100] containing protease inhibitor cocktail and phosphatase inhibitors (100 μM vanandate and 1 μM calyculin A). Lysates were then centrifuged to pellet insoluble material. A sample of cell lysate was removed before adding lysate to beads to allow analysis of total protein. The cleared lysates were transferred into tubes with 50 μl of GST-tagged protein and placed on a rotator at 4°C overnight. The beads were washed four times with ice-cold lysis buffer. After the final wash, the wash buffer was removed and SDS sample buffer was added. The sample was boiled and run on a 12% acrylamide gel and analyzed by immunoblotting.

In vivo tumorigenicity assays

Wild-type H1975 control (siCtrl) or CAR knockdown (siCAR-1 and siCAR-2) cell lines (3 × 106) were injected subcutaneously into the two posterior flanks of BALB/c nude mice (Charles River Laboratories). Nine 5-week-old female mice in total were used for control cell lines and five for each siRNA. Mice were followed daily, tumors were measured with a caliper in long and short axes, and volume was determined on the basis of the equation 0.4 × A × B2 (where A is the long axis of the tumor and B is the short axis of the tumor). At day 14 after injection, mice were culled using CO2 and tumors were removed aseptically with dissecting scissors and weighed. All animals were maintained under specific pathogen–free conditions and handled in accordance with the Institutional Committees on Animal Welfare of the UK Home Office Animals (Scientific Procedures) Act 1986. All animal experiments were approved by the Ethical Review Process Committee at King’s College London and carried out under license from the Home Office, UK.

Tissue samples, immunohistochemistry, and histopathological analysis

Tissue samples obtained from xenografts were fixed with formalin and paraffin-embedded. Immunohistochemical analyses were performed using 3-mm sections. Briefly, slides were dewaxed and antigen retrieval was performed using 0.1 M citrate buffer (pH 6.0) at 120°C for 10 min, followed by blocking in TBS–0.1% Tween 20 + 1% BSA + 1% fetal bovine serum. Primary antibodies were added overnight at 4°C. Peroxidase-conjugated (EnVision+) anti-rabbit and anti-mouse immunoglobulin reagents from Dako were used as secondary antibodies for 1 hour. Nonimmune (Santa Cruz Biotechnology) or preimmune rabbit serum was used as negative controls. Reactions were developed using diaminobenzidine as chromogenic substrate. Images from digitalized scans of the glass slide specimens were obtained at a ×20 magnification (0.45 μm/pixel resolution) using a Hamamatsu NanoZoomer 2.0 HT. All quantification was performed in ImageJ.

Statistical analysis

All statistical tests were performed using Students t tests or two-way analysis of variance (GraphPad Prism). Correlation analysis was performed using Pearson’s correlation coefficient (ImageJ).

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/11/515/eaaq1060/DC1

Fig. S1. CAR regulates proliferation in H1975 cells.

Fig. S2. CAR does not regulate EGFR signaling or degradation in response to EGF.

Fig. S3. KIF22 binds to CAR.

Fig. S4. KIF22 regulates cell proliferation.

Fig. S5. Blocking endocytosis does not alter A549 cell EGFR signaling.

REFERENCES AND NOTES

Acknowledgments: We thank M. Subba Reddy (CDFD, Hyderabad, India) and A. Wilde (University of Toronto) for the gifts of KIF22 plasmids. Funding: The authors acknowledge funding from King’s Health Partners, National Institute for Health Research (NIHR) Clinical Research Facility and NIHR Biomedical Research Centre based at Guy’s and St. Thomas’ National Health Service Foundation Trust, King’s College London, and the Biotechnology and Biological Sciences Research Council (BB/M503320/1). Author contributions: M.P. and G.S. conceived the project. R.P. conducted experiments and analyzed the data with help from B.L. E.O.-Z. conducted the xenograft studies and tissue analysis. M.P. wrote the manuscript with input from all other authors. All authors contributed to experimental design and had intellectual input into the project planning. Competing interests: The authors declare that they have no competing interests.
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