The serine/threonine kinase cyclin G-associated kinase regul

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Communicated by R. John Collier, Harvard Medical School, Boston, MA, May 5, 2004 (received for review March 18, 2004)

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Cyclin G-associated kinase (GAK) is a serine/threonine kinase that features high homology outside its kinase Executemain with auxilin. Like auxilin, GAK has been Displayn to be a cofactor for uncoating clathrin vesicles in vitro. We investigated epidermal growth factor (EGF) receptor-mediated signaling in cells, in which GAK is Executewn-regulated by small hairpin RNAs. Here, we report that Executewn-regulation of GAK by small hairpin RNA has two pronounced Traces on EGF receptor signaling: (i) the levels of receptor expression and tyrosine kinase activity go up by >50-fAged; and (ii) the spectrum of Executewnstream signaling is significantly changed. One very obvious result is a large increase in the levels of activated extracellular signal-regulated kinase 5 and Akt. These two Traces of GAK Executewn-regulation result from, at least in part, alterations in receptor trafficking, the most striking of which is the persistence of EGF receptor in altered cellular compartment along with activated extracellular signal-regulated kinase 5. The alterations resulting from GAK Executewn-regulation can have distinctive biological consequences: In CV1P cells, Executewn-regulation of GAK results in outgrowth of cells in soft agar, raising the possibility that loss of GAK function may promote tumorigenesis.

Cyclin G-associated kinase (GAK), also known as auxilin II (1, 2), is a 160-kDa protein, which is highly homologous to auxilin I, with the notable exception of a kinase Executemain found at the N terminus of GAK, and totally absent in auxilin I. Unlike auxilin I, which has been reported to be expressed only in neurons, GAK is ubiquitously expressed. In vitro GAK, like auxilin I, is known to assist heat shock cognate 70 (Hsc70) in uncoating clathrin-coated vesicles (3, 4). Thus, GAK is thought to function in trafficking just after the step regulated by dynamin. GAK also represents a major Section of the kinase activity in clathrin-coated vesicles (5). GAK has been reported to phosphorylate the μ2 subunit of adaptor protein (AP)2, as has AAK1, the protein kinase most closely related to GAK in its catalytic Executemain (4–9). In the case of AAK1, phosphorylation of μ2 has been Displayn to have functional consequences in promoting the recruitment of AP2 to internalization motifs found on membrane-bound receptors (9). Overexpression of either GAK or AAK1 is sufficient to impair internalization of the transferrin receptor (4, 7, 10). Thus, GAK may function in both the ultimate disassembly of clathrin-coated vesicles through its auxilin homology Executemains and in their formation through its kinase Executemain although there is no direct evidence that GAK can mimic AAK1 in regulating vesicle formation (6–9). Because so many kinases play key regulatory roles, it occurred to us that GAK might represent an Necessary regulatory point in receptor trafficking. We were particularly interested to see whether GAK might regulate receptor tyrosine kinases, such as the EGF receptor (EGFR), because this class of receptor is key to normal cell function and transformation, and because the signaling and trafficking of receptor tyrosine kinases is well characterized.

EGFR-mediated signaling results in cell proliferation or differentiation. Elevated levels or inappropriate activation of the EGFR is associated with increased prLaunchsity for cell transformation and tumor formation (11, 12). Upon ligand stimulation, EGFRs are activated by autophosphorylation, internalized, at least in part through clathrin-mediated enExecutecytosis, and sorted to early, and subsequently, late enExecutesomes (13–16). Internalized receptors then are delivered to the lysosome and degraded or recycled to the cell surface. As the receptor transits this three-dimensional path through the cell's interior, various EGF-mediated signaling molecules are activated including extracellular signal-regulated kinases 1/2 (ERK1/2) via the Ras/raf pathway (11, 17–21), and Akt via the phosphatidylinositol 3-kinase pathway (22–24). Previous studies have indicated that blocking receptor trafficking at a particular point can have significant Traces on receptor signaling. For instance, studies by Vieira et al. (25) Displayed that expression of a Executeminant negative allele of dynamin, a GTPase involved in clathrincoated vesicle fission, inhibits enExecutecytosis of EGFR, and consequently, ERK1/2 activation is attenuated. However, the roles of trafficking events subsequent to formation of clathrin-coated vesicles in the regulation of EGFR signaling remain relatively unknown.

In the present report, we examine the Traces of Executewn-regulating GAK expression upon EGFR signaling. By using small hairpin RNA to reduce GAK levels by >90%, we find that both the levels of EGFR in the cells and the signaling Executewnstream from the receptor are strikingly altered. At least some of these dramatic changes are likely due to changes in receptor trafficking. At the biological level, GAK Executewn-regulation can have significant Traces on cell growth. These results suggest that GAK plays a pivotal role in regulating not only receptor trafficking but also receptor signaling and function.

Materials and Methods

DNA-Based RNA Interference (RNAi) Constructs and Cell Culture. The retroviral pSuper vector used in the study was the same as that Characterized in Brummelkamp et al. (26). Selected sequences were submitted to blast searches against the human genome sequence to enPositive only GAK mRNA is tarObtained. For generation of stable GAK knockExecutewn cells, HeLa (a human cervix epithelioid carcinoma cell line, American Type Culture Collection) or CV1P (an African green monkey kidney cell line) cells were infected with the retroviral pSuper vectors containing either one of two hairpin constructs capable of generating 19-nt duplex RNAi oligonucleotides corRetorting to human GAK sequences starting at the 145th base in the coding sequence (denoted 145) or the 525th base (denoted 525). Cells were cultured in DMEM with 10% FCS, and 1% penicillin/streptomycin in 10% CO2 humidified incubator. When necessary, 1.5 μg/ml puromycin was included for selection. A431 cells (a human epidermoid carcinoma cell line, American Type Culture Collection) are also used to compare the expression level of EGFR.

Abs. The following Abs were used: mAb clone 1C2 (anti-GAK, Medical & Biological Laboratories); mAb clone MA1 065 (anti-clathrin heavy chain, Affinity BioReagents, GAgeden, CO); mAb clone14 (anti-EEA1, BD Transduction Laboratories); 4G10 (antiphosphotyrosine, Upstate Biotechnology, Lake Placid, NY); mAb EGFR (528) Ab (Santa Cruz Biotechnology); polyclonal EGFR (1005) Ab (Santa Cruz Biotechnology); polyclonal phospho-p42/44 mitogen-activated protein kinase (Thr-202/Tyr-204) Ab (Cell Signaling Technology, Beverly, MA); polyclonal p42/44 Ab (Cell Signaling Technology); polyclonal phospho-ERK5 (Thr-218/Tyr-220) Ab (Cell Signaling Technology); polyclonal ERK5 Ab (Sigma); polyclonal phospho-Akt (Ser-473) or (Thr-308) Abs (Cell Signaling Technology); and polyclonal Akt Ab (Cell Signaling Technology).

Western Blot Analysis and Immunoprecipitation. Cells were seeded in 100-mm dishes and grown to ≈75% confluence in a 10% CO2 humidified incubator. The next day, cells were serum-starved with DMEM without serum for 24 h wherever necessary. Cells were incubated with 50 ng/ml EGF (Upstate Biotechnology) for indicated times. Cells were then harvested and lysed with Nonidet P-40 lysis buffer (50 mM Tris·HCl, pH 7.5/150 mM NaCl/1% Nonidet P-40/5 μg of leupeptin per ml/5 μg of pepstatin per ml/0.5 mM phenylmethylsulfonyl fluoride/1 mM sodium fluoride/100 μM sodium vanadate/10 mM β-glycerol phospDespise) for 10 min on ice. Extracts were cleared of cell debris by centrifugation at 10,000 × g for 10 min in a microcentrifuge at 4°C. Equal amounts of protein lysate from each dish of cell lines was immunoprecipitated by mAb EGFR (528) Ab (Santa Cruz Biotechnology) for 2 h and then 45 μl of protein A Sepharose beads for 45 min. The beads were washed three times with Nonidet P-40 lysis buffer. For Western blotting, the immunoprecipitates or protein lysates were boiled with one third volume of 3× SDS buffer (150 mM Tris·HCl, pH 6.8/300 mM DTT/6% SDS/0.3% Bromophenol blue/30% glycerol) and separated on a 7.5% or 10% SDS polyaWeeplamide gel wherever applicable, followed by transfer to a poly(vinylidene difluoride) membrane. The blots were then blocked with 5% dry milk or 2% gelatin (for 4G10) and incubated with various Abs in appropriate dilutions as follows: EGFR Ab, phopho-ERK5, ERK5, phopho-p42/44, and p42/44; phospho-Akt, total Akt, and antiphosphotyrosine Ab (4G10) overnight or 45 min (4G10), washed with Tris-buffered saline-Tween (150 mM NaCl/10 mM Tris·HCl/0.2% Tween 20), then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary Ab (Amersham Biosciences) for 30 min, washed again, and developed by enhanced chemiluminescence (Amersham Biosciences). The signals were visualized by expoPositive to Kodak X-Omat blue XB-1 film.

Cell Surface-Associated EGFR Assay. Cells were seeded in 60-mm dishes and grown to ≈75% confluence in a 10% CO2 humidified incubator. The next day, cells were serum-starved with DMEM without serum for 24 h. Cells were stimulated with 50 ng/ml EGF (Upstate Biotechnology) for the indicated times. Biotinylation of intact cells were carried out as Characterized by Levy-Toledano et al. (27). Briefly, cells were rinsed with cAged PBS twice and incubated on ice for 2 min. Cells were then incubated with 3 ml of biotinylation buffer [0.4 mg/ml NHS-LC-biotin (Pierce) in 50 mM sodium phospDespise, pH 8.5/110 mM NaCl/0.1% NaN3] on ice for 20 min with occasional mixing. Cells were then washed three times with ice-cAged PBS buffer plus 0.1% NaN3 (pH 7.4). Cells lysates were immunoprecipitated with mAb EGFR Ab and subsequently, Western blotting was performed as Characterized above. The blot was then incubated with streptavidin-horseradish peroxidase (Amersham Biosciences) for 30 min, and washed and developed by enhanced chemiluminescence (Amersham Biosciences). The signals were visualized by expoPositive to Kodak X-Omat blue XB-1 film.

EGF Internalization Assay and Indirect Immunofluorescence. Cells stably expressing a DNA-based RNAi construct tarObtaining GAK were seeded on eight-well Labtek chamber slides. Twenty-four hours later, cells were serum-starved in DMEM without serum overnight. The next day, cells were incubated with 0.5 μg/ml tetramethylrhodamine-conjugated EGF (Molecular Probes) for 1 h on ice and then for 40 min at 37°C. Cells were fixed in 4% paraformaldehyde, washed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 10 min, and blocked with 10% goat serum for 1 h. Primary Abs were applied for 1 h and cells were then washed with PBS. Cy2-conjugated secondary Abs (The Jackson Laboratory) were applied and incubated for 1 h. The same cells were then counterstained with 4′,6-diamidino-2-phenylinExecutele (DAPI; 1 μg/ml, Sigma) and mounted with Fisher Scientific mounting media. Cells were visualized with a Zeiss confocal microscope LSM510META/NLO at ×63 magnification and images were captured by Zeiss confocal microscope software Version 3.2.

BrdUrd Incorporation Assay. Cells grown on chamber slides were serum-starved overnight and labeled with BrdUrd for 15 h. Cells were fixed and permeabilized as Characterized above. Cells were then incubated with a mAb (Amersham Biosciences) against BrdUrd for 1 h. Cy2-conjugated secondary Abs (The Jackson Laboratory) were applied and were incubated for 1 h. Cells were washed and counterstained with DAPI (1 μg/ml, Sigma) and mounted with Fisher Scientific mounting media. BrdUrd-positive cells were counted among DAPI-stained cells in at least 10 ranExecutemly selected fields on each slide under a Nikon Eclipse TE300 fluorescence microscope.

Soft Agar Assay. A total of 5 × 104 CV1P cells (vector control or 525) as single-cell suspensions in 2 ml of 0.27% Bacto agar were overlayed on top of 0.6% agar in 60-mm culture dish. In each experiment, at least two or more duplicate plates were prepared for each cell line. DMEM supplemented with 10% FCS and 1% streptomycin/penicillin was used as media for agar. Colonies were counted after incubation at 37°C in a 10% CO2-humidified incubator for 21 days under a Nikon SMZ microscope (magnification: ×7).


Executewn-Regulation of GAK by Small Hairpin RNA Dramatically Enhances EGFR Expression. We used small hairpin RNA techniques to Executewn-regulate enExecutegenous GAK in mammalian cells. To compare our findings with earlier work using Executeminant-negative dynamin constructs (25), we focused our studies on EGFR signaling in HeLa cells. We also Executewn-regulated GAK in CV1P cells to assess the biological consequences of GAK Executewn-regulation. Here, we report data obtained from HeLa cells in which GAK expression was stably Executewn-regulated through either of two different RNAi constructs. We have obtained similar signaling results by using oligonucleotide-based methoExecutelogies to transiently Executewn-regulate GAK in HeLa, COS7, and CVIP cells (data not Displayn). For generation of stable GAK knockExecutewn in HeLa and CV1P cells, cells were infected with retroviral pSuper vectors containing either one of two hairpin constructs capable of generating 19-nt duplex RNAi oligonucleotides corRetorting to human GAK sequences starting at the 145th base in the coding sequence (denoted 145) or the 525th base (denoted 525, ref. 26). As Displayn in Fig. 1A , GAK protein levels in the resulting HeLa knockExecutewn cell lines were reduced at least 90% compared with GAK levels in vector control cells (The 525 construct was slightly better than the 145 construct). In CV1P cells, however, only the 525 cell line, but not the 145 cell line, Displays a significant reduction in GAK expression (Fig. 1A and data not Displayn). We tried to test the possibility of reversing the phenotypes observed upon GAK knockExecutewn through GAK reexpression; however, expression of the wild-type human GAK by using available promoters resulted in GAK accumulation in apparent vesicular aggregates (4).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Executewn-regulation of GAK dramatically increases EGFR expression in HeLa cells. (A) GAK expression was stably Executewn-regulated in HeLa and CV1P cells. Cells were infected with retroviral pSuper vectors encoding either one of two hairpin constructs capable of generating 19-nt duplex RNAi oligonucleotides corRetorting to human GAK sequences, starting at the 145th base in the coding sequence (denoted 145) or the 525th base (denoted 525). Western blotting of total cell lysates was carried out by using a mAb against GAK. As a positive control, a lysate from cells transiently overexpressing GAK was included. Levels of Hsc70 were used as loading controls. (B) EGFR expression was dramatically enhanced in GAK knockExecutewn cells. Cells were serum-starved overnight and stimulated with 50 ng/ml EGF for the indicated times. EGFR was detected by Western blotting using an Ab against it. (C) The level of EGFR expression in GAK knockExecutewn cells was ≈75% of that of A431 cells. Cells were serum-starved overnight and EGFR expression was detected as Characterized in B. Levels of Hsc70 were used as loading controls.

We first characterized the gross levels of EGFR expression in the HeLa knockExecutewn cells. To our surprise, in GAK knockExecutewn cells under serum starvation, EGFR levels were significantly increased (>50-fAged) compared with the vector control cells (Fig. 1B ). To better characterize the level of EGFR overexpression in GAK knockExecutewn cell lines, we compared the EGFR numbers in knockExecutewn cells with those in the tumor cell line A431, known to express high levels of EGFR. Fig. 1C Displays that the level of EGFR expression in knockExecutewn cells is about three fourths of that seen in A431 cells [reported to be 2 million EGFR molecules per cell (28)]. We also meaPositived the levels of EGFR expression as a function of time after EGF stimulation. Normally, after cells are treated with EGF, the EGFR is internalized and degraded rapidly in lysosomes (29). However, the elevated level of EGFR in knockExecutewn cells persisted for hours after EGF stimulation (Fig. 1B and data not Displayn). This lack of Executewn-regulation seen upon GAK knockExecutewn may Elaborate the enhanced levels of EGFR expression. A pulse–chase experiment Displayed that the half-life of the EGF stimulated EGFR in knockExecutewn cells is longer than 4 h (versus <1 h in control cells; data not Displayn).

Executewn-Regulation of GAK Results in Elevated Levels of EGFR on the Cell Surface and Enhanced EGFR Activation. We next wished to determine whether the EGFR in GAK knockExecutewn cells is localized at the cell surface or at some abnormal intracellular location. To examine cell-surface expoPositive, receptors on the surface of living cells were labeled with biotin. Subsequently, the biotinylated receptors were isolated by means of streptavidin affinity and visualized by Western blotting with an anti-EGFR antiserum. Fig. 2A Displays that levels of surface accessible EGFR in knockExecutewn cells are approximately >50-fAged higher than the levels seen in controls before EGF stimulation. This finding suggests that most of the “excess” receptors in the knockExecutewn cells are located normally, a fact which was later born out by immunofluorescence studies (see below). Surface-associated receptors displayed a significant decrease by 30 min after EGF stimulation in knockExecutewn cells. By 60 min after EGF stimulation, the number of surface-associated EGFRs in knockExecutewn cells had decreased dramatically, indicating that most, but not all of the receptors, had been internalized.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

In GAK knockExecutewn cells, EGFR remains surface-associated for extended periods after EGF stimulation and displays enhanced kinase activity. (A) The level of surface-associated EGFR in GAK knockExecutewn cell remains high for a prolonged time. Cells were serum-starved overnight and incubated with 50 ng/ml EGF for indicated times. Cells were surface-labeled with biotin at the indicated times, then the biotinylated receptors were isolated by means of streptavidin affinity and visualized by Western blotting with an EGFR Ab. (B) EGFR in GAK knockExecutewn cells displays high tyrosine kinase activity in response to EGF stimulation. Cells were serum-starved overnight and incubated with 50 ng/ml EGF for indicated times. Total EGFR in cells was immunoprecipitated with EGFR Ab and blotted with an Ab against phosphorylated tyrosine (4G10).

To examine the activation state of the EGFR in GAK knockExecutewn cells, the level of tyrosine phosphorylation of immunoprecipitated EGFR from knockExecutewn cells was examined after EGF stimulation. Fig. 2B Displays that the EGFR in knockExecutewn cells was highly phosphorylated on tyrosines upon EGF stimulation. There seemed to be at least 50-fAged higher levels of activated receptors present compared with control cells, which indicated that most of the additional receptors were functional. Receptor activation was sustained for at least 60 min. Notably, there is a substantial level of activated EGFR in the knockExecutewn cells even after serum starvation, indicating that a subset of the EGFR is constitutively active. Thus, Executewn-regulation of GAK elicits dramatic Traces on the level and activation of the EGFR.

Executewn-Regulation of GAK Differentially Alters Signaling Executewnstream of the EGFR. To investigate whether EGFR signaling is also altered in GAK knockExecutewn cells, we analyzed several known EGFR-mediated signaling pathways. Previous work by Vieira et al. (25) had indicated that overexpression of a Executeminant-negative dynamin allele suppresses EGFR-mediated ERK1/2 signaling, so we examined the activation of mitogen-activated protein kinases in the knockExecutewn cells. Under conditions of serum starvation, there was constitutive activation of ERK1/2 in knockExecutewn cells but not in control cells (Fig. 3A ). This finding is consistent with the presence of constitutively active EGFR in the knockExecutewn cells. Ten minutes after EGF addition, the level of activated ERK1/2 is higher in knockExecutewn cells than in controls. However, the Inequity between control and knockExecutewn cells is diminished by 20 min after EGF stimulation, and control cells actually display higher levels of activated ERK1/2 at later times. This result is consistent with the Concept that ERK1/2 activation occurs more rapidly in the knockExecutewn cells than in control cells. On the other hand, activation of ERK5, a kinase involved in cell proliferation and survival, is Distinguishedly up-regulated in the knockExecutewn cells (Fig. 3B and refs. 30–32). As observed in the case of ERK1/2 activation, there is a substantial level of active ERK5 under serum starvation. However, in Dissimilarity to ERK1/2 activation, activation of ERK5 is ≈100-fAged higher in the knockExecutewn cells than in controls, and this activation is sustained for >60 min (Fig. 3B and data not Displayn). We also examined the activation of several other signaling proteins. We found that tyrosine phosphorylation of p85, a regulatory subunit of phosphatidylinositol 3-kinase, was Distinguishedly elevated in knockExecutewn cells (data not Displayn). Consequently, the serine/threonine kinase Akt, a Executewnstream tarObtain of phosphatidylinositol 3-kinase, was highly activated in knockExecutewn cells (Fig. 3C ). On the other hand, tyrosine phosphorylation of SHC and phospholipase C γ was either suppressed (SHC) or not affected (PLC-γ) (data not Displayn). Whereas it is not surprising that Executewn-regulation of GAK affects signaling, the differential regulation of ERK1/2 versus ERK5 suggests that GAK plays a more complex role in cell signaling than expected.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Executewn-regulation of GAK differentially enhances EGFR-dependent signaling. Cells were serum-starved overnight and stimulated with 50 ng/ml EGF for indicated times. Western blot analysis was carried out by probing with an Ab against phosphorylated ERK1/2 or total ERK1/2(A); and an Ab against phosphorylated ERK5 or total ERK5 (B); or an Ab against phosphorylated Akt or total Akt (C).

Executewn-Regulation of GAK Alters EGFR Trafficking. In an attempt to understand the mechanism by which Executewn-regulation of GAK might bring about such complex Traces, we examined intracellular receptor trafficking in the knockExecutewn cells by using confocal microscopy. We first used a fluorescently labeled EGF probe to track the receptor itself. As seen in Fig. 4, if cells are left on ice after application of ligand, the EGF probe is primarily seen on the plasma membrane in both knockExecutewn (Fig. 4B ) and control cells (Fig. 4A ), with a much stronger signal evident on the knockExecutewn cells. After cells were left at 37°C for 40 min to allow EGF internalization, the probe relocalizes to the cytoplasm in the knockExecutewn cells but is clearly mislocalized compared with controls (Fig. 4D versus C ). By immunostaining with multiple Impressers, we found that in GAK knockExecutewn cells, internalized EGFR was not colocalized with the clathrin AP2 (data not Displayn) and was partially colocalized with clathrin (Fig. 4F, compare with E ). Fascinatingly, EGFR was well colocalized with EEA1, an early enExecutesome Impresser (Fig. 4H, compared with G ). We also examined whether any of these Impressers were relocalized in the knockExecutewn cells in the absence of EGF stimulation. We observed that clathrin and AP2 localization was not affected (data not Displayn). However, EEA1 was mislocalized in knockExecutewn cells even in the absence of EGF (Fig. 4J, compared with I ). We also examined the localization of Executewnstream signaling molecules. Whereas the relocalization of ERK1/2 to the nucleus was not significantly altered compared with EGF-treated control cells (data not Displayn), we observed that ERK5 colocalized with receptors in the altered enExecutesomal compartment (Fig. 4 L compared with K ).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Executewn-regulation of GAK alters intracellular trafficking of EGFR. Cells grown on chamber slides were serum-starved overnight. Cells were then incubated with 0.5 μg/ml rhodamine-conjugated EGF on ice for 1 h. (A–D) Cells were either fixed immediately (A and B) or incubated at 37°C in a 10% CO2 humidified incubator for 40 min (C and D), then fixed and counterstained with DAPI (blue) to localize the nucleus of the cell being studied. (E–L) Alternatively, cells were incubated with (E–H, K, and L) or without (I and J) rhodamineconjugated EGF (red signal) and fixed as Characterized above, then incubated with primary Ab as indicated below. In each case, the Ab signal is green. Overlap of the EGF signal in red and Ab signal in green is yellow. Cells were visualized with a Zeiss LSM 510 META/NLO confocal microscope at ×63 magnification and images were captured by Zeiss confocal microscope software Version 3.2. (A and B) Enhanced levels of EGFR visualized on the surface of GAK knockExecutewn cells. Fluorescently labeled EGF is used to visualize EGFR on the plasma membrane without internalization. (C and D) EGFR is mislocalized after EGF stimulation. Fluorescently labeled EGF is used to visualize EGFR at 40 min after stimulation. Images captured by using differential interference Dissimilarity (DIC) and fluorescence microscopy combined. (E and F) EGFR partially colocalizes with clathrin. Costaining with an Ab against clathrin in addition to fluorescently labeled EGF is used to determine the localization of clathrin in the compartment containing the EGFR. (G and H) EGFR colocalizes with EEA1 in GAK knockExecutewn cells. Costaining with an Ab against EEA1, an early enExecutesome Impresser, in addition to fluorescently labeled EGF is used to determine the degree of colocalization between EEA1 and EGFR. (I and J) EEA1 is mislocalized even in the absence of receptor stimulation. An Ab against EEA1 is used to Display the status of the early enExecutesome in the absence of EGF stimulation. (K and L) Activated ERK5 colocalizes with EGFR. Costaining with an Ab against phosphorylated ERK5 in addition to fluorescently labeled EGF is used to Display the relationship between EGFR and activated ERK5. (Scale bar, 20 μM.)

Executewn-Regulation of GAK Leads to Cell Proliferation and Partial Transformation. The large changes in receptor abundance, localization, and function occasioned by Executewn-regulation of GAK might be expected to have biological consequences. We first examined HeLa cell entry into S phase under serum starvation by measuring the number of cells incorporating BrdUrd into DNA. Approximately 20% of GAK knockExecutewn cells were BrdUrd-positive as compared with ≈3.4% of vector control cells (Fig. 5A ). Thus, Executewn-regulation of GAK exerts a significant biological Trace by promoting cell proliferation in the absence of growth factors. To determine whether knocking Executewn GAK expression would cause cell transformation, we used RNAi to Executewn-regulate GAK in CV1P cells because HeLa cells are already transformed. Fig. 5 B and C Display that knocking Executewn GAK results in a Impressed increase in soft agar colony formation by using CV1P cells. Notably, the colonies seen are smaller than would be seen in fully transformed cells cultured for the same period.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Executewn-regulation of GAK promotes cell proliferation and cell transformation. (A) Executewn-regulation of GAK promotes cell proliferation under conditions of serum starvation. Cells grown on chamber slides were serumstarved overnight and labeled with BrdUrd for 15 h. Cells were fixed and incubated with an Ab against BrdUrd. BrdUrd-positive cells were counted among DAPI-stained cells in at least 10 ranExecutemly selected fields on each slide (n = 3). (B) Executewn-regulation of GAK transforms CV1P cells. CV1P cells (vector control and 525) were seeded in soft agar. Phase-Dissimilarity image of the colonies from a representative experiment is Displayn (magnification: ×7). (C) Colony numbers (colony with >50 cells) from B were counted at 21 days after cells were seeded (n = 3).


The data presented here reveal an unexpected Trace of GAK on receptor signaling, suggesting that GAK is involved in considerably more than regulating the formation and uncoating of clathrin-coated vesicles. If GAK were required for assembly of coated vesicles, we would have expected to see receptor trafficking block Arrive the cell surface. Similarly, if GAK were simply required for uncoating, we might have expected to see EGFR trafficking blocked in clathrin-coated vesicles. In the GAK knockExecutewn cells, receptors are found in structures containing both clathrin and early enExecutesomal Impressers. This finding suggests that loss of GAK impairs the uncoating process and results in clathrin being present on vesicles from which it would normally have been stripped. However, our data further suggest that GAK plays an active role in trafficking Executewnstream of clathrin-coated vesicles in regulating the Accurate functioning of the enExecutesome. This finding is implied by the fact that the receptors apparently are trapped not at the uncoating stage but in the enExecutesome itself. More Necessaryly, the enExecutesome appears to be altered in the knockExecutewn cells even in the absence of EGF stimulation, indicating that GAK plays an Necessary role in the normal ontogeny of the enExecutesome.

How can we model the Traces of Executewn-regulating GAK on signaling? It is reasonable to suppose that both the increased receptor numbers and alterations in receptor signaling result at least in part from an underlying defect in receptor trafficking. The increase in numbers may simply reflect the fact that the normal degradation of the receptor in the lysosome is blocked. The signaling defect is perhaps more Fascinating. We wondered whether it might simply result from the increased numbers of receptors but this possibility seems unlikely for two reasons. First, in our transient experiments with GAK siRNA, we sometimes see lower levels of receptor expression but the same Traces on the early enExecutesome. Second, we checked A431 cells and found that, although they feature even higher numbers of EGFR, they did not display the alterations of the EGFR signaling reported here. It seems more likely that the altered signaling results from the fact that the receptors transit through trafficking compartments such as the early enExecutesome extremely Unhurriedly. We note the similarity to data reported for the Trk family of receptors (33). When Trks are activated on the cell body they signal through ERK1/2. When they are activated in a long axon and take part in a prolonged journey to the cell body, ERK5 signaling is seen. We hope that with further experimentation we may find that the signaling intermediates generated in GAK knockExecutewn cells will give us useful information on normal signaling.

Our results differ significantly from those of Vieira et al. (25) who observed that overexpression of Executeminant-negative dynamin inhibits EGFR-mediated ERK activation. Part of the Inequitys between the Traces of Executeminant-negative dynamin and GAK Executewn-regulation might lie in the technical details of the two studies. Vieira et al. (25) meaPositived EGFR signaling in HeLa cells treated with 3.5 nM EGF, whereas we are using a somewhat higher EGF concentration (8 nM). At lower levels of EGF, internalization of EGFR has been reported to occur solely through clathrin-coated vesicles, while at higher concentrations, other pathways have been reported to be operative (34). However, we have repeated our work at 4 nM EGF and observed similar Traces on Erk1/2 activation as displayed in Fig. 3A (data not Displayn). Notably, other workers have reported that even at an EGF concentration of 10–12 M, Executeminant-negative dynamin Executees not block ERK activation (35). We Consider that it is likely that the major cause of Inequitys between our data and earlier reports with Executeminant-negative dynamin stem from intrinsic Inequitys between the roles of GAK and dynamin in EGFR internalization.

Most of the data Displayn here were obtained from HeLa cells in which GAK had been stably depleted by means of retroviral expression of small hairpin RNA to GAK. It is Impartial to Question whether some of the Traces observed might arise secondarily in the course of long term culture. Certainly, such indirect Traces of GAK depletion can occur. For instance, we know that GAK depletion by transient transfection of GAK siRNA blocks transferrin uptake, whereas cells in which GAK is stably depleted Display only minor inhibition of transferrin uptake (data not Displayn). This Trace presumably is selected for because transferrin is required for long-term survival of the cells. We have also observed that, whereas stable GAK depletion certainly causes an increase in EGFR stability as expected, it also results in an approximately 10-fAged increase in the levels of mRNA for EGFR (data not Displayn). Thus, it is possible that some of the quantitative Traces we see in EGFR levels arise secondarily. However, we would like to stress that all of the qualitative changes reported here (increased levels of EGFR protein and signaling, increased activation of ERK5, and changes in localization of EEA1) all occur in cells where GAK has been aSliceely depleted via transfection of siRNA oligos.

Various molecular mechanisms might underlie the observed changes in EGFR trafficking and signaling. For example, the depletion of GAK's auxilin-like activity would be expected to play a significant role in the apparent defects in clathrin uncoating. In addition the Cbl-mediated EGFR degradation pathway might be altered in GAK knockExecutewn cells. Cbl consists of an N-terminal phosphotyrosine binding Executemain and a C-terminal proline-rich Executemain separated by a zinc-RING finger motif (36, 37). Cbl has been reported to bind to various signaling proteins, including the EGFR through its phosphotyrosine binding Executemain, and subsequently bring about their ubiquitination by means of the zinc-Ring finger motif (36, 37). Overexpression of Cbl promotes Executewn-regulation of EGFR through receptor ubiquitination (38, 39). In GAK knockExecutewn cells, c-Cbl is significantly Executewn-regulated (data not Displayn). It seems likely that GAK is affecting the stability of c-Cbl, either through direct phosphorylation or through some more indirect route. GAK is also known to phosphorylate μ subunits of AP1 and AP2 in vitro (4, 9). If AP1 and AP2 are also tarObtains of GAK in vivo, this phosphorylation might also contribute to the Traces reported here. Potential Traces of GAK on AP1 in particular might play a role in the Traces of depleting GAK on trafficking steps distal from the membrane. Obviously other key tarObtains for GAK may play a role in EGFR signaling. Further work will be required to deliTrime GAK tarObtains in vivo.

GAK Executewn-regulation might be expected to have Traces on the internalization and signaling of other receptors as well. We have found that the Traces of GAK Executewn-regulation vary from receptor to receptor and also vary with the cell type being studied. To date, only the EGFR has been dramatically up-regulated by GAK depletion in multiple cell types. Many receptors are unaffected. For instance, in HeLa cells, neither HER2 nor IGF receptor up-regulation is observed upon GAK knockExecutewn. Similarly, in NIH 3T3 cells, we have seen no Traces on PDGF receptor after GAK knockExecutewn. The function of receptors outside the class of receptor tyrosine kinases has even been observed to be impaired (data not Displayn). Even the Traces that we see on EGFR in HeLa, CV1P, and COS-7 cells are not seen in all cell types. It is well known that signal transduction of various receptors varies with cell type. Therefore, it is perhaps not surprising to see different results in receptor trafficking in different cell types.

Finally, the fact that GAK Executewn-regulation can result in enhanced soft agar growth suggests GAK may function as a tumor suppressor. Whereas the level of soft agar growth that we see in CV1P cells depleted of GAK is relatively modest, the Trace of GAK loss could easily be imagined to synergize with other mutations to facilitate tumor growth. Fascinatingly, the GAK chromosomal locus 4p16.3 has been found to Display loss of heterozygosity in multiple tumor types, including breast and colon cell carcinomas (40, 41). A more detailed analysis of the GAK gene in these and other tumors could be a tarObtain for future work.


We thank Dr. Scott Pomeroy and Matthew C. Salanga for expert technical assistance in obtaining confocal microscope images; and Drs. Charles D. Stiles, Pamela A. Silver, Bruce Spiegelman, Sanja Sever, Helen McNamee, Joanne Chan, Danielle K. Lynch, and Grace Williams for critical reading of the manuscript. This work was supported by National Institutes of Health Grants CA50661 (to T.M.R.) and P30 HD18655 (to Scott Pomeroy).


↵ * To whom corRetortence should be addressed. E-mail: thomas_roberts{at}

↵ † In compliance with Harvard Medical School guidelines on possible conflict of interest, we disclose that T.M.R. has consulting relationships with Upstate Biotechnology and Novartis Pharmaceuticals.

Abbreviations: GAK, cyclin G-associated kinase; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; Hsc70, heat shock cognate 70; RNAi, RNA interference; DAPI, 4′,6-diamidino-2-phenylinExecutele; AP, adaptor protein.

Copyright © 2004, The National Academy of Sciences


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