The Serine Threonine Kinase Cyclin G-Associated Kinase Regulates

The serine͞threonine kinase cyclin G-associated kinase regulates epidermal growth factor receptor signaling Lei Zhang, Ole Gjoerup, and Thomas M. Roberts*† Department of Cancer Biology, Dana–Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, MA 02115 Communicated by R. John Collier, Harvard Medical School, Boston, MA, May 5, 2004 (received for review March 18, 2004) Cyclin G-associated kinase (GAK) is a serine͞threonine kinase that activated by autophosphorylation, internalized, at least in part features high homology outside its kinase domain with auxilin. through clathrin-mediated endocytosis, and sorted to early, and Like auxilin, GAK has been shown to be a cofactor for uncoating subsequently, late endosomes (13–16). Internalized receptors then clathrin vesicles in vitro. We investigated epidermal growth factor are delivered to the lysosome and degraded or recycled to the cell (EGF) receptor-mediated signaling in cells, in which GAK is down- surface. As the receptor transits this three-dimensional path regulated by small hairpin RNAs. Here, we report that down- through the cell’s interior, various EGF-mediated signaling mole- regulation of GAK by small hairpin RNA has two pronounced cules are activated including extracellular signal-regulated kinases effects on EGF receptor signaling: (i) the levels of receptor expres- 1͞2 (ERK1͞2) via the Ras͞raf pathway (11, 17–21), and Akt via the sion and tyrosine kinase activity go up by >50-fold; and (ii) the phosphatidylinositol 3-kinase pathway (22–24). Previous studies spectrum of downstream signaling is significantly changed. One have indicated that blocking receptor trafficking at a particular very obvious result is a large increase in the levels of activated point can have significant effects on receptor signaling. For in- extracellular signal-regulated kinase 5 and Akt. These two effects stance, studies by Vieira et al. (25) showed that expression of a of GAK down-regulation result from, at least in part, alterations in dominant negative allele of dynamin, a GTPase involved in clathrin- receptor trafficking, the most striking of which is the persistence of coated vesicle fission, inhibits endocytosis of EGFR, and con- EGF receptor in altered cellular compartment along with activated sequently, ERK1͞2 activation is attenuated. However, the roles extracellular signal-regulated kinase 5. The alterations resulting of trafficking events subsequent to formation of clathrin-coated from GAK down-regulation can have distinctive biological conse- vesicles in the regulation of EGFR signaling remain relatively quences: In CV1P cells, down-regulation of GAK results in out- unknown. growth of cells in soft agar, raising the possibility that loss of GAK In the present report, we examine the effects of down-regulating function may promote tumorigenesis. GAK expression upon EGFR signaling. By using small hairpin RNA to reduce GAK levels by Ͼ90%, we find that both the levels yclin G-associated kinase (GAK), also known as auxilin II (1, of EGFR in the cells and the signaling downstream from the C2), is a 160-kDa protein, which is highly homologous to receptor are strikingly altered. At least some of these dramatic auxilin I, with the notable exception of a kinase domain found changes are likely due to changes in receptor trafficking. At the at the N terminus of GAK, and totally absent in auxilin I. Unlike biological level, GAK down-regulation can have significant effects auxilin I, which has been reported to be expressed only in on cell growth. These results suggest that GAK plays a pivotal role neurons, GAK is ubiquitously expressed. In vitro GAK, like in regulating not only receptor trafficking but also receptor signal- auxilin I, is known to assist heat shock cognate 70 (Hsc70) in ing and function. uncoating clathrin-coated vesicles (3, 4). Thus, GAK is thought to function in trafficking just after the step regulated by dynamin. Materials and Methods GAK also represents a major portion of the kinase activity in DNA-Based RNA Interference (RNAi) Constructs and Cell Culture. The clathrin-coated vesicles (5). GAK has been reported to phos- retroviral pSuper vector used in the study was the same as that phorylate the ␮2 subunit of adaptor protein (AP)2, as has described in Brummelkamp et al. (26). Selected sequences were AAK1, the protein kinase most closely related to GAK in its submitted to BLAST searches against the human genome sequence catalytic domain (4–9). In the case of AAK1, phosphorylation of to ensure only GAK mRNA is targeted. For generation of stable ␮2 has been shown to have functional consequences in promot- GAK knockdown cells, HeLa (a human cervix epithelioid carci- ing the recruitment of AP2 to internalization motifs found on noma cell line, American Type Culture Collection) or CV1P (an membrane-bound receptors (9). Overexpression of either GAK African green monkey kidney cell line) cells were infected with the or AAK1 is sufficient to impair internalization of the transferrin retroviral pSuper vectors containing either one of two hairpin receptor (4, 7, 10). Thus, GAK may function in both the ultimate constructs capable of generating 19-nt duplex RNAi oligonucleo- disassembly of clathrin-coated vesicles through its auxilin ho- tides corresponding to human GAK sequences starting at the 145th mology domains and in their formation through its kinase base in the coding sequence (denoted 145) or the 525th base domain although there is no direct evidence that GAK can mimic (denoted 525). Cells were cultured in DMEM with 10% FCS, and ͞ AAK1 in regulating vesicle formation (6–9). Because so many 1% penicillin streptomycin in 10% CO2 humidified incubator. kinases play key regulatory roles, it occurred to us that GAK When necessary, 1.5 ␮g/ml puromycin was included for selection. might represent an important regulatory point in receptor trafficking. We were particularly interested to see whether GAK might regulate receptor tyrosine kinases, such as the EGF Abbreviations: GAK, cyclin G-associated kinase; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; Hsc70, heat shock cognate 70; RNAi, receptor (EGFR), because this class of receptor is key to normal RNA interference; DAPI, 4Ј,6-diamidino-2-phenylindole; AP, adaptor protein. cell function and transformation, and because the signaling and *To whom correspondence should be addressed. E-mail: thomas࿝[email protected]. trafficking of receptor tyrosine kinases is well characterized. edu. EGFR-mediated signaling results in cell proliferation or differ- † In compliance with Harvard Medical School guidelines on possible conflict of interest, we entiation. Elevated levels or inappropriate activation of the EGFR disclose that T.M.R. has consulting relationships with Upstate Biotechnology and Novartis is associated with increased propensity for cell transformation and Pharmaceuticals. tumor formation (11, 12). Upon ligand stimulation, EGFRs are © 2004 by The National Academy of Sciences of the USA 10296–10301 ͉ PNAS ͉ July 13, 2004 ͉ vol. 101 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403175101 Downloaded by guest on September 27, 2021 A431 cells (a human epidermoid carcinoma cell line, American chemiluminescence (Amersham Biosciences). The signals were Type Culture Collection) are also used to compare the expression visualized by exposure to Kodak X-Omat blue XB-1 film. level of EGFR. EGF Internalization Assay and Indirect Immunofluorescence. Cells Abs. The following Abs were used: mAb clone 1C2 (anti-GAK, stably expressing a DNA-based RNAi construct targeting GAK Medical & Biological Laboratories); mAb clone MA1 065 (anti- were seeded on eight-well Labtek chamber slides. Twenty-four clathrin heavy chain, Affinity BioReagents, Golden, CO); mAb hours later, cells were serum-starved in DMEM without serum clone14 (anti-EEA1, BD Transduction Laboratories); 4G10 (an- overnight. The next day, cells were incubated with 0.5 ␮g/ml tiphosphotyrosine, Upstate Biotechnology, Lake Placid, NY); mAb tetramethylrhodamine-conjugated EGF (Molecular Probes) for 1 h EGFR (528) Ab (Santa Cruz Biotechnology); polyclonal EGFR on ice and then for 40 min at 37°C. Cells were fixed in 4% (1005) Ab (Santa Cruz Biotechnology); polyclonal phospho-p42͞44 paraformaldehyde, washed with PBS, permeabilized with 0.5% mitogen-activated protein kinase (Thr-202͞Tyr-204) Ab (Cell Sig- Triton X-100 in PBS for 10 min, and blocked with 10% goat serum naling Technology, Beverly, MA); polyclonal p42͞44 Ab (Cell for 1 h. Primary Abs were applied for 1 h and cells were then washed Signaling Technology); polyclonal phospho-ERK5 (Thr-218͞Tyr- with PBS. Cy2-conjugated secondary Abs (The Jackson Labora- 220) Ab (Cell Signaling Technology); polyclonal ERK5 Ab tory) were applied and incubated for 1 h. The same cells were then (Sigma); polyclonal phospho-Akt (Ser-473) or (Thr-308) Abs (Cell counterstained with 4Ј,6-diamidino-2-phenylindole (DAPI; 1 ␮g/ Signaling Technology); and polyclonal Akt Ab (Cell Signaling ml, Sigma) and mounted with Fisher Scientific mounting media. Technology). Cells were visualized with a Zeiss confocal microscope LSM510META͞NLO at ϫ63 magnification and images were cap- Western Blot Analysis and Immunoprecipitation. Cells were seeded in tured by Zeiss confocal microscope software Version 3.2. Ϸ 100-mm dishes and grown to 75% confluence in a 10% CO2 humidified incubator. The next day, cells were serum-starved with BrdUrd Incorporation Assay. Cells grown on chamber slides were DMEM without

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