RESEARCH ARTICLE

The clathrin-binding and J-domains of GAK support the uncoatingand chaperoning of clathrin by Hsc70 in the brainBum-Chan Park*, Yang-In Yim*, Xiaohong Zhao, Maciej B. Olszewski, Evan Eisenberg and Lois E. Greene‡

ABSTRACTCyclin-G-associated kinase (GAK), the ubiquitously expressedJ-domain protein, is essential for the chaperoning and uncoating ofclathrin that is mediated by Hsc70 (also known as HSPA8). Adjacentto the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain througha PTEN-like domain. Knocking out GAK in fibroblasts causedinhibition of clathrin-dependent trafficking, which was rescued byexpressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene inmice rescued the lethality and the histological defects caused byknocking out GAK in the liver or in the brain. Furthermore, when bothGAK and auxilin (also known as DNAJC6), the neuronal-specifichomolog of GAK, were knocked out in the brain, mice expressing the62-kDa GAK fragment were viable, lived a normal life-span and hadno major behavior abnormalities. However, these mice were abouthalf the size of wild-type mice. Therefore, the PTEN-like domains ofGAK and auxilin are not essential for Hsc70-dependent chaperoningand uncoating of clathrin, but depending on the tissue, these domainsappear to increase the efficiency of these co-chaperones.

KEY WORDS: GAK, Auxilin, Chaperone, Clathrin

INTRODUCTIONA variety of physiological receptors are internalized throughclathrin-mediated endocytosis (CME) (Doherty and McMahon,2009; Kirchhausen et al., 2014). The endocytic cycle starts with thebinding of ligands to receptors in clathrin coated pits (CCPs).Dynamin then causes constriction of the necks of the pits, andultimately the scission of the CCPs releases clathrin-coated vesicles(CCVs) into the cytosol (Hinshaw, 2000). Finally, the clathrin coatis dissociated by Hsc70 (also known as HSPA8) before the fusion ofthe uncoated vesicles with the early endosome (Chappell et al.,1986). In addition to uncoating, biochemical studies have shownthat Hsc70 chaperones the dissociated clathrin (Jiang et al., 2000).The uncoating and chaperone activities of Hsc70 are dependent

on a co-chaperone, either the ubiquitously expressed GAK (alsoknown as auxilin 2) or the neuronally expressed auxilin (also knownas DNAJC6) (Eisenberg and Greene, 2007). Both GAK and auxilinshare the same multi-domain structure, except that GAK has anN-terminal kinase domain (Eisenberg and Greene, 2007; Lemmon,2001). Auxilin has three domains; an N-terminal PTEN-like domainand a C-terminal J-domain are bridged by a clathrin-bindingdomain. In vitro uncoating studies have shown that the clathrin-

binding domain and the J-domain, which binds to Hsc70, are bothnecessary and sufficient to support the uncoating of clathrin-coatedbaskets by Hsc70 (Holstein et al., 1996; Ma et al., 2002). In fact,even a C-terminal fragment of auxilin (Aux-C20), with just a singleclathrin-binding motif upstream of the J-domain, efficientlysupports the uncoating of clathrin baskets by Hsc70 in vitro (Maet al., 2002).

GAK and auxilin have been targeted to examine the role of Hsc70in clathrin-dependent trafficking both in tissue culture cells and inanimal models. When these co-chaperones are knocked down orknocked out in tissue culture cells, clathrin-mediated endocytosis(CME) and other clathrin-dependent pathways are inhibited (Hirstet al., 2008; Kametaka et al., 2007; Lee et al., 2005; Zhang et al.,2005). Clathrin and clathrin adaptors are mislocalized at the plasmamembrane and the trans-Golgi network (TGN) in cells that do notexpress GAK. In addition, instead of clathrin being readilydiffusible in the cytosol, the cytosolic clathrin is immobilized intocages (Hirst et al., 2008; Lee et al., 2005). As expected, given theimportance of these co-chaperones, their absence causes lethality inCaenorhabditis elegans, Drosophila and mice (Eun et al., 2008;Greener et al., 2001; Kandachar et al., 2008).

In contrast to the in vitro uncoating studies that show the kinase andthe PTEN-like domains of GAK are not essential for activity, animalmodel studies have yielded confusing and sometimes conflictingresults as to the function of these domains. Functional analysis ofGAK in zebrafish has been performed by injecting morpholinos intothe zygote (Bai et al., 2010). When 20 residues are excised from thekinase domain of GAK, there are defects in Notch signaling duringearly zebrafish development, whereas later in development, there is anincreased level of apoptosis in neuronal tissues, resulting inneurodegeneration. Interestingly, the same phenotype is obtainedwhen a nonsensemutation is introduced into the PTEN-like domain ofGAK, which if expressed, produces a GAK fragment without theclathrin-binding and J-domains. Based on these results, it appears thatthe kinase domain might have an essential role in GAK function, butthis has not beenvalidated byexpressing only the clathrin-binding andJ-domains of GAK in the morpholino-injected fish. This is especiallyimportant in light of the finding that morpholinos frequently do notgive the same phenotype as that obtained using gene editing to disruptproteins in zebrafish (Kok et al., 2015).

The importance of the rescue experiment in defining domainfunction is evident from studies using theDrosophilamodel system.Drosophila expresses only one auxilin ortholog that is structurallysimilar to GAK as it contains an N-terminal kinase domain. Anonsense mutation in the auxilin gene causes larval lethality,whereas missense mutations in either the kinase or PTEN-likedomains produce developmental defects in the eye and the wingowing to impaired Notch signaling (Eun et al., 2008, 2007;Hagedorn et al., 2006; Kandachar et al., 2008). These latter resultssuggest a role for the kinase and PTEN-like domains in Drosophilaauxilin, but surprisingly, expression of a truncated moleculeReceived 4 March 2015; Accepted 27 August 2015

Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, NationalInstitutes of Health, Bethesda, MD 20892, USA.*These authors contributed equally to this work

‡

Author for correspondence (greenel@helix.nih.gov)

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comprising only the clathrin-binding and J-domains of Drosophilaauxilin rescues these defects, indicating that the missense mutationsapparently cause partial misfolding of the full-length auxilinprotein. Therefore, there is no known function for the kinase andPTEN-like domains in Drosophila.A definitive role and highly specific role for the kinase domain of

GAK has been found in higher organisms. Engineering of mice toexpress a kinase-dead mutant of GAK from the GAK chromosomallocus results in newborn mice dying shortly after birth (Tabara et al.,2011). The neonatal mortality is caused by a defect in lungdevelopment. Interestingly, mouse embryonic fibroblasts (MEFs)derived from the above mice show normal distribution of clathrin,indicating that trafficking is not defective in these cells. In addition,our own studies using the GAK conditional knockout mice raisedthe possibility that the kinase domain has a specialized function inthe mouse brain (Lee et al., 2008). When GAK is knocked out in thebrain by expressing Cre recombinase from the nestin promoter, themice die within 4 days of birth, even though there is still expressionof auxilin in neuronal tissue. Histological analysis of the brains fromthe embryonic and newborn GAK-knockout mice shows grossmorphological defects. By contrast, auxilin-knockout mice, whichstill express GAK in neuronal tissue, are viable even though manymice die at birth (Yim et al., 2010). Because neuronal expression ofGAK, but not auxilin, is essential for mice viability, this raises thepossibility that the kinase domain of GAK has a specializedfunction in neurons as it does in the lung.Although a function for the PTEN-like domain has not been

established in animal models, this domain is necessary for a rapidwave of GAK and auxilin recruitment to newly budded CCVs, asdetermined by imaging green fluorescent protein (GFP)-labeledproteins in tissue culture cells (Lee et al., 2006; Massol et al., 2006).Similar to the PTEN protein, the PTEN-like domain binds tophospholipids, preferentially phosphatidylinositol monophosphates(Lee et al., 2006; Massol et al., 2006), but unlike PTEN, it does nothave tyrosine-protein phosphatase activity (Haynie and Ponting,1996). When mutations in the PTEN-like domain of auxilin aremade to weaken its affinity for lipids, the mutated auxilins are notrecruited to newly budded CCVs in tissue culture cells (Guan et al.,2010). Based on these results, the expression of GAK fragments thatlack the PTEN-like domain should markedly reduce the rate ofuncoating of CCVs in GAK-knockout MEFS, which in turn mightaffect the overall rate of CME. Therefore, if the rate of recruitment ofGAK and/or auxilin is important in the endocytic cycle, this shouldbe most apparent in neuronal tissue, a tissue which requires rapidendocytosis of synaptic vesicles during neuronal transmission.In this study, the physiological functions of the kinase and PTEN-

like domains of GAK were examined in the conditional GAK-knockout and conventional auxilin-knockout mice models, whichwere engineered in our laboratory, by expressing a C-terminalfragment of GAK, GAK-C62. First, the GAK-C62 fragment, whichcomprises only the clathrin-binding and J-domains, rescued all thedefects in clathrin chaperoning and uncoating that were caused byknocking out GAK in MEFs derived from the GAK-knockoutmouse. Second, by engineering a transgenic mouse that expressesthe GAK-C62 fragment, we found that this fragment rescued thelethality and morphological defects caused by knocking out GAKeither in the liver or the brain. Therefore, neither the kinase nor thePTEN-like domains were necessary for GAK function in thesetissues. Third, when both auxilin and GAK were knocked out in thebrain of the GAK-C62 transgenic mice, surprisingly, these micewere viable and lived a normal life-span, albeit they weresignificantly smaller than their siblings. Therefore, the PTEN-like

domain is not essential for auxilin and GAK function in the brain, atissue in which rapid endocytosis is required for fast recycling ofsynaptic vesicles during neuronal transmission, although it mightincrease the efficiency of endocytosis in the brain.

RESULTSRescue of clathrin localization and steady-state distributionof TfnR and M6PR by different GAK fragmentsUsing MEFs derived from the conditional GAK-knockout mouse,GAK was knocked out in these cells through infection withadenovirus expressing Cre recombinase, as described in theMaterials and Methods. At 5 days post infection, western blotanalysis of cell lysates showed a greater than 95% reduction in GAKlevels, whereas the levels of clathrin, AP1 and AP2 were unaffected(Fig. 1A). As expected, MEFs showed no detectable expression ofthe co-chaperone auxilin, which is highly homologous to GAK, butunlike GAK, is neuron-specific.

Because Cre recombinase excises GAK from its chromosomallocus, the GAK-knockout MEFs are an excellent tool to determinewhether the kinase and PTEN-like domains of GAK are necessaryto restore the clathrin uncoating and chaperoning functions ofHsc70. The GAK-knockout cells were transfected with differentGAK fragments in order to determine which fragments rescue GAKfunction (Fig. 1B). First, we examined the rescue of the clathrinpuncta on the plasma membrane, which was disrupted in the GAK-knockout cells (Fig. 1C). After transfecting the GAK-knockoutMEFs with different fragments, cells were co-stained for clathrinand AP2. Fig. 1D shows that, as expected, full-length GAK rescuedthe clathrin localization. The clathrin–AP2 puncta were also rescuedby expressing GAK-ΔK, GAK-C62 and even GAK-C20 (afragment that contains only one clathrin-binding motif and theJ-domain), but not by GAK-C11, which comprises only the J-domain of GAK. Therefore, the clathrin-binding and J-domainsappear to be necessary and sufficient for GAK co-chaperoneactivity. This was confirmed by expressing two different chimeras,which were engineered frommonomeric clathrin adaptors and the J-domain of GAK (Fig. 1B). The expression of either the AP180–C58J chimera, comprising the 58-kDa C-terminal clathrin-bindingdomain of AP180 fused to the J-domain of GAK, or the GGA1Jchimera, comprising full-length GGA1 fused to the J-domain ofGAK, restored the clathrin puncta on the plasma membrane in theGAK-knockout cells (Fig. 1D).

We next examined whether these same constructs were able torescue the clathrin-dependent trafficking in the GAK-knockoutMEFs by examining the localization of transferrin receptor (TfnR)and mannose 6-phosphate receptor (M6PR). These two cargos budfrom the membrane in CCVs, but differ in that TfnR andM6PR budfrom the plasma membrane and TGN, respectively. The M6PRtraffics through the endo-lysosomal pathway (Hirst et al., 1998) andthen is recycled back to the TGN through the retromer complex(Arighi et al., 2004). When budding of CCVs from the TGN isinhibited, the M6PR is transported to the plasma membrane througha default secretory pathway (Traub and Kornfeld, 1997). Therefore,immunostaining shows the presence of TfnR and M6PR on theplasma membrane in the GAK-knockout MEFs (Fig. 2A), whereasthese receptors have, predominantly, a juxtanuclear location incontrol cells. As shown in Fig. 2B and C, the juxtanuclearlocalization of TfnR andM6PR was restored by expressing differentGFP-labeled GAK fragments, including GAK-C62 and GAK-C20.

Because M6PR on the cell surface can be internalized throughCME (Le Borgne and Hoflack, 1997), we wanted to ensure that thejuxtanuclear rescue of M6PR localization was due to the budding of

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CCVs from the TGN and not from the plasma membrane.Therefore, we repeated this rescue experiment in GAK-knockoutMEFS that had been treated with small interfering (si)RNAoligonucleotides against AP2 to inhibit CME. The cells were thentransfected with the different GAK constructs, followed by stainingfor TfnR and M6PR. As shown in Fig. 2D, GAK-C62 and GAK-C20 rescued the juxtanuclear localization of M6PR (Fig. 2D), butdid not rescue TfnR internalization, indicating efficient knockdownof AP2. Therefore, the clathrin-binding and J-domains of GAK aresufficient to rescue clathrin-dependent trafficking.

Rescue of clathrin flashing and clathrin exchange by GAKfragmentsIt has been previously shown that the PTEN-like domain of eitherGAK or auxilin is necessary for a burst of recruitment of these co-chaperones to the CCVs, which occurs shortly after the scission ofthe CCPs by dynamin (Lee et al., 2006; Massol et al., 2006).Because these studies were performed in cells expressing

endogenous GAK (Lee et al., 2006; Massol et al., 2006), we havereexamined whether there is a burst of recruitment, which appears asa flash, of the different GFP-labeled GAK fragments in the GAK-knockout MEFs. After imaging the plasma membrane with totalinternal reflection fluorescence (TIRF) microscopy, Metamorphsoftware was used to analyze the frequency of GFP flashing on theplasma membrane. As shown in Fig. 3A, the rate of flashing of theGAK-ΔK fragment and the GAK-C62 fragment was 70% and 40%,respectively, of the rate obtained with the full-length molecule.There was no detectable flashing when the GAK-C20 fragment wasexpressed in the GAK-knockout cells. These results show thatalthough the PTEN-like domain is not essential for flashing, itgreatly enhances the rate of flashing.

Another function of GAK is that it is needed for the exchange ofclathrin between CCPs and the cytosol (Hirst et al., 2008; Lee et al.,2008, 2005). To determine which domains of GAK are necessary forthis function, GAK-knockout MEFs were co-transfected with GFP–clathrin-light-chain andmCherry-GAK fragments. Clathrin exchange

Fig. 1. Rescue of clathrin–AP2 puncta on the plasma membrane of GAK-knockout MEFs by expressing different GAK fragments and chimeras.(A) Western blot of lysates from control (Con) MEFs, GAK-knockout (GAK K/O) MEFs, and cow brain extract. Cell lysates were blotted for GAK, clathrin heavychain (CHC), auxilin (Aux), α-adaptin, γ-adaptin and β-actin (loading control). Cow brain extract was used a positive control for auxilin. (B) Schematic of differentconstructs used to rescue the defects in MEFs after knocking out GAK. The chimeras AP180–C58J and GGA1J comprise either the 58-kDa C-terminus ofAP180 or the full-length GGA1 fused with the J-domain of GAK. The indicated domains are as follows: CB, Clathrin-binding domain; J, J-domain; K, kinasedomain; P, PTEN-like domain. (C) Localization of clathrin and AP2 on the plasma membrane in control and GAK-knockout (GAK KO) MEFs. Fixed cells wereimmunostained for AP2 and clathrin. Images at low (left) and higher magnification (inset showing magnification of boxed region). (D) Effect of localization ofclathrin and AP2 on the plasma membranes in GAK-knockout MEFs expressing different GAK fragments or chimeras. Five days after treating cells withadenovirus expressing Cre recombinase, the cells were transfected with the indicated mCherry-labeled constructs (upper panel). Two days after transfection, thecells were fixed and stained for clathrin and AP2 (lower panels).

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was measured by photobleaching the GFP-labeled clathrin puncta,followed by determination of the rate of fluorescence recovery of theclathrin puncta. As shown in Fig. 3B, the rate of fluorescencerecovery in cells that had been transfected with either GAK-C62 orGAK-C20 was not significantly different from that of control MEFsthat still expressed endogenous GAK (Fig. 3B). In cells expressingthe chimeras – either AP180–C58J or GGA1J – clathrin exchangeoccurred at a slower rate. Our previous study shows that the AP180–C58J construct stoichiometrically supports the uncoating of clathrin

baskets by Hsc70, whereas the GAK fragments work catalytically(Ma et al., 2002). This might account for the slower rate of clathrinexchange in cells that express AP180–C58J than in cells that expressthe GAK fragments.

Rescue of transferrin internalization by GAK fragmentsThe rate of CME in the GAK-knockout cells that expressed differentGFP-labeled GAK fragments was determined from the uptake ofAlexa647-conjugated transferrin (Tfn) in 10 min, a time when Tfn

Fig. 2. Localization of TfnR and M6PR in control and GAK-knockout MEFs expressing different GAK constructs and chimeras. (A) Localization of TnfRand M6PR in control (Con) and GAK-knockout (GAK KO) MEFs. Cells were immunostained for TfnR and M6PR. (B) Effect of expressing different GFP-labeledGAK constructs and chimeras on the localization of TfnR in GAK-knockout MEFs. GAK-knockout cells that had been transfected with the indicatedconstructs were stained for TfnR. (C) Effect of expressing different GFP-labeled constructs and chimers on the localization of M6PR in GAK-knockout MEFs.GAK-knockout cells that had been transfected with the indicated constructs were stained for M6PR. (D) Effect of expressing GAK-C62 and GAK-C20 in GAK-knockout cells that had been treated with RNA oligonucleotides to knock down AP2. Cells were immunostained for TfnR and M6PR. The outlined cells in B–Dindicate the cells expressing the mCherry-labeled GAK fragments.

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internalization is still in the linear range. Cells were sorted based onthe fluorescence intensities of both GFP and Alexa647 to determinethe amount of Tfn internalized in the population of GFP-labeledcells. The Tfn uptake was then normalized to that of control MEFsthat still expressed endogenous GAK. As shown in Fig. 4A, theextent of Tfn internalization in cells expressing GAK-ΔK and GAK-C62 was comparable to that in control cells, whereas less Tfn wasinternalized in cells expressing GAK-C20 as well as thoseexpressing the AP180–C58J and GGA1J chimeras. These resultssuggest that neither the kinase nor the PTEN-like-domains of GAKaffect the rate of Tfn internalization.

To confirm the above conclusion, instead of using transienttransfection to express the different GAK constructs, stable MEFcell lines were made expressing Flag-tagged full length GAK,GAK-ΔK, GAK-C62 or GAK-C20. After deriving the stable celllines, the expression levels of the Flag-tagged constructs weredetermined after knocking out the chromosomal GAK with Crerecombinase. Lysates from the stable cell lines were run on SDS-PAGE gels, followed by probing with antibodies against GAK andFlag. The expression of Flag-tagged GAK, GAK-ΔK and GAK-C62 was not significantly different from that of the endogenousGAK protein (the ratio of expressed GAK constructs to endogenous

Fig. 4. Histogramof Tfn uptake inGAK-knockoutMEF cells transfectedwith different constructs. (A) Internalization of AlexaFluor647–Tfnwasmeasured inGAK-knockout cells that had been transfected with GFP, different GFP-labeled GAK fragments or GFP-labeled chimeras. Control (Con) cells, the MEFs derivedfrom the GAK-knockout mice that still expressed endogenous GAK, were transfected with GFP. Cells were sorted based on the intensity of GFP andAlexaFluor647 fluorescence. From the fluorescence-activated cell sorting (FACS) plot, the median fluorescence intensity of AlexaFluor674 for each population ofGFP-expressing cells was calculated. Data are from four independent experiments (mean±s.d.). GAK-FL, full-length GAK. (B) Internalization of Tfn in GAK-knockout cells that had been stably transfected with different GAK fragments. After establishing the stable cell lines, GAK was knocked out by treating cells withadenovirus expressingCre recombinase. Control MEFswere the stableMEF cell lines derived from theGAK-knockoutmice that still expressed endogenousGAK.In control cells and in the adenovirus-treated stable cell lines, AlexaFluor488–Tfn was internalized for either 5 min (open bars) or 10 min (shaded bars). Cells weresorted based on the intensity of the AlexaFluor488 fluorescence to determine the internalization of Tfn. Data are from three independent experiments (mean±s.d.).

Fig. 3. Recruitment of GAK fragments to the plasma membrane and rescue of clathrin exchange in GAK-knockout MEFs by the different constructs.(A) GAK-knockout cells were transfected with different GFP-labeled GAK fragments and then imaged using a TIRF microscope. GAK-FL, full-length GAK.The number of GAK flashes that occurred in a 25-µm2 area over a 6-s time period (a total of 60 images) was counted for each sample set on a minimum of 4 cells.*P<0.05 and ***P<0.001 are the statistical differences compared to the cells transfected with the GFP-labeled full-length GAK, determined using Student’s t-test.(B) Rate of clathrin exchange in GAK-knockout (GAK KO) cells that had been transfected with different mCherry-labeled constructs and GFP-tagged clathrinlight chain. Control (Con) cells were the MEFs derived from the GAK-knockout mice that still expressed endogenous GAK. At 48 h post transfection, a smallregion on the plasma membrane containing GFP-clathrin structures was photobleached, and imaging of the bleached area was performed to measure thefluorescence recovery. The fraction of initial fluorescence intensity was plotted versus time when data were analyzed, as described previously (Wu et al., 2001).For each construct, the plotted datawere the average from 10 to 15measurements. The data are representative of one of three sets of experiments that measuredthe fluorescence recovery of GFP–clathrin in cells that had been transfected with different mCherry-labeled GAK fragments.

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GAK was 1.1±0.2), whereas GAK-C20 was expressed at about halfthe level of that of the endogenous proteins (the ratio of GAK-C62to endogenous GAK was 0.6±0.1).Tfn uptake was measured in the different stable cell lines before

(control) and after infection with adenovirus that encoded Crerecombinase. Flow cytometry was used to quantify the amount ofAlexa488–Tfn internalized within 5 min (open bars) and 10 min(shaded bars). Fig. 4B shows that the Tfn uptake in cells that stablyexpressed GAK, GAK-ΔK or GAK-C62 was about 80% of theuptake of the controls both at 5 and 10 min. The GAK-C20-expressing cells showed reduced Tfn uptake compared to the control,especially at 10 min, whichmight be owing to the reduced expressionlevel of this fragment. These results appear to show that GAK-C62rescues CME as well as other clathrin-dependent pathways in GAK-knockout MEFs. Therefore, the kinase and PTEN-like domains donot appear to have an essential function for the co-chaperone activityof GAK in MEFs.

Transgenic GAK-C62 miceHaving established that GAK-C62 restored chaperoning of clathrinby Hsc70 in the GAK-knockout MEFS, we next examined whether

this truncated fragment, which lacks both the kinase and PTEN-likedomains, is able to rescue the lethality caused by knocking out GAKin the mouse. It has been previously shown that if the chromosomalGAK is mutated such that a kinase dead GAK is expressed, the miceall die shortly after birth owing to a defect in lung development(Tabara et al., 2011). Therefore, we decided to engineer a transgenicmouse to ubiquitously express the GAK-C62 fragment from theROSA 26 promoter (Kisseberth et al., 1999). In all of our breedingschemes, we ensured that mice expressing GAK-C62 wereheterozygous for this transgene (GAK-C62+/−). The expression ofGAK-C62 in different tissues from the transgenic mice wasconfirmed by western blotting. As shown in Fig. 5A, GAK-C62was expressed at comparable levels in the different tissues, whichwas expected based on the expression pattern of ROSA 26 (Giel-Moloney et al., 2007).

The GAK-C62 transgenic micewere crossed to the GAKfl/fl mice,and their offspring were then bred with mice expressing Crerecombinase from different tissue-specific promoters. First, weexamined whether GAK-C62 rescues the lethality and liverdysfunction that is observed when GAK is knocked out in theliver (Lee et al., 2008). As previously reported, when GAK wasknocked out in the liver by expressing Cre recombinase from thealbumin promoter, the newborn mice were jaundiced, smaller thantheir littermates, and died several days after birth. When the 3-week-old offspring of mating GAK-C62+/−,GAKfl/fl mice with AlbCre+/−,GAKfl/−mice were genotyped, there were mice with the GAK-C62-expressing GAK-knockout (GAK-C62/GAK-knockout) genotype(GAK-C62+/−,AlbCre+/−,GAKfl/fl). In fact, genotyping many littersshowed that the percentage of the GAK-C62/GAK-knockout micewas consistent with Mendelian genetics. Western blots of liverlysates were used to confirm that these mice were expressing GAK-C62, but not endogenous GAK (Fig. 5B). In addition, the westernblot analysis provides a measure of the relative level of GAK-C62relative to endogenous GAK. The expression level of GAK-C62was, at most, twice (1.8±0.2, n=4) that of the full-length protein. Asexpected, the level of GAK-C62 was unaffected by knocking outendogenous GAK.

The mice that expressed GAK-C62, but not endogenous GAK,were healthy and lived a normal life span. To better characterize thephenotype of the rescued mice, histological liver sections wereexamined from wild-type, GAK-knockout and GAK-C62/GAK-knockout mice. As shown in Fig. 5C, the hematoxylin and eosin(H&E)-stained liver sections from the GAK-C62/GAK-knockoutmice showed normal differentiation and biliary duct formation,unlike the liver from the GAK-knockout. Furthermore,immunostaining showed that the localization of clathrin andTGN38 at the Golgi in the GAK-C62/GAK-knockout miceappeared similar to that in the wild-type mice, instead of beingmislocalized as observed in the GAK-knockout mouse (Lee et al.,2008).

Next, we examined whether GAK-C62 rescues the lethality that iscaused by knocking out GAK in the brain by expressing Crerecombinase from the nestin promoter (Lee et al., 2008). Here again,we genotyped the mice at 3 weeks of age and found mice with theGAK-C62/GAK-knockout genotype (GAK-C62+/−,NesCre+/−,GAKfl/fl). Genotyping of many litters showed that the percentageof the GAK-C62/GAK-knockout mice was consistent withMendelian genetics. Western blot analysis of brain lysatesconfirmed that these mice expressed GAK-C62 but notendogenous GAK (Fig. 6A). Before knocking out GAK, therelative expression level of GAK-C62 to endogenous GAK was1.7±0.3 (n=5), which is similar to the ratio we obtained in the liver.

Fig. 5. Transgenic expression of GAK-C62 rescues the lethality that iscaused by knocking out GAK in the mouse liver. (A) Western blot of GAK-C62 (C62) in lysates prepared from different organs isolated from 1-week-oldGAK-C62 control mouse (GAK-C62+/−). The following organs were used:Br, brain; Li, liver; Lu, lung, He, heart; Ki, kidney. GAK-C62 was probed usingan antibody against GAK. Actin was loaded as an internal loading control.(B) Immunoblotting of GAK and GAK-C62 in liver lysates from 3-week-oldwild-type (WT) mice, GAK-C62-expressing control mice (C62/Con) and GAK-C62-expressing GAK-knockout mice (C62/GAK KO). GAK and GAK-C62(C62) were probed using an antibody against GAK. Actin was used as aninternal loading control. (C) Histological analysis of livers from wild-type mice,GAK-knockout mice and GAK-C62/GAK-knockout mice. H&E andimmunostaining of liver sections from newborn mice [postnatal day (P)0.5].Cells were stained for clathrin heavy chain (CHC) and TGN38. The genotypesare as follows: WT (GAK+/+), C62/Con (GAK-C62+/−,GAKfl/fl), GAK KO(AlbCre+/−,GAKfl/fl), C62/GAK KO (GAK-C62+/−,AlbCre+/−,GAKfl/fl).

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There was no compensation in the amount of GAK-C62 or auxilinexpressed when GAK was knocked out in neuronal tissue.The GAK-C62/GAK-knockout mice lived a normal life-span

with no obvious behavioral defects. H&E staining of brain sectionsfrom the GAK-C62/GAK-knockout mice showed no prominent lossof cells in the cerebellar cortex (Fig. 6B), unlike the brains from theGAK-knockout mice that were not expressing GAK-C62. TheGAK-C62/GAK-knockout mice were on average 25% smaller thancontrol mice (Fig. 6D), similar to the reduction in size observed inthe conventional auxilin-knockout mouse. These results show thatthe kinase and PTEN-like domains of GAK do not have aspecialized function in the brain. However, the above results donot address whether the PTEN-like domain has an essential role inthe brain because auxilin is still expressed in the GAK-C62/GAK-knockout mice. Therefore, to examine whether this domain has anessential function in neurons, we engineered a mouse that expressedGAK-C62 but neither GAK nor auxilin in neuronal cells bybreeding the GAK conditional knockout mice and the auxilinconventional knockout mice with the GAK-C62 transgenic mouse

and then crossing the offspring to mice expressing Cre recombinasefrom the nestin promoter. Genotyping the mice at 3 weeks of ageshowed that there were mice expressing GAK-C62 that were alsodouble knockout for GAK and auxilin (GAK-C62/doubleknockout) (GAK-C62+/−,Aux−/−,NesCre+/−,GAKfl/fl). Becausethe conventional auxilin-knockout mouse already has multipledefects – including high neonatal mortality, infertility problems andlow body weight (Yim et al., 2010) – we were surprised that theGAK-C62/double-knockout mice were viable. As expected, theGAK-C62/double-knockout mice had the same phenotypic defectsas the auxilin-knockout mice, but in addition, the mice weredistinguished by being much smaller than littermates with differentgenotypes (Fig. 6C). At 3 weeks of age, when the mice wereweaned, the weight of the GAK-C62/double-knockout mice wereabout half the weight of the control mice that expressed both auxilinand full-length GAK, along with GAK-C62, in the brain (Fig. 6D).The small size of the transgenic GAK-C62/double-knockout micepersisted into adulthood, which shows that insufficient supply ofmother’s milk did not cause their small size.

Fig. 6. Expression of GAK-C62 rescues the lethality that is caused by knocking out GAK in mice both in the absence of auxilin expression.(A) Immunoblotting of GAK, GAK-C62 (C62) and auxilin in brain lysates from 3-week-old micewith the indicated genotypes. Actin was used as an internal loadingcontrol. (B) Ultrastructure of the brain of embryonic mice with altered expression of the co-chaperones GAK and auxilin. Brains were sectioned from mice[embryonic day (E)15.5] with the following genotypes: wild type (WT), GAK-knockout (GAK KO), GAK-C62/GAK-knockout (C62/GAK KO), GAK-C62/doubleknockout (C62/DKO). H&E staining of the coronal section of the cerebral cortex. CP, cortical plate; VZ, ventricular zone. (C) Body size comparison of GAK-C62transgenicmicewith the indicated genotypes at 3 weeks of age. Aux KO, auxilin knockout; DKO, GAK and auxilin double knockout; GAK knockout, GAK KO.Micegenotypes are given below. (D) Comparison of the relative body weights of GAK-C62 transgenic mice with the indicated genotypes. The body weight of 3-week-old mice was measured from 10 mice for each genotype. Error bars show mean±s.e.m. (E) Western blot of brain lysates from 3-week-old GAK-C62 transgenicmice with the indicated genotypes. The blots were probed with the indicated antibodies. Genotypes are as follows: wild type (GAK+/+); GAK-C62-expressingcontrol mice, C62/Con (GAK-C62+/−,GAKfl/fl); GAK KO (NesCre+/−,GAKfl/fl); C62/GAK KO (GAK-C62+/−,NesCre+/−,GAKfl/fl); C62/Aux KO (GAK-C62+/−,Aux−/−,);C62/DKO (GAK-C62+/−,NesCre+/−,GAKfl/fl,Aux−/−).

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In addition to genotyping the mice, western blot analysis of brainlysates from the GAK-C62/double-knockout mice confirmed thelack of GAK and auxilin expression. As noted above, beforeknocking out GAK, the level of GAK-C62 was, at most, twice thelevel of endogenous GAK. Knocking out GAK and/or auxilin didnot affect the expression of GAK-C62. Western blot analysis wasused to compare the levels of other endocytic proteins in brainlysates to determine whether there was a change in their expression.In particular, we were interested in whether synaptojanin orendophilin was upregulated in the GAK-C62/double-knockoutmice. Both the synaptojanin- and endophilin-knockout mice hadmore CCVs (Cremona et al., 1999; Milosevic et al., 2011), whichsuggests that these proteins play a role in dissociating the clathrincoat. As shown in the western blot analysis, there was no detectableupregulation of synaptojanin 1 or endophilin 1 in the GAK-C62/double-knockout mouse (Fig. 6E). Therefore, GAK-C62 issufficient to support the interaction of clathrin with Hsc70 in thebrain, which shows that the kinase and PTEN-like domains are notessential for synaptic function in the mouse.

DISCUSSIONThe numerous studies on the function of members of the auxilinfamily have shown that these proteins, which are conserved fromyeast to man, have an essential role in the uncoating andchaperoning of clathrin by Hsc70. A further understanding ofthese co-chaperones has come from functional analysis of theirindividual domains. At the C-terminus of GAK and auxilin, there isa J-domain, which binds to Hsc70, and adjacent to the J-domain isthe clathrin-binding domain. The clathrin-binding and J-domains ofauxilin and GAK have been shown to be necessary and sufficient tosupport clathrin uncoating in biochemical studies (Holstein et al.,1996; Ma et al., 2002). The roles of the other domains – the PTEN-like domain, which is present in both GAK and auxilin, and thekinase domain, which is present only in GAK – are less wellunderstood. In this study, we addressed whether the kinase andPTEN-like domains are important in mammalian cells and inmammals. Specifically, we examined the role of these domains inour GAK-knockout mouse model and MEFs derived from thesemice.By expressing different GAK fragments in the GAK-knockout

MEFs, we showed that a fragment comprising the clathrin-bindingand J-domains of GAK (GAK-C62) restored clathrin uncoating andchaperoning when GAK was knocked out in the MEFs, inagreement with other studies (Eun et al., 2008; Kandachar et al.,2008). Specifically, similar to the control MEFs, the GAK-knockout MEFs expressing GAK-C62 restored the localization ofclathrin, the steady-state distribution of TfnR and M6PR, the rate ofclathrin exchange, and the rate of Tfn uptake. Compared to full-length GAK, there was a marked reduction in the frequency ofGAK-C62 flashing on the plasma membrane, which is a measure ofits bulk recruitment onto newly budded CCVs, which occurs justbefore uncoating (Lee et al., 2006). Evidently, this reduction inrecruitment, which is expected to decrease the rate of uncoating,does not manifest itself in a decreased rate of Tfn uptake. Theseresults show that the kinase and PTEN-like domains are not requiredfor clathrin uncoating or chaperoning in MEFs. However, theymight modulate the function of GAK. For example, expression of akinase-dead mutant of GAK in cells in which GAK levels wereknocked down with RNA interference caused a reduction in the rateof maturation of cathepsin D (Kametaka et al., 2007), a process inwhich cathepsin D traffics in a clathrin-dependent pathway from theTGN to the lysosome.

Other studies using mouse models have provided mixed results asto the function of the kinase and PTEN-like domains of GAK andauxilin. Interestingly, auxilin from yeast and C. elegans does notcontain a kinase domain or a PTEN-like domain, which shows thatthese domains are not necessary for the co-chaperone function ofauxilin in these organisms (Lemmon, 2001). In Drosophila, onlyone member of the auxilin family is expressed, which contains adomain structure similar to that of GAK. Because missensemutations in the kinase and PTEN-like domain of Drosophilaauxilin cause developmental defects through impaired notchsignaling (Eun et al., 2008, 2007; Hagedorn et al., 2006;Kandachar et al., 2008), at first, it seems as if these domains areessential. However, because these missense mutations, as well asnonsense mutations that cause larval lethality, can be rescued byexpressing a fragment comprising just the clathrin-binding andJ-domains (Eun et al., 2008; Kandachar et al., 2008), it appears thatthe missense mutations cause overall misfolding of the full-lengthprotein rather than simply disrupting the kinase and PTEN-likedomains (Eun et al., 2008).

In higher organisms, the two members of the auxilin family differin their expression patterns; GAK is expressed ubiquitously, andauxilin is expressed only in neurons. When morpholinos are used toexcise a small region of the kinase domain of GAK in zebrafish, thebrain has developmental defects due to impairment of notchsignaling (Bai et al., 2010), just as observed in Drosophila (Eunet al., 2008; Hagedorn et al., 2006). Surprisingly, a similarphenotype in the zebrafish is obtained when morpholinos are usedto generate a nonsense mutation in the PTEN-like domain of GAK(Bai et al., 2010). However, there has been no attempt to rescue thezebrafish defects with a fragment comprising just the clathrin-binding and J-domains of GAK so it is unclear whether, as inDrosophila (Eun et al., 2008; Kandachar et al., 2008), thesedomains are sufficient for GAK function in zebrafish. Nor it is clearwhy the effects of mutating GAK are not rescued by auxilin, whichis also present in neurons; perhaps auxilin is not expressed early indevelopment or maybe it does not localize to the same part of theneuron as GAK.

In the mouse, it has been well established that the kinase domainof GAK is essential for lung development (Tabara et al., 2011), butit is not clear whether it has an important function in other tissues orin the adult mouse. It is also unclear whether the PTEN-like domainof GAK and auxilin are important in the mouse to rapidly recruitthese co-chaperones to the plasma membrane to promote fastuncoating of the CCVs. In a model of clathrin uncoating by Hsc70,proposed by the Kirchhausen laboratory (Massol et al., 2006), thehydrolysis of phospholipids on the membrane of the CCVs bysynaptojanin regulates the recruitment of GAK and auxilin. Thisrecruitment is dependent on the binding of the PTEN-like domainsof these co-chaperones to phospholipids on the newly buddedCCVs (Guan et al., 2010). Therefore, it might be expected that thisdomain is required to achieve rapid endocytosis.

In the present study, to better understand the function of thekinase and PTEN-like domains, we engineered a transgenic GAK-C62 mouse to determine whether this fragment rescues the defectscaused by tissue-specific knockouts in the GAK- and auxilin-knockout mice models (Lee et al., 2008; Yim et al., 2010). First, wefound that GAK-C62 rescued the lethality that is caused byknocking out GAK in the liver. The rescued mice were healthy withno apparent histological or physiological defects, which indicate anon-essential function for the kinase and PTEN-like domain in thistissue. Similar results were obtained when we knocked out GAK inthe brain. This shows that not only is the kinase domain not required

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in the brain but, in addition, the lethality that we previouslyobserved when GAK was knocked out was either due to lack ofexpression of auxilin during the early stages of brain development orelse due to different subcellular localization of GAK and auxilin(Lee et al., 2008).Knocking out the neuronally expressed auxilin in mice produces

multiple defects, even though the neurons express GAK (Yim et al.,2010). The auxilin-knockout mice have a high incidence ofperinatal mortality, low body weight and infertility (Yim et al.,2010). Furthermore, electron microscopy studies show thatsynapses from the auxilin-knockout mice have an increase in thenumber of clathrin-coated structures, whereas electrophysiologystudies have shown an impairment of clathrin-mediated endocytosisin the synaptic vesicles of neurons from the knockout mice (Yimet al., 2010). In this study, we used the auxilin-knockout mice,along with the GAK-knockout mice, to generate a mouse thatexpresses only GAK-C62 in neuronal tissue in order to examinewhether the PTEN-like domain has an essential function in thebrain. Because the PTEN-like domain has been proposed to benecessary for the rapid recruitment of auxilin to the plasmamembrane (Guan et al., 2010), we expected profound defects in theGAK-C62/double-knockout mice due to the lack of rapidendocytosis in order to recycle the synaptic vesicles duringneurotransmission. Surprisingly, the major defect of the GAK-C62/double knockout was their small size, which suggests that ahypothalamus defect causes a reduction in the secretion of growthhormone from the pituitary gland in these animals (Slabaugh et al.,1981). Otherwise, these mice were viable with no apparentbehavioral defects and lived a normal life span.Although our data show that the PTEN-like domains are not

essential in the mice brain and liver, missense mutations in thePTEN-like domain of auxilin has been reported to cause juvenileParkinson’s disease (Edvardson et al., 2012). In a possibly relatedeffect, GAK, through its PTEN-like domain, forms a complex withleucine-rich kinase 2 (LRRK2), a kinase that when mutated isassociated with a high risk of the development of Parkinson’sdisease, along with mutations in Bag5 and Rab7L1 (Beilina et al.,2014). This multi-protein complex is needed to maintain theintegrity of the TGN. Therefore, these results suggest that thePTEN-like domains of GAK and auxilin have other functions thatare not yet understood, aside from facilitating the recruitment ofthese co-chaperones to phospholipids.

MATERIALS AND METHODSCell culture and Ad-Cre infectionMEFs derived from mice with GAK fl/fl (Lee et al., 2008) were maintainedin Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing4.5 g/l glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mML-glutamine, and 10% fetal bovine serum (FBS; Invitrogen). Foradenoviral infection, cells plated 1 day before infection were incubated infresh DMEM culture medium with adenovirus expressing Cre recombinase(Ad-Cre; Vector Biolabs) at a multiplicity of infection (MOI) of 200 in thepresence of 5 µg/ml polybrene (Sigma-Aldrich), as described previously(Olszewski et al., 2014). The stable cell lines were treated with adenovirus asdescribed above to knockout the endogenous GAK. To knockdown AP2,oligonucleotide duplexes (Dharmacon) against α-adaptin were transfectedusing Lipofectamine RNAiMAX reagent (Invitrogen).

Stable cell lines expressing full-length and different GAK fragmentswere generated by infecting the GAK-knockout MEFs with differentpBabe retroviral expression vectors. The stable cell lines were selected for4–6 weeks with puromyocin. The stable cell lines were then treated withadenovirus expressing Cre recombinase to knockout GAK geneexpression.

Immunofluorescence microscopyMEFs plated onto Lab-Tek glass chamber slides (Nalge Nunc) were fixed in4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA)and then immunostained with the following primary antibodies: rabbitpolyclonal antibody against clathrin heavy chain (Abcam); rabbit polyclonalantibodies against TGN38 (AbD Serotec), cation-independent M6PR (fromDr Linton Traub, University of Pittsburgh, Pittsburgh, PA), mousemonoclonal antibodies against GM130 (clone 35; BD Biosciences),α-adaptin (clone AP6; Affinity BioReagents), and TfnR (Invitrogen).Cells were immunostained with fluorescently labeled secondary antibodies(Invitrogen and Jackson ImmunoResearch Laboratories).

Western blottingProtein lysates from cells or tissues were run on 4–12% NuPAGE gels(Invitrogen) and transferred onto nitrocellulose membranes (Invitrogen)through Trans Blot SD system (Bio-Rad, Hercules, CA). The proteins wereimmunoblotted with the following antibodies: rabbit polyclonal antibodiesagainst GAK (Greener et al., 2000), auxilin (Yim et al., 2010), synaptonin 1(Dr Pietro de Camilli, Yale University, New Haven, CT), and endophilin 1(Dr Pietro de Camilli); mouse monoclonal antibodies to β-actin (cloneAC-15; Abcam), dynamin (clone Hudy1 from Invitrogen), FLAG antibody(Sigma-Aldrich), clathrin heavy chain (clone 23, BD Biosciences),α-adaptin (clone 8, BD Biosciences) and γ-adaptin (clone 88, BDBiosciences). Protein bands were detected by using a classicchemiluminescent method with the ECL blotting substrate (ThermoFisher Scientific) and horseradish peroxidase (HRP)-conjugatedsecondary antibodies (Jackson ImmunoResearch Laboratories), or with animaging method using infrared secondary antibodies, such as IRDye 680and 800CW (Li-Cor Bioscience, Lincoln, NE) followed by scanning in theOdyssey infrared detection system (Li-Cor Bioscience).

Plasmids and transfectionThe construction of GFP-tagged full-length GAK, GFP-tagged GAKtruncation constructs (GAK-ΔK, GAK-C62, GAK-C20, GAK-C11) andGFP-LCa (encoding GFP-tagged clathrin light chain A) has beendescribed previously (Lee et al., 2005). The GAK fragments were alsosubcloned into pmCherry-C1 vector (Clontech). To generate chimericconstructs, referred to as AP180-C58J and GGA1J, DNA fragments of a58-kDa C-terminal domain of AP180 and a full-length GGA1 proteinwere amplified from pQE30-AP180-C58J (Ma et al., 2002) and pEGFP-GGA1 (Puertollano et al., 2003), respectively. The J-domain of GAKwas cloned in frame at the C-terminus of GGA1. The chimeras werecloned into either pEGFP or pmCherry vectors (Invitrogen). To make thestable cell lines, the pBabe retro viral expression vector (Cell Biolabs)was used to express the full-length and fragments of GAK. Transienttransfections were performed using FuGENE HD reagent (Roche) as perthe manufacturer’s instructions. To transfect the cells, FuGENE HDreagent (Roche) in Opti-MEM (Invitrogen) was mixed with plasmidDNA at a 6:2 ratio.

Fluorescent recovery after photobleaching and TIRF imaging ofcellsCells were imaged using the Zeiss LSM 510 confocal microscopeimaging system (Carl Zeiss MicroImaging) with a 40× or a 63×, 1.4 NAobjectives.

GFP–clathrin in normal MEFs or GAK-knockout MEFs with or withoutexpression of rescue constructs was imaged and photobleached using a488-nm laser light with a 40×, 1.4 NA objective. A defined region wasphotobleached at high laser power resulting in 50–80% reduction in thefluorescence intensity. Scanning at low laser power monitored thefluorescence recovery after photobleaching (FRAP). Data were analyzedas described previously (Wu et al., 2001).

For TIRF imaging, the Olympus microscopewas used with a 60×1.45 NAoil-immersion objective using the 488-nm line of an argon laser (MellesGriot, Carlsbad, CA). Images were collected using a Photometrics CoolSnap HQ CCD camera (Photometrics, Tucson, AZ). Streams of 120 frameswere exposed and acquired at 10 frames/s for 120 s.

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Tfn internalizationMEFs were incubated in DMEM containing 0.2% bovine serum albumin(DMEM-BSA) for 1 h to deplete serum. The cells were transferred tofresh DMEM-BSA medium containing 10 µg/ml AlexaFluor-conjugatedTfn and incubated for 10 min. The cells were then washed with coldDMEM-BSA, incubated with cold acid buffer (0.2 M acetic acid, 0.5 MNaCl) to dissociate surface Tfn and then fixed with 4% PFA.AlexaFluor-labeled Tfn was analyzed with flow cytometry using aFACS Calibur instrument.

Engineering and breeding of transgenic mouseThe GAK-C62 fragment was expressed from the ROSA 26 promoter. Thefragment was cloned into the AgeI and EcoRI restriction sites that are presentin the multiple-cloning site of the pBroad3 vector (InvivoGen). The vectorwas linearized using the PacI restriction enzyme (New England Biolabs)and then purified by using gel extraction. The vector was then injected intofertilized C57BL/6 mouse eggs, followed by their implantation into pseudo-pregnant recipient mice. The genotype of the transgenic mouse wasdetermined by using PCR analysis with genomic DNA isolated from the tailof the mice.

To determine whether GAK-C62 rescued the GAK-knockoutphenotypes, the GAK-C62 transgenic mouse was bred with a GAKfl/fl

mouse to produce GAK-C62+/−,GAKfl/fl. These mice were then bred withAlbCre+/− and NesCre+/− mice to knockout GAK in the liver and brain,respectively. The transgenic Cre mice lines were purchased from JacksonLaboratory. To generate the GAK-C62 double knockout of neuronal GAKand auxilin, GAK-C62+/−,NesCre+/−,GAKfl/fl were bred to the Aux−/−,GAKfl/fl to generate Gak-C62+/−. GAKfl/fl,Aux−/−,NesCre+/− mice. Micewere genotyped by using PCR analysis with genomic DNA isolated fromthe mice tail. In all of the GAK-C62 rescue experiments, the mice werealways heterozygous for GAK-62.

All mice were handled in accordance with National Institutes of Healthguide for the care and use of laboratory animals.

Histological studiesThe liver and brain were dissected from the mice followed by fixation in10% phosphate-buffered formalin for a day at room temperature. The organswere paraffin embedded and sectioned (5 µm) by HistoServ (Germantown,MD). H&E staining and immunostaining were performed as describedpreviously (Lee et al., 2008; Yim et al., 2010).

AcknowledgementsWewould like to acknowledge the following National Heart, Lung, and Blood Institutecore facilities – transgenic core, light microscopy core and flow cytometry core.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsB.-C.P., Y.-I.Y., X.Z. and M.B.O. performed experiments and analyzed data. L.E.G.and E.E. planned experiments and wrote the manuscript.

FundingThis work was supported by the Intramural Research Program in the National Heart,Lung, and Blood Institute (NHLBI) at the National Institutes of Health (NIH).Deposited in PMC for release after 12 months.

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RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3811-3821 doi:10.1242/jcs.171058

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