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Address correspondence to Sandra Schmid, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 784-2311. Fax: (858) 784-9126. email: slschmid{at}scripps.edu
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Abstract |
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Key Words: endocytosis; AP-2; clathrin; AAK1; kinase
Abbreviations used in this paper: AAK1, adaptor-associated kinase; CCV, clathrin-coated vesicle; CME, clathrin-mediated endocytosis; siRNA, small interfering RNA; Tfn, transferrin; TfnR, Tfn receptor; tTA, tetracycline transactivator.
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Introduction |
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Phosphorylation of AP-2 complexes regulates their recruitment to the plasma membrane (Fingerhut et al., 2001), their interaction with cargo molecules (Ricotta et al., 2002), and their assembly with clathrin (Wilde and Brodsky, 1996). Several kinases copurify with clathrin-coated vesicles (CCVs), including (1) casein kinase II, which appears to phosphorylate clathrin light chains (Korolchuk and Banting, 2002); (2) an unknown kinase(s) that phosphorylates the and ß2 adaptins and may regulate their plasma membrane recruitment (Fingerhut et al., 2001); and (3) two related kinases, GAK/auxilin 2 (Umeda et al., 2000) and the newly discovered adaptor-associated kinase (AAK1) (Conner and Schmid, 2002), that phosphorylate µ2. In vitro phosphorylation of µ2 by AAK1 increases AP-2 affinity for tyrosine-based internalization motifs roughly 25-fold (Ricotta et al., 2002). Additionally, AAK1 inhibits AP-2stimulated transferrin (Tfn) internalization in perforated cell assays that reconstitute early steps in CCV formation (Conner and Schmid, 2002). Here, we establish that full-length AAK1 interacts with and perturbs AP-2 function in vivo. Surprisingly, the disruption of AP-2 function by AAK1 overexpression reveals a more cargo-specific role for AP-2 in clathrin-dependent receptor-mediated endocytosis.
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Results and discussion |
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In vitro kinase assays did not reveal any major AAK1 targets other than µ2 in either cytosolic or membrane fractions (Conner and Schmid, 2002). Moreover, µ2 phosphorylation is known to be required for endocytosis in vivo (Olusanya et al., 2001). Thus, we expected that overexpression of full-length AAK1 constructs inhibited AP-2 function by shifting the balance of µ2 into a phosphorylated (WT AAK1) or dephosphorylated (K74A or D176A AAK1) state. However, immunoprecipitation of AP-2 complexes from whole cell lysates, following in vivo labeling, did not show any significant alteration in µ2 phosphorylation in cells overexpressing either WT or kinase-dead AAK1 (Fig. 3 A). Although we cannot rule out the existence of a specifically localized subpopulation of phosphorylated µ2, these data suggest that µ2 phosphorylation activity of AAK1 in vivo is tightly regulated. Unexpectedly, a significant decrease in phosphorylation of the large AP-2 subunits was observed. Thus, rather than altering the µ2 phosphorylation state, the observed receptor internalization block appears to result from the kinase activityindependent binding of full-length AAK1 to AP-2, which exerts a more global effect on AP-2 function.
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Postulating that AAK1 overexpression sequesters AP-2 in the cytosol, we determined the distribution of AP-2 in cytosol and membrane fractions. Surprisingly, WT AAK1 overexpression did not appear to alter the AP-2 distribution between soluble and particulate pools compared with control cells, or that of cells overexpressing K74A, D176A, AID, or AID constructs (Fig. 3 D; and data not depicted). Although these fractionation experiments cannot eliminate the possibility that cytosolic AP-2 forms sedimentable structures following AAK1 overexpression, or that AAK1 overexpressions causes the mislocalization of AP-2 to other disperse membranes within the cytosol, these observations suggest that AAK1 overexpression inhibits endocytosis of the Tfn receptor (TfnR) and LRP by functionally sequestering AP-2 complexes and preventing their clustering on the plasma membrane.
To extend our analysis of AAK1 function in CME, we used small interfering RNAs (siRNAs) to knock down AAK1 expression in cells. Transfection of two different siRNAs that specifically target AAK1 reduced AAK1 expression by 80% in either A549 or HeLa cells. However, in neither case did we observe an alteration in Tfn internalization, AP-2 distribution, or µ2 phosphorylation (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200304069/DC1). There are several possible conclusions that can be drawn based on the inability to detect an effect of AAK1 depletion on µ2 phosphorylation. One possibility is that AAK1 is not the endogenous µ2 kinase. However, given that AAK1 copurifies with CCVs and colocalizes with endocytic clathrin-coated pits in both neuronal and nonneuronal cells (Conner and Schmid, 2002), and that the AAK1 phosphorylation site on µ2 is required for endocytosis in vivo (Umeda et al., 2000; Ricotta et al., 2002), we think this is unlikely. Instead, our results may reflect functional redundancy with other kinases of the Ark1/Prk1 family. This prospect would be consistent with the functional redundancy observed in yeast between the Ark1p and Prk1p proteins that regulate actin dynamics and endocytosisa yeast phenotype is only observed following disruption of both the Ark1 and Prk1 genes (Cope et al., 1999). Indeed, GAK, another Ark1/Prk1 family member is known to be associated with CCVs and to phosphorylate µ2 in vitro (Korolchuk and Banting, 2002; Umeda et al., 2000). Other AAK1-related kinases also exist in the mammalian genome (Conner and Schmid, 2002). Moreover, it is also possible that the levels of AAK1 that remain following siRNA treatment are sufficient to support normal levels of µ2 phosphorylation.
The µ2 subunit of AP-2 specifically recognizes tyrosine-based internalization motifs, whereas other AP-2 subunits are believed to function in endocytosis by directing clathrin assembly into curved lattices and by recruiting other essential cofactors to the coated pit (Kirchhausen, 1999). Therefore, we expected that clathrin-coated pit assembly would also be disrupted in cells overexpressing inhibitory AAK1 constructs that functionally sequester AP-2. Surprisingly, clathrin recruitment into coated pits was not altered in WT AAK1overexpressing cells relative to controls (Fig. 4 A). Previous studies have established that EGF and TfnRs are internalized in the same coated pits (Lamaze et al., 1993). We therefore asked if AAK1 overexpression had any effect on EGF uptake. Surprisingly, neither WT nor K74A AAK1 overexpression had any effect on the internalization of EGF compared with controls (Fig. 4 , B and C). High concentrations of EGF are known to saturate the clathrin-mediated pathway for EGFR endocytosis (Jiang and Sorkin, 2003); therefore, care was taken to use low concentrations (2 ng/ml) of 125I-labeled EGF for these assays. As an additional control for CME, cells infected with recombinant K44A dynamin-1 adenovirus showed the expected EGF internalization defect (Damke et al., 1994). We cannot rule out that the small amounts of AP-2 remaining at the cell surface are selectively associated with coated pits engaged in EGF uptake. However, our results are completely consistent with recent findings reporting that siRNA-mediated AP-2depleted cells are capable of forming clathrin-coated pits that are competent for the internalization of the EGFR and an LDLR chimera, but defective in TfnR endocytosis (Motley et al., 2003). Thus, we conclude that the functional sequestration of AP-2 by AAK1 overexpression demonstrates an unexpected cargo-selective requirement for this coat constituent in CME.
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Materials and methods |
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Polyclonal antibodies against the COOH-terminal AID fragment of AAK1 expressed in E. coli and the NH2-terminal AID fragment expressed in baculovirus-infected Tn5 cells were generated in this laboratory as previously described (Conner and Schmid, 2002).
Endocytosis assays
Except where noted, tTA HeLa cells were cultured and infected in the presence of G418 to maintain expression of the tTA that is required for protein overexpression. tTA HeLa cells were infected with recombinant adenoviruses as previously described (Altschuler et al., 1998). Viral loads showing nearly 100% infection and uniform protein overexpression were used, as determined by immunolocalization. Internalization of biotinylated Tfn was assayed as previously described (Carter et al., 1993), assessing internalization by inaccessibility to avidin. Identical results were obtained by assessing resistance to MesNa (unpubished data). For EGF internalization, virus-infected cells were serum starved for 1.52 h in binding buffer (DMEM, 1% BSA) before the internalization assay. Cells were then detached from dishes in PBS/5 mM EDTA, rinsed with ice cold binding buffer, resuspended in binding buffer containing 2 ng/ml 125I-EGF, and incubated on ice for 1 h. Cells were then washed with binding buffer and aliquoted. One aliquot was kept on ice to measure total ligand binding, and the rest were transferred to 37°C for the indicated times, returned to ice to stop endocytosis, and then acid washed (0.5 M NaCl, 0.2 M acetic acid, pH 2.8) for 5 min on ice to remove surface bound ligand. Cells were pelleted, the supernatant containing released ligand was aspirated, and the samples were measured for internalization with a gamma counter.
Other assays
Kinase assays and in vitro protein interaction tests were performed essentially as described (Conner and Schmid, 2002). In vivo labeling and immunoprecipitation of AP-2 was performed as described using the mAb AP.6 (Wilde and Brodsky, 1996).
Online supplemental material
Fig. S1 shows immunofluorescence assays for RAP-GST endocytosis in control and WT AAK1overexpressing cells. Fig. S2 shows the time course of siRNA-mediated AAK1 reduction by immunoblot analysis as well as single round Tfn internalization assays after siRNA treatment with control and AAK1-specific oligonucleotides. Supplemental materials and methods include information regarding AAK1 site-directed mutagenesis, the generation of AAK1 adenovirus and baculovirus constructs, RAP internalization, and siRNA treatments. All supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200304069/DC1.
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Acknowledgments |
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S.L. Schmid and S.D. Conner were supported by National Institutes of Health grants (R37-MH61345 and GM20632-01, respectively). This is The Scripps Research Institute manuscript number 15361-CB.
Submitted: 14 April 2003
Accepted: 1 August 2003
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