Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis
Sean D Conner, Sandra L Schmid, Sean D Conner, Sandra L Schmid
Abstract
The mu 2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that mu 2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896-900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of alpha-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the mu subunit in vitro, and stage-specific assays for endocytosis show that mu phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous mu 2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.
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References
- Ahle, S., A. Mann, U. Eichelsbacher, and E. Ungewickell. 1988. Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 7:919–929.
- Bar-Zvi, D., and D. Branton. 1986. Clathrin-coated vesicles contain two protein kinase activities. Phosphorylation of clathrin beta-light chain by casein kinase II. J. Biol. Chem. 261:9614–9621.
- Bauerfeind, R., K. Takei, and P. De Camilli. 1997. Amphiphysin I is associated with coated endocytic intermediates and undergoes stimulation-dependent dephosphorylation in nerve terminals. J. Biol. Chem. 272:30984–30992.
- Brodsky, F.M. 1985. Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites. J. Cell Biol. 101:2047–2054.
- Carter, L.L., T.E. Redelmeier, L.A. Woollenweber, and S.L. Schmid. 1993. Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis. J. Cell Biol. 120:37–45.
- Chen, H., V.I. Slepnev, P.P. Di Fiore, and P. De Camilli. 1999. The interaction of epsin and Eps15 with the clathrin adaptor AP-2 is inhibited by mitotic phosphorylation and enhanced by stimulation-dependent dephosphorylation in nerve terminals. J. Biol. Chem. 274:3257–3260.
- Chin, D.J., R.M. Straubinger, S. Acton, I. Nathke, and F.M. Brodsky. 1989. 100-kDa polypeptides in peripheral clathrin-coated vesicles are required for receptor-mediated endocytosis. Proc. Natl. Acad. Sci. USA. 86:9289–9293.
- Cope, M.J., S. Yang, C. Shang, and D.G. Drubin. 1999. Novel protein kinases Ark1p and Prk1p associate with and regulate the cortical actin cytoskeleton in budding yeast. J. Cell Biol. 144:1203–1218.
- Cousin, M.A., T.C. Tan, and P.J. Robinson. 2001. Protein phosphorylation is required for endocytosis in nerve terminals: potential role for the dephosphins dynamin I and synaptojanin, but not AP180 or amphiphysin. J. Neurochem. 76:105–116.
- Fingerhut, A., K. von Figura, and S. Honing. 2001. Binding of AP2 to sorting signals is modulated by AP2 phosphorylation. J. Biol. Chem. 276:5476–5482.
- Georgieva-Hanson, V., W.J. Schook, and S. Puszkin. 1988. Brain coated vesicle destabilization and phosphorylation of coat proteins. J. Neurochem. 50:307–315.
- Hannan, L.A., S.L. Newmyer, and S.L. Schmid. 1998. ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: a dual role for Hsc70. Mol. Biol. Cell. 9:2217–2229.
- Harlow, E., and D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 726 pp.
- Keen, J.H., and M.M. Black. 1986. The phosphorylation of coated membrane proteins in intact neurons. J. Cell Biol. 102:1325–1333.
- Kirchhausen, T. 1999. Adaptors for clathrin-mediated traffic. Annu. Rev. Cell Dev. Biol. 15:705–732.
- Kirchhausen, T. 2000. Clathrin. Annu. Rev. Biochem. 69:699–727.
- Liu, J.P., A.T. Sim, and P.J. Robinson. 1994. Calcineurin inhibition of dynamin I GTPase activity coupled to nerve terminal depolarization. Science. 265:970–973.
- McLauchlan, H., J. Newell, N. Morrice, A. Osborne, M. West, and E. Smythe. 1998. A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits. Curr. Biol. 8:34–45.
- Morgan, J.R., K. Prasad, W. Hao, G.J. Augustine, and E.M. Lafer. 2000. A conserved clathrin assembly motif essential for synaptic vesicle endocytosis. J. Neurosci. 20:8667–8676.
- Morris, S.A., A. Mann, and E. Ungewickell. 1990. Analysis of 100-180-kDa phosphoproteins in clathrin-coated vesicles from bovine brain. J. Biol. Chem. 265:3354–3357.
- Musacchio, A., C.J. Smith, A.M. Roseman, S.C. Harrison, T. Kirchhausen, and B.M. Pearse. 1999. Functional organization of clathrin in coats: combining electron cryomicroscopy and x-ray crystallography. Mol. Cell. 3:761–770.
- Nathke, I.S., J. Heuser, A. Lupas, J. Stock, C.W. Turck, and F.M. Brodsky. 1992. Folding and trimerization of clathrin subunits at the triskelion hub. Cell. 68:899–910.
- Newmyer, S., and S. Schmid. 2001. Dominant-interfering hsc70 mutants disrupt multiple stages of the clathrin-coated vesicle cycle in vivo. J. Cell Biol. 152:607–620.
- Ohno, H., J. Stewart, M.C. Fournier, H. Bosshart, I. Rhee, S. Miyatake, T. Saito, A. Gallusser, T. Kirchhausen, and J.S. Bonifacino. 1995. Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science. 269:1872–1875.
- Ohno, H., M.C. Fournier, G. Poy, and J.S. Bonifacino. 1996. Structural determinants of interaction of tyrosine-based sorting signals with the adaptor medium chains. J. Biol. Chem. 271:29009–29015.
- Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Phosphorylation of threonine 156 of the μ2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo. Curr. Biol. 11:896–900.
- Owen, D.J., Y. Vallis, M.E. Noble, J.B. Hunter, T.R. Dafforn, P.R. Evans, and H.T. McMahon. 1999. A structural explanation for the binding of multiple ligands by the alpha-adaptin appendage domain. Cell. 97:805–815.
- Page, L.J., and M.S. Robinson. 1995. Targeting signals and subunit interactions in coated vesicle adaptor complexes. J. Cell Biol. 131:619–630.
- Pauloin, A., I. Bernier, and P. Jolles. 1982. Presence of cyclic nucleotide-Ca2+ independent protein kinase in bovine brain coated vesicles. Nature. 298:574–576.
- Pauloin, A., and C. Thurieau. 1993. The 50 kDa protein subunit of assembly polypeptide (AP) AP-2 adaptor from clathrin-coated vesicles is phosphorylated on threonine-156 by AP-1 and a soluble AP50 kinase which co-purifies with the assembly polypeptides. Biochem. J. 296:409–415.
- Pauloin, A., J. Loeb, and P. Jolles. 1984. Protein kinase(s) in bovine brain coated vesicles. Biochim. Biophys. Acta. 799:238–245.
- Pauloin, A., C. Thurieau, and P. Jolles. 1988. Cyclic phosphorylation/dephosphorylation cascade in bovine brain coated vesicles. Biochim. Biophys. Acta. 968:91–95.
- Rapoport, I., M. Miyazaki, W. Boll, B. Duckworth, L.C. Cantley, S. Shoelson, and T. Kirchhausen. 1997. Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes. EMBO J. 16:2240–2250.
- Ricotta, D., S.D. Comer, S.L. Schmid, K. von Figura, and S. Höning. 2002. Phosphorylation of the AP2 μ subunit by AAK1 mediates high affinity binding to the membrane protein sorting signals. J. Cell Biol. 156:791–795.
- Robinson, P.J., J.P. Liu, K.A. Powell, E.M. Fykse, and T.C. Sudhof. 1994. Phosphorylation of dynamin I and synaptic-vesicle recycling. Trends Neurosci. 17:348–353.
- Salcini, A.E., S. Confalonieri, M. Doria, E. Santolini, E. Tassi, O. Minenkova, G. Cesareni, P.G. Pelicci, and P.P. Di Fiore. 1997. Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module. Genes Dev. 11:2239–2249.
- Schibler, U., M. Tosi, A.C. Pittet, L. Fabiani, and P.K. Wellauer. 1980. Tissue-specific expression of mouse alpha-amylase genes. J. Mol. Biol. 142:93–116.
- Schmid, S.L. 1997. Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu. Rev. Biochem. 66:511–548.
- Shih, W., A. Gallusser, and T. Kirchhausen. 1995. A clathrin-binding site in the hinge of the beta 2 chain of mammalian AP-2 complexes. J. Biol. Chem. 270:31083–31090.
- Slepnev, V.I., G.C. Ochoa, M.H. Butler, D. Grabs, and P.D. Camilli. 1998. Role of phosphorylation in regulation of the assembly of endocytic coat complexes. Science. 281:821–824.
- Smythe, E., L.L. Carter, and S.L. Schmid. 1992. Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors. J. Cell Biol. 119:1163–1171.
- Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76:4350–4354.
- Turner, K.M., R.D. Burgoyne, and A. Morgan. 1999. Protein phosphorylation and the regulation of synaptic membrane traffic. Trends Neurosci. 22:459–464.
- Warnock, D.E., L.J. Terlecky, and S.L. Schmid. 1995. Dynamin GTPase is stimulated by crosslinking through the C-terminal proline-rich domain. EMBO J. 14:1322–1328.
- Wendland, B., and S.D. Emr. 1998. Pan1p, yeast eps15, functions as a multivalent adaptor that coordinates protein–protein interactions essential for endocytosis. J. Cell Biol. 141:71–84.
- Wilde, A., and F.M. Brodsky. 1996. In vivo phosphorylation of adaptors regulates their interaction with clathrin. J. Cell Biol. 135:635–645.
- Zeng, G., and M. Cai. 1999. Regulation of the actin cytoskeleton organization in yeast by a novel serine/threonine kinase Prk1p. J. Cell Biol. 144:71–82.
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