J. Cell Biol., Volume 141, Number 2, April 20, 1998 349-358

Cytoplasmic Tail Phosphorylation of the -Factor Receptor Is Required for Its Ubiquitination and Internalization

Linda Hicke,^* Bettina Zanolari, and Howard Riezman

^* Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208; and  Department of Biochemistry, Biozentrum, University of Basel, Basel, Switzerland CH-4056

G protein-coupled (GPC) receptors are phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation. The -factor pheromone receptor (Ste2p) of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding -factor. Ubiquitination triggers Ste2p internalization into the endocytic pathway. Here we demonstrate that phosphorylation of Ste2p promotes downregulation by positively regulating ubiquitination and internalization. Serines and a lysine are essential elements of the Ste2p SINNDAKSS internalization signal that can mediate both constitutive and ligand-stimulated endocytosis. The SINNDAKSS serines are required for receptor phosphorylation which, in turn, facilitates ubiquitination of the neighboring lysine. Constitutive phosphorylation is required to promote constitutive internalization, and is also a prerequisite for ligand-induced phosphorylation at or near the SINNDAKSS sequence. Mutants defective in yeast casein kinase I homologues are unable to internalize -factor, and do not phosphorylate or ubiquitinate the receptor, indicating that these kinases play a direct or indirect role in phosphorylating the receptor. Finally, we provide evidence that the primary function of phosphorylation controlled by the SINNDAKSS sequence is to trigger receptor internalization, demonstrating that phosphorylation-dependent endocytosis is an important mechanism for the downregulation of GPC receptor activity.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

• Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama, A., Date, T., Matsuura, Y., Miyamura, T., Wakita, T., Suzuki, T. (2008). Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core Protein Is Critical for the Production of Infectious Virus Particles. J. Virol. 82: 7964-7976 [Abstract] [Full Text]  
• Lee, S. C., Schmidtke, S. N., Dangott, L. J., Shaw, B. D. (2008). Aspergillus nidulans ArfB Plays a Role in Endocytosis and Polarized Growth. Eukaryot Cell 7: 1278-1288 [Abstract] [Full Text]  
• Geldner, N., Robatzek, S. (2008). Plant Receptors Go Endosomal: A Moving View on Signal Transduction. Plant Physiol. 147: 1565-1574 [Full Text]  
• Li, J.-G., Haines, D. S., Liu-Chen, L.-Y. (2008). Agonist-Promoted Lys63-Linked Polyubiquitination of the Human {kappa}-Opioid Receptor Is Involved in Receptor Down-Regulation. Mol. Pharmacol. 73: 1319-1330 [Abstract] [Full Text]  
• Estrella, L. A., Krishnamurthy, S., Timme, C. R., Hampsey, M. (2008). The Rsp5 E3 Ligase Mediates Turnover of Low Affinity Phosphate Transporters in Saccharomyces cerevisiae. J. Biol. Chem. 283: 5327-5334 [Abstract] [Full Text]  
• Diestel, S., Schaefer, D., Cremer, H., Schmitz, B. (2007). NCAM is ubiquitylated, endocytosed and recycled in neurons. J. Cell Sci. 120: 4035-4049 [Abstract] [Full Text]  
• Zhou, J., Livak, M. F. A., Bernier, M., Muller, D. C., Carlson, O. D., Elahi, D., Maudsley, S., Egan, J. M. (2007). Ubiquitination is involved in glucose-mediated downregulation of GIP receptors in islets. Am. J. Physiol. Endocrinol. Metab. 293: E538-E547 [Abstract] [Full Text]  
• Geldner, N., Hyman, D. L., Wang, X., Schumacher, K., Chory, J. (2007). Endosomal signaling of plant steroid receptor kinase BRI1. Genes Dev. 21: 1598-1602 [Abstract] [Full Text]  
• Dupre-Crochet, S., Figueroa, A., Hogan, C., Ferber, E. C., Bialucha, C. U., Adams, J., Richardson, E. C. N., Fujita, Y. (2007). Casein Kinase 1 Is a Novel Negative Regulator of E-Cadherin-Based Cell-Cell Contacts. Mol. Cell. Biol. 27: 3804-3816 [Abstract] [Full Text]  
• Shenoy, S. K. (2007). Seven-Transmembrane Receptors and Ubiquitination. Circ. Res. 100: 1142-1154 [Abstract] [Full Text]  
• Kota, J., Melin-Larsson, M., Ljungdahl, P. O., Forsberg, H. (2007). Ssh4, Rcr2 and Rcr1 Affect Plasma Membrane Transporter Activity in Saccharomyces cerevisiae. Genetics 175: 1681-1694 [Abstract] [Full Text]  
• Toret, C. P., Drubin, D. G. (2006). The budding yeast endocytic pathway. J. Cell Sci. 119: 4585-4587 [Full Text]  
• Mero, P., Zhang, C. Y., Huang, Z.-Y., Kim, M.-K., Schreiber, A. D., Grinstein, S., Booth, J. W. (2006). Phosphorylation-independent Ubiquitylation and Endocytosis of Fc{gamma}RIIA. J. Biol. Chem. 281: 33242-33249 [Abstract] [Full Text]  
• Staub, O., Rotin, D. (2006). Role of Ubiquitylation in Cellular Membrane Transport. Physiol. Rev. 86: 669-707 [Abstract] [Full Text]  
• Gadura, N., Robinson, L. C., Michels, C. A. (2006). Glc7-Reg1 Phosphatase Signals to Yck1,2 Casein Kinase 1 to Regulate Transport Activity and Glucose-Induced Inactivation of Saccharomyces Maltose Permease. Genetics 172: 1427-1439 [Abstract] [Full Text]  
• Kim, K., Galletta, B. J., Schmidt, K. O., Chang, F. S., Blumer, K. J., Cooper, J. A. (2006). Actin-based Motility during Endocytosis in Budding Yeast. Mol. Biol. Cell 17: 1354-1363 [Abstract] [Full Text]  
• Levin, D. E. (2005). Cell Wall Integrity Signaling in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 69: 262-291 [Abstract] [Full Text]  
• Gruhler, A., Olsen, J. V., Mohammed, S., Mortensen, P., Faergeman, N. J., Mann, M., Jensen, O. N. (2005). Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway. Mol. Cell. Proteomics 4: 310-327 [Abstract] [Full Text]  
• Schmitz, C., Kinner, A., Kolling, R. (2005). The Deubiquitinating Enzyme Ubp1 Affects Sorting of the ATP-binding Cassette-Transporter Ste6 in the Endocytic Pathway. Mol. Biol. Cell 16: 1319-1329 [Abstract] [Full Text]  
• Johnston, H. D., Foote, C., Santeford, A., Nothwehr, S. F. (2005). Golgi-to-Late Endosome Trafficking of the Yeast Pheromone Processing Enzyme Ste13p Is Regulated by a Phosphorylation Site in its Cytosolic Domain. Mol. Biol. Cell 16: 1456-1468 [Abstract] [Full Text]  
• Chen, S. H., Chen, S., Tokarev, A. A., Liu, F., Jedd, G., Segev, N. (2005). Ypt31/32 GTPases and Their Novel F-Box Effector Protein Rcy1 Regulate Protein Recycling. Mol. Biol. Cell 16: 178-192 [Abstract] [Full Text]  
• Leithe, E., Rivedal, E. (2004). Ubiquitination and Down-regulation of Gap Junction Protein Connexin-43 in Response to 12-O-Tetradecanoylphorbol 13-Acetate Treatment. J. Biol. Chem. 279: 50089-50096 [Abstract] [Full Text]  
• Abdel-Sater, F., El Bakkoury, M., Urrestarazu, A., Vissers, S., Andre, B. (2004). Amino Acid Signaling in Yeast: Casein Kinase I and the Ssy5 Endoprotease Are Key Determinants of Endoproteolytic Activation of the Membrane-Bound Stp1 Transcription Factor. Mol. Cell. Biol. 24: 9771-9785 [Abstract] [Full Text]  
• Liang, W., Fishman, P. H. (2004). Resistance of the Human {beta}1-Adrenergic Receptor to Agonist-induced Ubiquitination: A MECHANISM FOR IMPAIRED RECEPTOR DEGRADATION. J. Biol. Chem. 279: 46882-46889 [Abstract] [Full Text]  
• Zhang, Z., Wu, J.-Y., Hait, W. N., Yang, J.-M. (2004). Regulation of the Stability of P-Glycoprotein by Ubiquitination. Mol. Pharmacol. 66: 395-403 [Abstract] [Full Text]  
• Babu, P., Deschenes, R. J., Robinson, L. C. (2004). Akr1p-dependent Palmitoylation of Yck2p Yeast Casein Kinase 1 Is Necessary and Sufficient for Plasma Membrane Targeting. J. Biol. Chem. 279: 27138-27147 [Abstract] [Full Text]  
• Yin, D., Gavi, S., Wang, H.-y., Malbon, C. C. (2004). Probing Receptor Structure/Function with Chimeric G-Protein-Coupled Receptors. Mol. Pharmacol. 65: 1323-1332 [Abstract] [Full Text]  
• Hao, N., Yildirim, N., Wang, Y., Elston, T. C., Dohlman, H. G. (2003). Regulators of G Protein Signaling and Transient Activation of Signaling: EXPERIMENTAL AND COMPUTATIONAL ANALYSIS REVEALS NEGATIVE AND POSITIVE FEEDBACK CONTROLS ON G PROTEIN ACTIVITY. J. Biol. Chem. 278: 46506-46515 [Abstract] [Full Text]  
• Gitan, R. S., Shababi, M., Kramer, M., Eide, D. J. (2003). A Cytosolic Domain of the Yeast Zrt1 Zinc Transporter Is Required for Its Post-translational Inactivation in Response to Zinc and Cadmium. J. Biol. Chem. 278: 39558-39564 [Abstract] [Full Text]  
• Wu, C., Arcand, M., Jansen, G., Zhong, M., Iouk, T., Thomas, D. Y., Meloche, S., Whiteway, M. (2003). Phosphorylation of the MAPKKK Regulator Ste50p in Saccharomyces cerevisiae: a Casein Kinase I Phosphorylation Site Is Required for Proper Mating Function. Eukaryot Cell 2: 949-961 [Abstract] [Full Text]  
• Wang, Y., Ge, Q., Houston, D., Thorner, J., Errede, B., Dohlman, H. G. (2003). Regulation of Ste7 Ubiquitination by Ste11 Phosphorylation and the Skp1-Cullin-F-box Complex. J. Biol. Chem. 278: 22284-22289 [Abstract] [Full Text]  
• Babu, P., Bryan, J. D., Panek, H. R., Jordan, S. L., Forbrich, B. M., Kelley, S. C., Colvin, R. T., Robinson, L. C. (2003). Plasma membrane localization of the Yck2p yeast casein kinase 1 isoform requires the C-terminal extension and secretory pathway function. J. Cell Sci. 115: 4957-4968 [Abstract] [Full Text]  
• Aguilar, R. C., Watson, H. A., Wendland, B. (2003). The Yeast Epsin Ent1 Is Recruited to Membranes through Multiple Independent Interactions. J. Biol. Chem. 278: 10737-10743 [Abstract] [Full Text]  
• Klapisz, E., Sorokina, I., Lemeer, S., Pijnenburg, M., Verkleij, A. J., van Bergen en Henegouwen, P. M. P. (2002). A Ubiquitin-interacting Motif (UIM) Is Essential for Eps15 and Eps15R Ubiquitination. J. Biol. Chem. 277: 30746-30753 [Abstract] [Full Text]  
• Heese-Peck, A., Pichler, H., Zanolari, B., Watanabe, R., Daum, G., Riezman, H. (2002). Multiple Functions of Sterols in Yeast Endocytosis. Mol. Biol. Cell 13: 2664-2680 [Abstract] [Full Text]  
• Wang, Y., Dohlman, H. G. (2002). Pheromone-dependent Ubiquitination of the Mitogen-activated Protein Kinase Kinase Ste7. J. Biol. Chem. 277: 15766-15772 [Abstract] [Full Text]  
• Howard, J. P., Hutton, J. L., Olson, J. M., Payne, G. S. (2002). Sla1p serves as the targeting signal recognition factor for NPFX(1,2)D-mediated endocytosis. J. Cell Biol. 157: 315-326 [Abstract] [Full Text]  
• Parrish, W., Eilers, M., Ying, W., Konopka, J. B. (2002). The Cytoplasmic End of Transmembrane Domain 3 Regulates the Activity of the Saccharomyces cerevisiae G-Protein-Coupled {alpha}-Factor Receptor. Genetics 160: 429-443 [Abstract] [Full Text]  
• Tamai, K. K., Shimoda, C. (2002). The novel HECT-type ubiquitin-protein ligase Pub2p shares partially overlapping function with Pub1p in Schizosaccharomyces pombe. J. Cell Sci. 115: 1847-1857 [Abstract] [Full Text]  
• Marchal, C., Dupre, S., Urban-Grimal, D. (2002). Casein kinase I controls a late step in the endocytic trafficking of yeast uracil permease. J. Cell Sci. 115: 217-226 [Abstract] [Full Text]  
• Marchese, A., Benovic, J. L. (2001). Agonist-promoted Ubiquitination of the G Protein-coupled Receptor CXCR4 Mediates Lysosomal Sorting. J. Biol. Chem. 276: 45509-45512 [Abstract] [Full Text]  
• Wang, G., McCaffery, J. M., Wendland, B., Dupré, S., Haguenauer-Tsapis, R., Huibregtse, J. M. (2001). Localization of the Rsp5p Ubiquitin-Protein Ligase at Multiple Sites within the Endocytic Pathway. Mol. Cell. Biol. 21: 3564-3575 [Abstract] [Full Text]  
• Galan, J.-M., Wiederkehr, A., Seol, J. H., Haguenauer-Tsapis, R., Deshaies, R. J., Riezman, H., Peter, M. (2001). Skp1p and the F-Box Protein Rcy1p Form a Non-SCF Complex Involved in Recycling of the SNARE Snc1p in Yeast. Mol. Cell. Biol. 21: 3105-3117 [Abstract] [Full Text]  
• Dunn, R., Hicke, L. (2001). Domains of the Rsp5 Ubiquitin-Protein Ligase Required for Receptor-mediated and Fluid-Phase Endocytosis. Mol. Biol. Cell 12: 421-435 [Abstract] [Full Text]  
• Dosil, M., Schandel, K. A., Gupta, E., Jenness, D. D., Konopka, J. B. (2000). The C Terminus of the Saccharomyces cerevisiae alpha -Factor Receptor Contributes to the Formation of Preactivation Complexes with Its Cognate G Protein. Mol. Cell. Biol. 20: 5321-5329 [Abstract] [Full Text]  
• Feng, Y., Davis, N. G. (2000). Akr1p and the Type I Casein Kinases Act prior to the Ubiquitination Step of Yeast Endocytosis: Akr1p Is Required for Kinase Localization to the Plasma Membrane. Mol. Cell. Biol. 20: 5350-5359 [Abstract] [Full Text]  
• Wiederkehr, A., Avaro, S., Prescianotto-Baschong, C., Haguenauer-Tsapis, R., Riezman, H. (2000). The F-box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae. J. Cell Biol. 149: 397-410 [Abstract] [Full Text]  
• Feng, Y., Davis, N. G. (2000). Feedback Phosphorylation of the Yeast a-Factor Receptor Requires Activation of the Downstream Signaling Pathway from G Protein through Mitogen-Activated Protein Kinase. Mol. Cell. Biol. 20: 563-574 [Abstract] [Full Text]  
• Panek, H., Conibear, E, Bryan, J., Colvin, R., Goshorn, C., Robinson, L. (2000). Identification of Rgp1p, a novel Golgi recycling factor, as a protein required for efficient localization of yeast casein kinase 1 to the plasma membrane. J. Cell Sci. 113: 4545-4555 [Abstract]  
• Lill, N. L., Douillard, P., Awwad, R. A., Ota, S., Lupher, M. L. Jr., Miyake, S., Meissner-Lula, N., Hsu, V. W., Band, H. (2000). The Evolutionarily Conserved N-terminal Region of Cbl Is Sufficient to Enhance Down-regulation of the Epidermal Growth Factor Receptor. J. Biol. Chem. 275: 367-377 [Abstract] [Full Text]  
• Pruyne, D, Bretscher, A (2000). Polarization of cell growth in yeast. J. Cell Sci. 113: 571-585 [Abstract]  
• Decottignies, A., Owsianik, G., Ghislain, M. (1999). Casein Kinase I-dependent Phosphorylation and Stability of the Yeast Multidrug Transporter Pdr5p. J. Biol. Chem. 274: 37139-37146 [Abstract] [Full Text]  
• Blomenröhr, M., Heding, A., Sellar, R., Leurs, R., Bogerd, J., Eidne, K. A., Willars, G. B. (1999). Pivotal Role for the Cytoplasmic Carboxyl-Terminal Tail of a Nonmammalian Gonadotropin-Releasing Hormone Receptor in Cell Surface Expression, Ligand Binding, and Receptor Phosphorylation and Internalization. Mol. Pharmacol. 56: 1229-1237 [Abstract] [Full Text]  
• Cotlin, L. F., Siddiqui, M. A., Simpson, F., Collawn, J. F. (1999). Casein Kinase II Activity Is Required for Transferrin Receptor Endocytosis. J. Biol. Chem. 274: 30550-30556 [Abstract] [Full Text]  
• DeZwaan, T. M., Carroll, A. M., Valent, B., Sweigard, J. A. (1999). Magnaporthe grisea Pth11p Is a Novel Plasma Membrane Protein That Mediates Appressorium Differentiation in Response to Inductive Substrate Cues. Plant Cell 11: 2013-2030 [Abstract] [Full Text]  
• Beck, T., Schmidt, A., Hall, M. N. (1999). Starvation Induces Vacuolar Targeting and Degradation of the Tryptophan Permease in Yeast. J. Cell Biol. 146: 1227-1238 [Abstract] [Full Text]  
• Marsh, M., McMahon, H. T. (1999). The Structural Era of Endocytosis. Science 285: 215-220 [Abstract] [Full Text]  
• Robinson, L. C., Bradley, C., Bryan, J. D., Jerome, A., Kweon, Y., Panek, H. R. (1999). The Yck2 Yeast Casein Kinase 1 Isoform Shows Cell Cycle-specific Localization to Sites of Polarized Growth and Is Required for Proper Septin Organization. Mol. Biol. Cell 10: 1077-1092 [Abstract] [Full Text]  
• Pitcher, C., Höning, S., Fingerhut, A., Bowers, K., Marsh, M. (1999). Cluster of Differentiation Antigen 4 () Endocytosis and Adaptor Complex Binding Require Activation of the Endocytosis Signal by Serine Phosphorylation. Mol. Biol. Cell 10: 677-691 [Abstract] [Full Text]  
• Murakami, A., Kimura, K., Nakano, A. (1999). The Inactive Form of a Yeast Casein Kinase I Suppresses the Secretory Defect of the sec12 Mutant. IMPLICATION OF NEGATIVE REGULATION BY THE Hrr25 KINASE IN THE VESICLE BUDDING FROM THE ENDOPLASMIC RETICULUM. J. Biol. Chem. 274: 3804-3810 [Abstract] [Full Text]  
• Vickery, R. G., von Zastrow, M. (1999). Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes. J. Cell Biol. 144: 31-43 [Abstract] [Full Text]  
• Gammie, A. E., Stewart, B. G., Scott, C. F., Rose, M. D. (1999). The Two Forms of Karyogamy Transcription Factor Kar4p Are Regulated by Differential Initiation of Transcription, Translation, and Protein Turnover. Mol. Cell. Biol. 19: 817-825 [Abstract] [Full Text]  
• Breuer, P., Braulke, T. (1998). Stabilization of Mutant 46-kDa Mannose 6-Phosphate Receptors by Proteasomal Inhibitor Lactacystin. J. Biol. Chem. 273: 33254-33258 [Abstract] [Full Text]  
• Dube, P., Konopka, J. B. (1998). Identification of a Polar Region in Transmembrane Domain 6 That Regulates the Function of the G Protein-Coupled alpha -Factor Receptor. Mol. Cell. Biol. 18: 7205-7215 [Abstract] [Full Text]  
• Budd, D. C., McDonald, J. E., Tobin, A. B. (2000). Phosphorylation and Regulation of a Gq/11-coupled Receptor by Casein Kinase 1alpha. J. Biol. Chem. 275: 19667-19675 [Abstract] [Full Text]  
• Marchal, C., Haguenauer-Tsapis, R., Urban-Grimal, D. (2000). Casein Kinase I-dependent Phosphorylation within a PEST Sequence and Ubiquitination at Nearby Lysines Signal Endocytosis of Yeast Uracil Permease. J. Biol. Chem. 275: 23608-23614 [Abstract] [Full Text]  
• Flanary, P. L., DiBello, P. R., Estrada, P., Dohlman, H. G. (2000). Functional Analysis of Plp1 and Plp2, Two Homologues of Phosducin in Yeast. J. Biol. Chem. 275: 18462-18469 [Abstract] [Full Text]  
• Petaja-Repo, U. E., Hogue, M., Laperriere, A., Bhalla, S., Walker, P., Bouvier, M. (2001). Newly Synthesized Human delta Opioid Receptors Retained in the Endoplasmic Reticulum Are Retrotranslocated to the Cytosol, Deglycosylated, Ubiquitinated, and Degraded by the Proteasome. J. Biol. Chem. 276: 4416-4423 [Abstract] [Full Text]  
• Dubois, T., Kerai, P., Zemlickova, E., Howell, S., Jackson, T. R., Venkateswarlu, K., Cullen, P. J., Theibert, A. B., Larose, L., Roach, P. J., Aitken, A. (2001). Casein Kinase I Associates with Members of the Centaurin-alpha Family of Phosphatidylinositol 3,4,5-Trisphosphate-binding Proteins. J. Biol. Chem. 276: 18757-18764 [Abstract] [Full Text]  
• Hu, M. C., Fan, L., Crowder, L. A., Karim-Jimenez, Z., Murer, H., Moe, O. W. (2001). Dopamine Acutely Stimulates Na+/H+ Exchanger (NHE3) Endocytosis via Clathrin-coated Vesicles. DEPENDENCE ON PROTEIN KINASE A-MEDIATED NHE3 PHOSPHORYLATION. J. Biol. Chem. 276: 26906-26915 [Abstract] [Full Text]  
• Burchett, S. A., Scott, A., Errede, B., Dohlman, H. G. (2001). Identification of Novel Pheromone-response Regulators through Systematic Overexpression of 120 Protein Kinases in Yeast. J. Biol. Chem. 276: 26472-26478 [Abstract] [Full Text]  
• Dunn, R., Hicke, L. (2001). Multiple Roles for Rsp5p-dependent Ubiquitination at the Internalization Step of Endocytosis. J. Biol. Chem. 276: 25974-25981 [Abstract] [Full Text]  
• deHart, A. K.A., Schnell, J. D., Allen, D. A., Hicke, L. (2002). The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast. J. Cell Biol. 156: 241-248 [Abstract] [Full Text]  
• Howard, J. P., Hutton, J. L., Olson, J. M., Payne, G. S. (2002). Sla1p serves as the targeting signal recognition factor for NPFX(1,2)D-mediated endocytosis. J. Cell Biol. 157: 315-326 [Abstract] [Full Text]  

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents