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• W1997347495 abstract "The two clathrin-associated adaptor complexes AP1 and AP2 are known to participate in the formation of clathrin-coated vesicles at the trans-Golgi network and at the plasma membrane. During this process adaptors are involved in the sequestration of vesicle cargo by binding to the sorting signals within the cytoplasmic domains of the cargo proteins and in the recruitment of the clathrin coat. After budding of the clathrin-coated vesicles, the clathrin and adaptors dissociate from the vesicles. Here we show that in vitro binding of AP2 to sorting signals, which is one of the initial steps in receptor-mediated endocytosis, is modulated by adaptor phosphorylation. AP2 was phosphorylated by incubating purified AP2 in the presence of ATP and dephosphorylated by incubation with alkaline phosphatase. Affinity for tyrosine-, leucine-based and noncanonical sorting motifs was 15–33 times higher for phosphorylated than for dephosphorylated AP2. Also the binding of AP2 to membranes was regulated by adaptor phosphorylation/dephosphorylation and was about 8-fold higher for phosphorylated than for dephosphorylated AP2. Moreover, AP2 isolated from cytosol is higher phosphorylated than membrane-extracted and exhibits a 5-fold higher binding affinity than AP2 extracted from membranes. Taken together these data point to a cycle of phosphorylation/dephosphorylation as a mechanism for regulating the reversible association of AP2 with membranes and sorting signals during the process of receptor-mediated endocytosis. The two clathrin-associated adaptor complexes AP1 and AP2 are known to participate in the formation of clathrin-coated vesicles at the trans-Golgi network and at the plasma membrane. During this process adaptors are involved in the sequestration of vesicle cargo by binding to the sorting signals within the cytoplasmic domains of the cargo proteins and in the recruitment of the clathrin coat. After budding of the clathrin-coated vesicles, the clathrin and adaptors dissociate from the vesicles. Here we show that in vitro binding of AP2 to sorting signals, which is one of the initial steps in receptor-mediated endocytosis, is modulated by adaptor phosphorylation. AP2 was phosphorylated by incubating purified AP2 in the presence of ATP and dephosphorylated by incubation with alkaline phosphatase. Affinity for tyrosine-, leucine-based and noncanonical sorting motifs was 15–33 times higher for phosphorylated than for dephosphorylated AP2. Also the binding of AP2 to membranes was regulated by adaptor phosphorylation/dephosphorylation and was about 8-fold higher for phosphorylated than for dephosphorylated AP2. Moreover, AP2 isolated from cytosol is higher phosphorylated than membrane-extracted and exhibits a 5-fold higher binding affinity than AP2 extracted from membranes. Taken together these data point to a cycle of phosphorylation/dephosphorylation as a mechanism for regulating the reversible association of AP2 with membranes and sorting signals during the process of receptor-mediated endocytosis. clathrin-coated vesicle(s) trans-Golgi network normal rat kidney Madin-Darby-Bovine-Kidney polyacrylamide gel electrophoresis high pressure liquid chromatography 4-morpholineethanesulfonic acid Trafficking of membrane proteins within the secretory and endocytic route of eukaryotic cells is characterized by the sorting of the membrane proteins at sites where transport pathways diverge and the packaging of the membrane proteins into vesicles occurs. The adaptors AP1 and AP2 appear to play a key role in the sorting and packaging of membrane proteins in clathrin-coated vesicles (CCVs).1 AP1-containing CCVs are formed at the TGN and facilitate the transport of cargo from the TGN to endosomes, whereas AP2-containing CCVs function in receptor-mediated endocytosis at the plasma membrane. The adaptors are heterotetrameric complexes composed of two 100-kDa subunits (designated γ and β1 in AP1 and α and β2 in AP2; for review see Ref.1Lewin D. Mellman I. Biochim. Biophys. Acta. 1998; 1401: 129-145Crossref PubMed Scopus (50) Google Scholar). The α-, β1-, and β2-subunits have been implicated in clathrin binding (2Goodman O.B. Keen J.H. J. Biol. Chem. 1995; 270: 23768-23773Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 3Shih W. Gallusser A. Kirchhausen T. J. Biol. Chem. 1995; 270: 31083-31090Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 4Traub L.M. Kornfeld S. Ungewickell E. J. Biol. Chem. 1995; 270: 4933-4942Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Recognition of the membrane proteins is mediated by the medium subunits μ1 and μ2 (5Ohno H. Aguilar R. Yeh D. Taurus D. Saito T. Bonifacino J. J. Biol. Chem. 1998; 273: 25915-25921Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), but also the β-subunits have been implicated in binding to membrane targets (6Beltzer J.P. Spiess M. EMBO J. 1991; 10: 3735-3742Crossref PubMed Scopus (81) Google Scholar, 7Rapoport I. Chen Y. Cupers P. Shoelson S. Kirchhausen T. EMBO J. 1998; 17: 2148-2155Crossref PubMed Scopus (258) Google Scholar). Tyrosine- and leucine-based sorting motifs in cytoplasmic tails of membrane proteins can serve as adaptor binding sites, and the tyrosine and leucine residues have been shown to be critical for the sorting of the membrane proteins, as well as for the adaptor binding (for review see Ref. 8Heilker R. Spiess M. Crottet P. BioAssays. 1999; 21: 558-567Crossref PubMed Scopus (121) Google Scholar). For noncanonical sequences mediating either sorting or adaptor binding, this correlation, however, remains to be established. After formation of the CCVs, the clathrin coat is removed by the uncoating ATPase (hsc70) in a reaction depending on ATP hydrolysis (9Braell W.A. Schlossman D.M. Schmid S.L. Rothman J.E. J. Cell Biol. 1984; 99: 734-741Crossref PubMed Scopus (93) Google Scholar, 10Höning S. Kreimer G. Robenek H. Jockusch B.M. J. Cell Sci. 1994; 107: 1185-1196PubMed Google Scholar, 11Hannan L. Newmeyer S. Schmid S. Mol. Biol. Cell. 1998; 9: 2217-2229Crossref PubMed Scopus (58) Google Scholar). The adaptors are removed by a yet uncharacterized reaction. Thus adaptors associate with specific membranes such as the TGN or the plasma membrane and dissociate from the vesicles budding from these membranes. The mechanisms that control the association of adaptors with and their dissociation from membranes are unknown. In principle three different mechanisms can be envisaged. First, the adaptors may become specifically modified before they bind to or dissociate from membranes. Second the targets in membranes, to which adaptors bind, are subject to a covalent modification prior to binding or to dissociation. Third, a change of the internal milieu of CCVs,e.g. by an ion pump, may translate into a conformational change of the target e.g. by altering its oligomeric state. Several components of CCVs are subject to phosphorylation, including clathrin, adaptors, and cytoplasmic tails of cargo proteins, which serve as membrane targets (12Hill B.L. Drickamer K. Brodsky F.M. Parham P. J. Biol. Chem. 1988; 263: 5499-5501Abstract Full Text PDF PubMed Google Scholar, 13Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar, 14Pitcher C. Höning S. Fingerhut A. Bowers K. Marsh M. Mol. Biol. Cell. 1999; 10: 677-691Crossref PubMed Scopus (135) Google Scholar). Furthermore several kinases are associated with CCVs and purified adaptors (15Pauloin A. Thurieau C. Biochem. J. 1993; 296: 409-415Crossref PubMed Scopus (28) Google Scholar, 16Bar-Zvi D. Mosley T. Branton D. J. Biol. Chem. 1988; 263: 4408-4415Abstract Full Text PDF PubMed Google Scholar, 17Morris S. Mann A. Ungewickell E. J. Biol. Chem. 1990; 265: 3354-3357Abstract Full Text PDF PubMed Google Scholar). The phosphorylation of membrane-associated and cytosolic AP2 has been shown to be different, with cytosolic AP2 being the higher phosphorylated species (13Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar). These observations point to a role of adaptor phosphorylation for regulating its binding to membranes. In the present study we analyzed the effect of phosphorylation and dephosphorylation of purified adaptors on the binding to peptides representing adaptor binding sequences, and we also analyzed AP2 binding to membranes. We observed that phosphorylation of AP2 enhances the association constant for in vitro binding to various AP2 binding motifs and the recruitment of AP2 to membranes, whereas dephosphorylation has the opposite effect. The phosphorylation of the α-, β2-, and μ2-subunit is catalyzed by kinases copurifying with AP2. Furthermore, cytosolic AP2 is higher phosphorylated and has a higher binding affinity as compared with membrane-extracted AP2. Taken together these data suggest that a cycle of phosphorylation and dephosphorylation of AP2 is regulating the binding of AP2 to membranes. NRK and MDBK cells were obtained from ATCC (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum. Antibodies specific for subunits of AP2 were obtained from Sigma (anti-γ and anti-α) and from Transduction Laboratories (anti-α and anti-β). The antibodies specific for μ2 and ς2 were kindly provided by M. S. Robinson (Cambridge, United Kingdom). Clathrin-coated vesicles were prepared from porcine brain according to Keen et al. (18Keen J.H. Willingham M.C. Pastan I.H. Cell. 1979; 16: 303-312Abstract Full Text PDF PubMed Scopus (299) Google Scholar). Coat proteins were extracted from CCVs with 0.5 m Tris-HCl, pH 7.5, for 1 h under rotation. After centrifugation at 100,000 × g for 30 min, the material was applied to a Superose-6 column (1.6 × 55 cm) connected to a fast protein liquid chromatography system (Amersham Pharmacia Biotech) at a flow rate of 0.3 ml/min. The column was equilibrated in 0.5m Tris-HCl, pH 7.0, 0.2 mm dithiothreitol. 1.5-ml fractions were collected, and adaptor-containing fractions were identified by SDS-PAGE. For the separation of AP1 from AP2, the adaptor-containing fractions were pooled and applied to hydroxyapatite chromatography according to Manfredi and Bazari (19Manfredi J.J. Bazari W.L. J. Biol. Chem. 1987; 262: 12182-12188Abstract Full Text PDF PubMed Google Scholar). The purity of AP1 and AP2 was analyzed by SDS-PAGE and Western blotting using monoclonal antibodies to the α-subunit of AP2 or the γ-subunit of AP1. Peptides were synthesized using amino acids protected with Fmoc (N-(9-fluorenyl)methoxycarbonyl) and activated with benzotriazol-1-yl-oxytripyrolidinophosphonium hexafluorophosphate and a 9050 peptide synthesizer (Millipore). After cleavage from the resin and the protecting groups, peptides were purified by reverse phase HPLC using Delta Pac C-18 column (Millipore) and an elution from 0–50% acetonitrile in 0.1% trifluoroacetic, water for 50 min. Purity was confirmed by HPLC, UV spectrometry, and mass spectrometry. Peptides corresponding to the tail sequences of the following membrane proteins were used in this study: from lysosomal acid phosphatase (RMQAQPPGYRHVADGQDHA), from the 67 residues comprising MPR46 tail the residues 2–16 (RLVVGAKGMEQFPHL) and 49–67 (GDDQLGEESEERDDHLLPM), and from invariant chain (MDDQRDLISNNEQLP-MLGRRPGAPESKCSR). The latter one was produced as a glutathione S-transferase fusion protein and was used as described (20Hofmann M. Höning S. Rodionov D. Dobberstein B. von Figura K. Bakke O. J. Biol. Chem. 1999; 274: 36153-36158Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). AP1 and AP2 were dialyzed into Buffer A (20 mm Hepes-NaOH, pH 7.0, 150 mm NaCl, 10 mm KCl, 2 mmMgCl2, 0.2 mm dithiothreitol) and incubated with various amounts of alkaline phosphatase (Roche Molecular Biochemicals) for 30 min at 37 °C. AP2 was dialyzed against 50 mmHepes-KOH, pH 7.4, 5 mm MgCl2, 2 mmMnCl2 and incubated with 1 μl of [γ-32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech) for 15 min at room temperature. The reaction was stopped by the addition of sample buffer and boiling for 5 min at 95 °C. The probes were analyzed by SDS-PAGE, autoradiography, and Western blotting with antibodies against the α-, β2- and μ2-subunits. For experiments including kinase inhibitors, a mixture of threonine and serine kinase inhibitors was used (catalog number 539572; Calbiochem). For phosphorylation with unlabeled ATP, AP2 was dialyzed into Buffer A and incubated for 30 min at 37 °C in the presence of 2 mmATP. To test for phosphatases copurifying with AP2, the adaptor was first incubated with [γ-32P]ATP for 30 min at 37 °C, was then treated with kinase inhibitors, and further incubated for up to 2 h at 37 °C. Subsequently, the samples were resolved on SDS-PAGE and analyzed after autoradiography. AP2 that was incubated in parallel with unlabeled ATP was analyzed for tail binding by using the biosensor. For phosphorylation with unlabeled ATP, AP2 was dialyzed into Buffer A and incubated for 30 min at 37 °C in the presence of 2 mm ATP. NRK cells were grown to confluency on a 15-cm dish. The cells were washed two times with phosphate-buffered saline, scraped in Buffer B (25 mm Hepes-KOH, pH 7.2, 125 mm KAc, 5 mm MgAc), and lysed by passing the suspension through a 22×g syringe 10 times. The cell lysate was centrifuged for 10 min at 1000 × g to remove intact cells and nuclei. The supernatant was removed and centrifuged for 30 min at 100,000 ×g to separate membranes (pellet) from the cytosol (supernatant). The membrane pellet was incubated for 1 h with 0.5m Tris-HCL, pH 7.5, to remove endogenous AP2. Subsequently the membranes were recovered by centrifugation at 100,000 ×g, solubilized in Buffer B, and stored in aliquots at −70 °C until use. AP2 was dialyzed in Buffer B, pre-incubated with alkaline phosphatase or ATP, and incubated with NRK membranes for 30 min on ice. The membranes were recovered by centrifugation at 100,000 × g for 15 min, solubilized in 2× sample buffer, resolved by SDS-PAGE, and analyzed by Western blotting with an antibody against the α-subunit of AP2. The interaction between AP1, AP2, or MDBK cytosolic and membrane fractions and the cytoplasmic tail peptides was analyzed in real time by surface plasmon resonance using a BIAcore 2000 biosensor (BIAcore AB). The peptides were coupled to a CM5 sensor chip via their primary amino groups following the manufacturer's instructions (immobilized ligands). Peptides with an isoelectric point below 3.5 could not be immobilized to the CM5 sensor chip via amino coupling because of their low pI. These peptides were immobilized to the sensor surface via the thiol group of a cysteine residue that was synthesized at the amino terminus (for details see Ref. 21Höning S. Sandoval I. von Figura K. EMBO J. 1998; 17: 1304-1314Crossref PubMed Scopus (246) Google Scholar). AP1 and AP2 (analytes) were injected at a flow rate of 20 μl/min unless stated otherwise. The adaptors were used at concentrations ranging from 50–350 nm to avoid mass transport effects that can occur if the analyte concentration is low. For cytosolic and membrane-extracted adaptors, concentrations were used ranging from 5–11 nmbecause of the limited amount of adaptors. Association (1–2 min) was followed by dissociation (2 min) during which Buffer A was perfused. Subsequently bound APs were removed by a short pulse injection (15 s) of 10 mm NaOH, 0.5% SDS. The rate constants (ka for association andkd for dissociation) of the interaction between tail peptides and the adaptor complexes were calculated by using the evaluation software of the BIAcore 2000. Association was determined 15–20 s after switching from buffer flow to adaptor solution to avoid distortions because of injection and mixing. The dissociation rate constants were determined 5–10 s after switching to buffer flow. The association rate constant ka, the dissociation rate constant kd, and the calculation of the equilibrium rate constant, KD =kd/ka, were determined by assuming a first order kinetic A + B = AB. Further details are described elsewhere (22Höning S. Sosa M. Hille-Rehfeld A. von Figura K. J. Biol. Chem. 1997; 272: 19884-19890Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 23Jonsson U. Fagerstam L. Ivarsson B. Johnsson B. Karlsson R. Lundh K. Lofas S. Persson B. Roos H. Ronnberg I. Biotechniques. 1991; 11: 620-627PubMed Google Scholar). MDBK cells were harvested in 100 mm Mes, pH 6.8, 1 mm EGTA, 0.5 mm MgCl2, and 0.2 mm dithiothreitol and lysed by passing the suspension through a 22×g syringe 10 times. The cell lysate was centrifuged at 1000 × g to remove intact cells and nuclei. The supernatant was removed and centrifuged for 30 min at 100,000 × g to separate membranes from the cytosol. The pellet was extracted with 0.5 m Tris, pH 7.5, and centrifuged for 30 min at 100,000 × g. For gel filtration the cytosol and the membrane extract (50-μl aliquots) were passed over a Superdex-200 column connected to a SMART™ system (Amersham Pharmacia Biotech), equilibrated, and eluted with Buffer A at a flow rate of 40 μl/min. Fractions of 50 μl were collected and analyzed by Western blotting using an antibody against the α-subunit of AP2. The AP2-positive fractions were combined and stored at −70 °C. AP2 was dialyzed against Buffer A and incubated with alkaline phosphatase for 30 min at 37 °C. AP2 was precipitated with methanol/chloroform (24Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3161) Google Scholar), subjected to isoelectric focusing according to Braun et al. (25Braun M.A. Waheed A. von Figura K. EMBO J. 1989; 8: 3633-3640Crossref PubMed Scopus (134) Google Scholar), transferred onto nitrocellulose, and analyzed by Western blotting with antibodies against the four subunits of AP2. AP1 and AP2 are phosphoproteins (13Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar, 16Bar-Zvi D. Mosley T. Branton D. J. Biol. Chem. 1988; 263: 4408-4415Abstract Full Text PDF PubMed Google Scholar,17Morris S. Mann A. Ungewickell E. J. Biol. Chem. 1990; 265: 3354-3357Abstract Full Text PDF PubMed Google Scholar), and both kinases and phosphatases copurify with adaptor complexes (15Pauloin A. Thurieau C. Biochem. J. 1993; 296: 409-415Crossref PubMed Scopus (28) Google Scholar, 17Morris S. Mann A. Ungewickell E. J. Biol. Chem. 1990; 265: 3354-3357Abstract Full Text PDF PubMed Google Scholar, 18Keen J.H. Willingham M.C. Pastan I.H. Cell. 1979; 16: 303-312Abstract Full Text PDF PubMed Scopus (299) Google Scholar). To test whether dephosphorylation of adaptors affects their binding to the cytoplasmic tails of transmembrane cargo proteins of CCVs, AP1 and AP2 purified from pig brain were treated with alkaline phosphatase. Following this treatment, the binding of adaptors to a peptide derived from the cytoplasmic tail of MPR46 was analyzed by surface plasmon resonance. MPR46 is a cargo molecule of AP1 CCVs at the TGN and of AP2 CCVs at the plasma membrane. The peptide representing residues 49–67 of the cytoplasmic tail of MPR46 is known to bind AP1 and AP2 with high affinity (22Höning S. Sosa M. Hille-Rehfeld A. von Figura K. J. Biol. Chem. 1997; 272: 19884-19890Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). A corresponding peptide in which the critical dileucine motif was mutated to a pair of alanines served as a control for AP2 binding. Treatment with phosphatase had only a minor effect on the affinity of AP1, whereas the affinity of AP2 was decreased about 20-fold from a KD of 16 nm to a KD of 366 nm. The decrease in affinity was because of a lower association rate constant (see Fig.1 and TableI). The effect of alkaline phosphatase treatment was dependent on the concentration of alkaline phosphatase (Fig. 2) and sensitive to the inhibition by 1 mm pyrophosphate or 1 mm vanadate (data not shown).Table IRate constants for the binding of AP1 and AP2 to the MPR46 tail peptide 49–67PhosphataseAP1AP2k ak dK Dk ak dK D1/M × s1/snm1/M × s1/snm−15 × 1043.9 × 10−32625 × 1044.1 × 10−316+8.9 × 1043.4 × 10−3381.5 × 1045.5 × 10−3366Purified AP1 and AP2 were incubated for 30 min at 37 °C in the absence or presence of 2 units of alkaline phosphatase. Subsequently, binding of AP1 and AP2 to the MPR46 tail peptide 49–67 was recorded with a biosensor as described under Fig. 1. The rate constants for association (k a), dissociation (k d), and the equilibrium rate constant (K D = k d/k a) were determined from the sensorgrams shown in Fig. 1. Open table in a new tab Figure 2Dose-dependent effect of alkaline phosphatase on AP2 binding. AP2 was incubated with various amounts of alkaline phosphatase prior to recording AP2 binding to the MPR46 tail peptide 49–67 as described in the legend to Fig. 1. The equilibrium rate constants for the AP2 binding to the tail peptide was determined (KD =kd/ka), and the values are plotted against the amount of phosphatase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Purified AP1 and AP2 were incubated for 30 min at 37 °C in the absence or presence of 2 units of alkaline phosphatase. Subsequently, binding of AP1 and AP2 to the MPR46 tail peptide 49–67 was recorded with a biosensor as described under Fig. 1. The rate constants for association (k a), dissociation (k d), and the equilibrium rate constant (K D = k d/k a) were determined from the sensorgrams shown in Fig. 1. In bovine kidney cells all subunits of AP2 except for the ς2-subunit are known to exist as phosphoproteins (13Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar). Isoelectric focusing of AP2 prior to and after phosphatase treatment confirmed these data for AP2 subunits from pig brain. The α-, β2-, and μ2-subunit exist as multiple forms, whereas the ς2-subunit exists as a single form. Phosphatase treatment shifted the pattern of the α-, β2-, and μ2-subunits toward more basic forms, whereas it did not affect the isoelectric point of ς2 (shown for α, β2, and ς in Fig.3). We conclude from these data that three of the four subunits of AP2 are phosphorylated and that dephosphorylation of AP2 reduces its high affinity in vitrobinding to a peptide derived from a transmembrane cargo protein of CCVs. AP2 recognizes in cytoplasmic tails of transmembrane proteins tyrosine- and leucine based- and noncanonical sorting motifs (8Heilker R. Spiess M. Crottet P. BioAssays. 1999; 21: 558-567Crossref PubMed Scopus (121) Google Scholar, 26Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-297Abstract Full Text PDF PubMed Scopus (258) Google Scholar). The tail peptide 49–67 of MPR46 contains a leucine-based and a noncanonical AP2 binding motif that can substitute each other (22Höning S. Sosa M. Hille-Rehfeld A. von Figura K. J. Biol. Chem. 1997; 272: 19884-19890Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). To determine whether dephosphorylation of AP2 reduces binding to either of these motifs, binding to the lysosomal acid phosphatase (LAP) tail peptide, the invariant chain tail peptide, and the MPR46 tail peptide 2–16 was determined. These peptides are known to bind AP2 via a tyrosine-based signal (LAP, 27), a leucine-based signal (invariant chain, 20), or a noncanonical sorting motif MPR46, 22). Alkaline phosphatase treatment reduced the affinity to any of the three classes of AP2 binding motifs by 4- to 15-fold (TableII). As for the MPR46 peptide 49–67 this decrease in affinity resulted almost exclusively from lower association rate constants (not shown).Table IIAP2 binding to tail peptides containing different sorting signalsAP2 treatmentK D (nm)NoATPPhosphataseLAP6423544Inv. chain250681011MPR46 (2–16)2210335MPR46 (49–67)177330Peptides corresponding to the cytoplasmic tails of LAP, invariant chain, and MPR46 were immobilized on a sensor surface and probed for AP2 binding as described above. Prior to tail peptide binding, AP2 was kept untreated or incubated with ATP (2 mm) or alkaline phosphatase (2 units) for 30 min at 37 °C. The equilibrium rate constants (K D) for the AP2 interaction with the various tail peptides were calculated from the BIAcore sensorgrams. The decrease or increase of the K D caused by preincubation with ATP or alkaline phosphatase resulted from changes of the association rate constants (not shown). Open table in a new tab Peptides corresponding to the cytoplasmic tails of LAP, invariant chain, and MPR46 were immobilized on a sensor surface and probed for AP2 binding as described above. Prior to tail peptide binding, AP2 was kept untreated or incubated with ATP (2 mm) or alkaline phosphatase (2 units) for 30 min at 37 °C. The equilibrium rate constants (K D) for the AP2 interaction with the various tail peptides were calculated from the BIAcore sensorgrams. The decrease or increase of the K D caused by preincubation with ATP or alkaline phosphatase resulted from changes of the association rate constants (not shown). Endogenous kinases associated with purified AP2 are known to phosphorylate AP2 subunits (17Morris S. Mann A. Ungewickell E. J. Biol. Chem. 1990; 265: 3354-3357Abstract Full Text PDF PubMed Google Scholar,19Manfredi J.J. Bazari W.L. J. Biol. Chem. 1987; 262: 12182-12188Abstract Full Text PDF PubMed Google Scholar). When AP2 purified from pig brain was incubated in the presence of [γ-32P]ATP, incorporation of32P into the α-, β2-, and μ2-subunits was observed (Fig. 4). To test the influence of AP2 phosphorylation and dephosphorylation more precisely, we purified AP2 and devided the preparation into three aliquots. One aliquot was treated with alkaline phosphatase, one was incubated in the presence of ATP to allow phosphorylation by copurified kinases, and one aliquot served as a nontreated control. One-third of each sample was subsequently analyzed for binding to the MPR46 tail peptide 49–67 (Fig. 5 B), whereas the remaining sample was subjected to isoelectric focusing, followed by Western blotting and detection of the AP2 β-subunit (Fig.5 A). As shown in Fig. 5 A, the purified AP2 β-subunit (mock) exists as two major forms with pIs of 6.5 and 7.2 (Fig. 5 A, lane 1). Incubation with alkaline phosphatase resulted in a shift of almost all β2 to the basic form (lane 2). When incubated in the presence of ATP, β2 was converted to more acidic forms (lane 3). A similar result was obtained for the α-subunit (data not shown). This indicates that the bulk of isolated AP2 represents a partially phosphorylated form (dash), which is converted by dephosphorylation or phosphorylation into more basic (open triangle) or acidic (filled triangle) forms, respectively. In conclusion, dephosphorylation of AP2 with alkaline phosphatase or phosphorylation by copurifying kinases changes the phosphorylation state of the entire AP2 pool and not just of a fraction of it. When the same aliquots were analyzed for binding to the MPR46 tail peptide 49–67 (see Fig. 5 B and Table II), the phosphorylation of AP2 by endogenous kinases led to a 2.5-fold enhanced association rate to the peptide 49–67 of MPR46 (Fig. 5 B,+ATP) as compared with untreated AP2 (mock). On the other hand, the phosphatase-treated AP2 sample exhibited a 20-fold decreased affinity for the tail peptide, clearly demonstrating that the phosphorylation status of AP2 is a critical determinant for the binding to sorting signals. Phosphorylation also enhanced the affinity of AP2 to pe343 ptides representing tyrosine- and leucine-based and noncanonical AP2 binding motifs 2- to 4-fold (Table II). The presence of 150 μg/ml polylysine during the phosphorylation reaction affected neither the incorporation of 32P into nor the affinity of AP2 (not shown). We conclude from these data that in vitro phosphorylation of AP2 by associated kinases enhances the affinity of AP2 to the different classes of AP2 binding motifs. When phosphorylated AP2 was incubated in the presence of general protein kinase inhibitors for 30 min at 37 °C, no dephosphorylation was detectable (not shown). This suggests that in contrast to kinases, endogenous phosphatases are not associated with purified AP2. The experiments described above had demonstrated the dependence of in vitro binding of AP2 to immobilized peptides by the phosphorylation status of AP2. Next we assayed whether phosphorylation or dephosphorylation of AP2 affects its binding to membranes. A membrane fraction was prepared from NRK cells and stripped from endogenous adaptors by treatment with 0.5m Tris, pH 7.5. The purified AP2 was incubated for 30 min at 37 °C in the presence of ATP, and subsequently the AP2 was placed on ice and incubated for 30 min in the presence of membranes. Membrane-associated AP2 was then pelleted and quantified by Western blotting (Fig. 6). Incubation of AP2 in the presence of ATP increases the amount of membrane-bound AP2 as compared with a control in which AP2 was incubated in the absence of ATP by 4.1 ± 1.0-fold (n = 5). Under the experimental conditions used, the ATP that was used to stimulate the phosphorylation of AP2 may also affect the phosphorylation status of some membrane components even at 4 °C. To rule out this possibility, AP2 was cleared from ATP by centrifugation through a high molecular weight cut-off filter and then incubated with the membranes. The stimulation of AP2 binding was similar as before (data not shown), indicating that it is the phosphorylation of AP2 that increases the AP2 association with membranes. The amount of membrane binding of AP2 was lowered by 2.0 ± 1.1-fold (n = 4) when the complex was treated with alkaline phosphatase for 30 min at 37 °C and then incubated on ice 30 min with m" @default.
• W1997347495 created "2016-06-24" @default.
• W1997347495 creator A5012975261 @default.
• W1997347495 creator A5013229171 @default.
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• W1997347495 date "2001-02-01" @default.
• W1997347495 modified "2023-09-28" @default.
• W1997347495 title "Binding of AP2 to Sorting Signals Is Modulated by AP2 Phosphorylation" @default.
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