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Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins
BBRC Biochemical and Biophysical Research Communications 330 (2005) 695–700 www.elsevier.com/locate/ybbrc
Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins A. Alberdi *, T. Sartor, M.A. Sosa Instituto de Histologı´a y Embriologı´a, Facultad de Ciencias Me´dicas, Universidad Nacional de Cuyo, Mendoza (5500), Argentina Received 21 February 2005 Available online 17 March 2005
Abstract Phosphorylation of proteins appears as a key process in early steps of clathrin coated vesicle formation. Here, we report that treatment of post-nuclear fraction with alkaline phosphatase induced redistribution of a subunits of AP-2 adaptor complex to cytosol and this effect was higher in the a2 subunit. A high serine phosphorylation status of a subunits correlated with the higher affinity of AP-2 to membranes. Using a simple binding assay, where membranes were incubated with either purified adaptors or cytosols, we observed an inhibitory effect of tyrphostin, a tyrosine kinase inhibitor, on the binding of AP-2 to membranes, but also an unexpected decrease induced by the phosphatase inhibitor cyclosporine. We also show an inhibitory effect of ATP mediated by cytosolic proteins, although it could not be related to the phosphorylation of AP-2, suggesting an action upstream a cascade of phosphorylations that participate in the regulation of the assembly of AP-2 to membranes. 2005 Elsevier Inc. All rights reserved. Keywords: Vesicular traffic; Clathrin adaptors; Protein phosphorylation; Membranes; ATP; Brain
Clathrin-mediated endocytosis (CME) is the major way of entry into eukaryotic cells and the best-understood mechanism of macromolecule internalization [1]. In brain, CME is required for recycling of membrane proteins after the release of neurotransmitters during synapses [2]. As the first step of CME, coat proteins are recruited to the cytoplasmic side of the plasma membrane, forming clathrin coated pits (CCP), in zones where receptors and their ligands are concentrated. The assembly of the coat in CCP is regulated by specific adaptors through interactions with both cytoplasmic domain of receptors and clathrin. After budding of clathrin coated vesicles (CCV) from the plasma membrane, the coat disassembles, and the clathrin and adaptors are released to the cytoplasm for another round of endocytosis. This process ends when the vesicle
*
Corresponding author. E-mail address: [email protected] (A. Alberdi).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.038
fuses selectively with the proper membrane acceptor [1]. Among the four adaptor complexes described so far, AP-2 is known to play a central role in sorting and packaging of receptors during endocytosis. This complex is a heterotetramer composed by two 100-kDa (named a and b2), one 50-kDa (l2), and one 17-kDa (r2) subunits [3]. Two subtypes of a subunits (named a1/a2 or aA/aC) exist in neurons, and whether they play different roles in the central nervous system is still unknown. Covalent modifications on the coat proteins have been proposed to regulate the assembly–disassembly cycles onto clathrin coated pits. Because l2, b2, and a subunits are phosphoproteins [4], it has been proposed that the mechanisms of AP-2 phosphorylation may also participate in regulating CCV formation [5,6]. For instance, phosphorylation of l2 is crucial for internalization in vitro and in vivo [1]. Despite the two phosphorylation sites has been found on the b2 subunit, only the serine localized in the hinge region has a regulatory role in clathrin binding [4,7]. Although a subunit is also phos-
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phorylated in its hinge region [4,5], little is known about the importance of this covalent modification. In this work, by using an in vitro binding assay, we attempted to know: first, if phosphorylation of adaptor subunits or other cytosolic proteins regulates the assembly–disassembly cycles of AP-2; if so, which types of kinases and/or phosphatases may be involved, and then if the nucleotide ATP exerts a regulatory effect in the AP-2 phosphorylation.
Materials and methods Antibodies and reagents. Monoclonal antibodies against a- and cadaptin, phosphoserine, and phosphotyrosine were from Sigma Chemicals (St. Louis, MO). Kinase- or phosphatase-inhibitors were all from Calbiochem. Brains from freshly slaughtered bovines were obtained from a local slaughterhouse in Mendoza (Argentina). Sprague– Dawley rats were provided by the breeding colony of the Institute de Histologia y Embriologia, Universidad Nacional de Cuyo (Argentina). Distribution of proteins between membranes and cytosol. Adult rat brains were homogenized (1:5 weight/volume) in 0.01 M Tris–HCl, pH 7.2, 0.25 M sucrose, 0.1%(v/v) EDTA, 1 mM PMSF, 1 mM leupeptin, and 1 mM aprotinin (buffer H). The homogenate was centrifuged at 1500g for 10 min, and the post-nuclear supernatant (PNS) was collected and subjected to further centrifugation at 10,000g for 30 min. The supernatant (cytosol) and pellet (membranes) were collected and studied for AP-2 content. In other experiments, PNS were pre-incubated at 37 C for 1 h with 2.5–10 U alkaline phosphatase in buffer H. Pellets or supernatants were solubilized in sample buffer and analysed by immunoblot for the presence of APs, and proteins were phosphorylated in either serine or tyrosine residue. Preparation of coated vesicles and cytosol. Adaptors were prepared from clathrin coated vesicles (CCVs) purified from bovine brain as previously described [8], and fractions enriched in AP-1 and AP-2 were stored in buffer B (1 M Tris–HCl, pH 7.0, containing 0.2 mM EDTA, 0.2%(v/v) b-mercaptoethanol, 0.2 mM PMSF, and 0.02%(v/v) NaN3) at 4 C up to one week. Samples were dialysed against 25 mM Hepes/ KOH buffers, pH 7.0, and supplemented with 125 mM potassium acetate and 5 mM magnesium acetate (buffer C) before using in binding assays. The stripped vesicles were washed sequentially, once with buffer B, three times with buffer C, and then resuspended in the buffer C and stored at 70 C until use. Binding assays. Binding assays were carried out according to Sosa et al. [8]. Briefly, 25 lg of membranes (stripped vesicles) was mixed with either 25 lg of purified APs or 50 lg cytosols in a final volume of 250 ll buffer C. After 15 min incubation at 37 C, 500 ll of buffer C was added, and the tubes were centrifuged at 12,000g for 10 min. The pellets were washed once with 500 ll buffer C, and prepared for SDS/ PAGE and Western blot. On this typical binding assay, phosphatase or kinase inhibitors and/or an ATP-regenerating system (1 mM ATP, 5 mM creatine phosphate, and 10 U/ml creatine phosphokinase) were added to the corresponding experiments. Preparation of cytosol. The post-nuclear supernatants of the bovine brains were subjected to further centrifugation at 1000g for 60 min, supplemented with 1 mM PMSF, 5 lg/ml leupeptin, and 0.1 TIU/ml aprotinin, and stored at 70 C up to use. Samples were dialysed against buffer C before use in binding assays. Fractionation of cytosol. Bovine cytosol was applied to a 70 · 2 cm Sephacryl S-500 column (Pharmacia Biotech) previously equilibrated with buffer B, and 2 ml fractions were collected and analysed by SDS/ PAGE and Western blot. Those fractions devoid of clathrin and APs were pooled into four groups: fractions 1–4, containing proteins ranged between 30–80, 30–60, 20–50, and <30 kDa, respectively. Each
pool was concentrated by ultrafiltration under Nitrogen atmosphere (PM10 membrane, AMICON, Witten, FRG) and stored at 4 C up to use. Before use in binding assays, samples were dialysed against buffer C. In vitro phosphorylation of proteins. Purified APs and/or cytosolic fractions were incubated with 1 mM ATP and 1 ll [c-32P]ATP (3000 Ci/mM) in buffer C for 15 min at room temperature. Reactions were stopped by addition of SDS Laemmli sample buffer [9] and analysed in 7.5% SDS/PAGE gels. The gels were stained with Coomassie blue dye, dried, and the phosphorylated proteins were detected by autoradiography on Kodax X-Omat films. Electrophoresis and Western blotting. Samples were boiled in SDS Laemmli sample buffer [9] and run out on 8% SDS/PAGE gels. The gels were blotted onto nitrocellulose membranes (Sartorius, Go¨ttingen, Germany) and then the membranes were blocked with 5% milk in PBST (10 mM NaH2PO4–Na2HPO4, pH 7.2, 0.05%(v/v) Tween 20), and incubated with the corresponding monoclonal antibody diluted in PBS-T, washed with PBS-T, incubated with a biotin-conjugated antimouse antibody (Sigma, in PBS-T), washed as before, and incubated with peroxidase-conjugated Streptavidin (Sigma, in PBS-T). After washings, the protein bands were detected by enhanced chemiluminescence method (ECL, Amersham), using Kodak X-Omat film. Bands were quantified by densitometric scanning of the films with NIH Image 1.60, and the results were analysed with Graph Pad Prism (Graph Pad Software, San Diego, CA, USA) and KyPlot software (Kyens Lab, Tokyo, Japan).
Results Redistribution of AP-2 by treatment with alkaline phosphatase As a first approach, by immunoblot analysis using anti-a (1 and 2) or b1/b2 adaptins, we measured the distribution of the different AP-2 subunits between membranes and cytosol from a post-nuclear fraction of rat brain, treated or not with alkaline phosphatase. The distribution between the two a subtypes at steady state was different, since a2 was predominantly bound to membranes, whereas a1 was similarly distributed in both, membranes and cytosol (Fig. 1). When the post-nuclear supernatants were treated with alkaline phosphatase, we observed a redistribution of both a-subtypes towards the cytosol, although this was significant for a2. Interestingly, b2 subunit did not follow the redistribution of a2 to the cytosols (Fig. 1), indicating that a2 may alternatively interact with b subunits other than b2, forming hybrid tetrameric complexes. The distribution of c and b1 subunits (AP-1 complex from TGN to late endosomes) was similar to that of a2 subunit in controls. However, they were not redistributed to the cytosol by the treatment with alkaline phosphatase (Fig. 1). Phosphorylation status of a subunit correlates with their distribution to membranes To further explore if redistribution of a subunits to cytosol by treatment with alkaline phosphatase was due to dephosphorylation of these proteins, we analysed
A. Alberdi et al. / Biochemical and Biophysical Research Communications 330 (2005) 695–700
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(PH in Fig. 2). No phosphorylation was detected in tyrosine residues of these proteins (data not shown). ATP as a possible mediator of phosphorylation
Fig. 1. Western blot analysis of AP complex distribution between membranes and cytosol by the treatment with alkaline phosphatase. Post-nuclear fractions from rat brains were incubated or not with alkaline phosphatase (Alk.Ph.) at 37 C for 1 h and centrifuged. The resultant pellets (membranes) or supernatants (cytosol) were solubilized in sample buffer, and analysed by SDS/PAGE and Western blot for the presence of AP subunits (anti-a and -b-AP-2, and anti-b and -cAP1). The graphs show the values of densitometric scanning (OD) of AP-2 and AP-1 bands on the blots.
the phosphorylation status of a1 and a2, before and after the treatment using specific antibodies against either phosphorylated serine or tyrosine residue. As shown in Fig. 2, proteins phosphorylated in serine overlapped with both a subtypes, and underwent partial dephosphorylation by the treatment with alkaline phosphatase. We also observed that the rate of dephosphorylation may be related more to a2 than a1 detachment from the membranes (compare to Fig. 1). When samples were incubated with inhibitors of alkaline phosphatase, both subtypes were overphosphorylated
Fig. 2. Western blot analysis of rat brain membranes and detection of serine phosphorylated proteins and a AP-2 subunits. Post-nuclear supernatants from rat brains were incubated in the presence or absence of alkaline phosphatase and protein kinase inhibitors (DPH), or phosphatase inhibitors (PH). DPH treatment included 10 U of alkaline phosphatase and 30 lM KN93, 1 lM calphostine, staurosporine, and tyrphostin. PH treatment included 100 nM okadaic acid, 1 mM sodium orthovanadate, 100 lM calyculine, and 1 lM cyclosporine. After 1 h incubation at 37 C, samples were centrifuged and the resulting pellets were analysed by Western blot with anti-a-AP-2 (A). The blots were stripped by incubation for 30 min at 50 C in a buffer containing 2%(v/v) SDS, 100 mM 2-mercaptoethanol, and 50 mM Tris (pH 6.7). Blots were then washed extensively with PBS-T and subsequently probed with anti-phosphoserine antibody (B). Bands were quantified by densitometric scanning (OD). CO, control. This figure is representative of three independent experiments (n = 3).
Accumulated evidences showed that the protein phosphorylation mediated by ATP regulates several steps of CCV formation in neuronal cells [10]. The role of ATP in assembly–disassembly cycles of coat proteins is still controversial; while some studies showed that ATP favours the recruitment of AP-2 to membranes [11–13], others consider that ATP is essential for the release of AP-2 from the membranes [14–16]. In a previous report, we demonstrated that ATP inhibits the binding of AP-2 to membranes and this effect was boosted when assayed with cytosols [8]. We here attempted to elucidate whether ATP is an inhibitor of binding, and if it exerts its action via protein phosphorylation. To give a new contribution to the knowledge on building the coat in clathrin coated vesicles, we took advantage of the in vitro binding assay developed by other authors [8]. Purified APs from bovine brain were incubated with membranes in a typical binding assay, either in the presence or the absence of ATP-regeneration system (ATPrs) and in the presence or the absence of cytosol. We confirmed that ATP inhibits the binding of AP-2 to membranes and this effect was enhanced by the presence of cytosol, in a concentration-dependent fashion. Interestingly, the inhibitory effect mediated by ATP-cytosol on a2 was higher than that on a1 (Fig. 3). As a first step towards the identification of ATP-cytosolic factors that interfere with binding of AP-2 to membranes, we carried out a gel filtration from brain cytosols and pooled four fractions depleted of APs (Fig. 4A). One aliquot of each fraction was added to the binding assay of purified APs to membranes, either in the absence or in the presence of ATPrs. As observed in Fig. 4B, the fractions 2 and 3 (at lesser extent) inhibited the binding of AP-2 to membranes in the presence of the nucleotide. As it occurred with the whole cytosols,
Fig. 3. In vitro binding assay of purified APs to vesicle membranes. Stripped vesicles (sv) were incubated with purified APs, in the presence or absence of bovine brain cytosol (Cyt) and in the presence or absence of an ATP-regeneration system at indicated concentrations. Assays were analysed by SDS/PAGE and Western blot, and blots were processed for immunodetection of a subunit of AP-2 and quantified by densitometric scanning of the films (OD). This figure is representative of three independent assays (n = 3).
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Fig. 4. In vitro binding assay of purified APs to stripped vesicles in the presence of cytosolic fractions. (A) Fractionation of cytosol by filtration chromatography. Bovine brain cytosol was applied to a filtration chromatography column and 2 ml fractions were collected. Fractions devoid of clathrin and APs were pooled into four groups: fractions 1–4 and each pool was concentrated by ultrafiltration. Samples of each pool were analysed with SDS/PAGE and gels (7.5%) were stained with Coomassie blue. (B) Stripped vesicles (sv) were incubated with purified APs in the presence of cytosolic fractions (Fr. 1–4) obtained by gel filtration chromatography, in the presence or absence of an ATP-regeneration system at indicated concentrations. Assay was analysed by SDS/PAGE and Western blot, and blots were processed for immunodetection of a subunit of AP-2 and quantified by densitometric scanning of the films (OD). This figure is representative of three independent assays (n = 3). (C) Phosphorylation of APs in vitro. Pure APs (lane 1) or combined with the indicated cytosolic fractions (lanes 2–9) were incubated with [c-32P]ATP and then analysed by SDS/ PAGE. Bands were detected by autoradiography.
the isoform a2 was more sensitive than a1 to the inhibitory effect of ATP. The inhibition mediated by ATP-cytosol is not related to AP-2 phosphorylation If we consider that the inhibitory effect of ATP was due to an increase of AP-2 subunit phosphorylation, cytosolic fractions with inhibitory activity would exhibit higher capability to phosphorylate AP-2 subunits. To assess this possibility, we incubated purified adaptins with aliquots of each cytosolic fraction and [c32P]ATP. We observed that proteins in the range of l subunits were phosphorylated by all the cytosolic fractions and to a similar extent, although with little phosphorylation on proteins ranging between 100 and 110 kDa (Fig. 4C). We observed neither self-phosphorylation of cytosolic proteins or APs. Kinases and/or phosphatases may participate in the complex machinery for the assembly of AP-2 From the results detailed above, we wondered if cytosolic kinases and/or phosphatases were located sequentially and participated in a fairly complex cascade of phosphorylations/dephosphorylations to regulate the AP-2 assembly–disassembly cycle. To explore this, we
pre-incubated cytosols with inhibitors for either phosphatase or kinase prior to the binding assays and evaluated the incidence on the interactions between AP-2 and membranes. Fig. 5A shows that tyrphostin, a tyrosine kinase inhibitor, was able to abolish the binding of AP-2 to membranes, and that this effect was enhanced by ATP. In this case, the effect was more significant for a1 than for a2 subunit. KN93, a well-known inhibitor for calcium-calmodulin-kinase type II (CAMKII), did not significantly affect the binding of AP-2 to membranes (Fig. 5B). According to this, protein phosphorylation favours the binding of AP-2 to membranes, and tyrosine kinases may be involved in some steps of this process. Redistribution of AP-2 to cytosol induced by alkaline phosphatase treatment (Fig. 1) supports this hypothesis. In this way, we may expect that inhibitors of phosphatase could enrich the binding of AP-2 to membranes. Surprisingly, when cytosol was pre-incubated with cyclosporine, an inhibitor of calcineurin, the binding of AP-2 to membranes was reduced (Fig. 5C). Likewise, an additional inhibitory effect was observed when cyclosporine was added in the presence of ATP regeneration system. Although the binding of AP-2 was also reduced by the addition of the ATPase inhibitor sodium orthovanadate, the effect was at lesser extent (Fig. 5D), and no inhibition was observed with okadaic acid (data not shown).
A. Alberdi et al. / Biochemical and Biophysical Research Communications 330 (2005) 695–700
Fig. 5. In vitro binding assay of cytosolic AP-2 to vesicle membranes after incubation with protein kinase and protein phosphatase inhibitors. Binding assays were performed as previously. Cytosols (50 lg) were incubated with stripped vesicles (sv, 20 lg) in the presence or absence of an ATP-regeneration system at indicated concentrations. Some cytosols were pre-incubated prior to binding with the KN93 (30 mM), tyrphostin (1 mM), sodium orthovanadate (1 mM), or cyclosporine (1 lM) inhibitors. After binding, the samples were prepared, and subjected to SDS/ PAGE and Western blot with antibodies for AP-2. Graphs show the values of densitometric scanning (OD) of a2- and a1-AP-2 bands from blots. Bars represent the mean of independent experiments ± standard error mean (SEM). The number of experiences (n) is indicated. Data were analysed with One-Way ANOVA followed by TukeyÕs multiple comparisons test. Signification level: p < 0.05.
Discussion The results presented in this study, combined with those from others [5,6,17], support that cycles of phosphorylation and dephosphorylation of proteins may regulate rounds of assembly–disassembly of AP-2 in clathrin coated vesicle formation. We also attempted to explain the role of ATP, related to phosphorylation and dephosphorylation of proteins, and to the interaction between AP-2 and membranes. Accumulated evidences suggest that the protein phosphorylation that utilizes ATP regulates several steps of CCV formation in neuronal cells (for review see [10]). However, in vitro and in vivo studies have shown opposite effects of ATP on recruitment and release of AP-2 to and from membranes [11–16]. ATP also participates in clathrin disassembly by a mechanism that involves ATPase activity of Hsc70 and auxilin [15]. In this study, the facts that the treatment with alkaline phosphatase induced a redistribution of AP-2 to cytosol and that tyrphostin inhibited the binding of AP-2 to membranes suggest that phosphorylation of proteins favours that interaction, in agreement with previous reports [5,6,17–19]. However, although a1 and a2 subunits of AP-2 are phosphorylated in serine residues, we consider that this covalent modification is not deter-
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mining for the interaction with membranes, but they may form part of a complex cascade of phosphorylations instead, aimed to enhance that interaction. This was based on the decrease of the binding of AP-2 to membranes by a tyrosine kinase inhibitor (Fig. 5B) and that AP-2 subunits were not phosphorylated in tyrosine. Recently, it was shown that b2 is phosphorylated in tyrosine by EGFR kinase [7]. If phosphorylation of b2 regulates binding of AP-2 to membranes, this may justify the inhibitory effect of tyrphostin. However, the fact that b2 was scarcely redistributed to the cytosol by treatment with alkaline phosphatase indicates that phosphorylation of this subunit does not regulate the binding of AP-2 to membranes. Since ATP favoured phosphorylation of proteins (including l subunits), but it also inhibited the binding of AP-2 to membranes (Fig. 3), we speculate that ATP may be a substrate donor that induces inactivation of certain tyrosine kinases by phosphorylation, in a putative complex cascade of phosphorylations. According to this, a phosphatase (as calcineurin) may activate certain kinases by dephosphorylation and justify the decrease of binding by cyclosporine (Fig. 5D). In support of this, a previous report showed that calcineurin upregulates the CCV assembly in nerve terminals [10]. It is also possible that certain tyrosine kinases (inhibitable by tyrphostin) exert an action downstream the cascade, activating other kinases by phosphorylation (e.g., serine kinases). However, we cannot discard the possibility that, in our model, the proposed tyrosine kinase may phosphorylate other accessory proteins (as dephosphins) that aid in the assembly of AP-2 [10]. On the other hand, we observed that assembly of both a1 and a2 to membranes might be differently regulated. This is supported by the fact that at steady state, a2 is mostly bound to brain membranes meanwhile a1 is distributed to the cytosol. Thus, redistribution of a2 to cytosol by the alkaline phosphatase treatment became more dramatic than for a1. The existence of two a subtypes in neuronal tissue is still poorly understood and from our results we propose that both may belong to different populations of clathrin coated vesicles. Since dephosphorylation by alkaline phosphatase, or incubation with either ATP or inhibitors, did not completely abolish the binding of AP-2, it is also possible that certain AP-2 complexes assemble into clathrin coated pits by a phosphorylation-independent mechanism. One possibility for this alternative binding may be via the phosphoinositide-binding sites of a- [20] and l2 subunits [21]. This could provide an initial targeting of certain AP-2 complexes since the plasma membrane is enriched in phosphatidylinositol 4,5-biphosphate [20]. In conclusion, from the results presented here we suggest that a cascade of protein phosphorylations consist-
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ing in the coordinated action of kinases and phosphatases to activate proteins may be responsible for enhancing the assembly of AP-2 to membranes at the early steps of clathrin coated vesicle formation. Further studies should be done in order to identify the possible kinases involved in this process and the targets for those enzymes.
References [1] S.D. Conner, S.L. Schmid, Differential requirements for AP-2 in clathrin-mediated endocytosis, J. Cell Biol. 162 (2003) 773– 779. [2] J. Hirst, M.S. Robinson, Clathrin and adaptors, Biochim. Biophys. Acta 1404 (1998) 173–193. [3] T. Kirchhausen, Adaptors for clathrin-mediated traffic, Annu. Rev. Cell Dev. Biol. 15 (1999) 705–732. [4] A. Wilde, F.M. Brodsky, In vivo phosphorylation of adaptors regulates their interaction with clathrin, J. Cell Biol. 135 (1996) 635–645. [5] A. Fingerhut, K. von Figura, S. Honing, Binding of AP2 to sorting signals is modulated by AP2 phosphorylation, J. Biol. Chem. 276 (2001) 5476–5482. [6] S.D. Conner, S.L. Schmid, Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis, J. Cell Biol. 156 (2002) 921–929. [7] F. Huang, X. Jiang, A. Sorkin, Tyrosine phosphorylation of the b2 subunit of clathrin adaptor complex AP-2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking, J. Biol. Chem. 278 (2003) 43411–43417. [8] M.A. Sosa, F. Bertini, K. von Figura, A. Hille-Rehfeld, Is an ATPase involved in uncoating of plasma membrane adaptor complex AP-2? FEBS Lett. 407 (1997) 173–176. [9] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685.
[10] M.A. Cousin, P.J. Robinson, The dephosphins: dephosphorylation by calcineurin triggers synaptic vesicle endocytosis, Trends Neurosci. 24 (2001) 659–665. [11] E. Smythe, L.L. Carter, S.L. Schmid, Cytosol- and clathrindependent stimulation of endocytosis in vitro by purified adaptors, J. Cell Biol. 119 (1992) 1163–1171. [12] M.N. Seaman, C.L. Ball, M.S. Robinson, Targeting and mistargeting of plasma membrane adaptors in vitro, J. Cell Biol. 123 (1993) 1093–1105. [13] M.A. West, N.A. Bright, M.S. Robinson, The role of ADPribosylation factor and phospholipase D in adaptor recruitment, J. Cell Biol. 138 (1997) 1239–1254. [14] L.A. Hannan, S.L. Newmyer, S.L. Schmid, ATP- and cytosoldependent release of adaptor proteins from clathrin-coated vesicles: a dual role for Hsc70, Mol. Biol. Cell 9 (1998) 2217–2229. [15] A. Umeda, A. Meyerholz, E. Ungewickell, Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation, Eur. J. Cell Biol. 79 (2000) 336–342. [16] J.R. Morgan, K. Prasad, S. Jin, G.J. Augustine, E.M. Lafer, Uncoating of clathrin-coated vesicles in presynaptic terminals: roles for Hsc70 and auxilin, Neuron 32 (2001) 289–300. [17] P. Ghosh, S. Kornfeld, AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation, J. Cell Biol. 160 (2003) 699–708. [18] D. Ricotta, S.D. Conner, S.L. Schmid, K. von Figura, S. Honing, Phosphorylation of the AP2 l subunit by AAK1 mediates high affinity binding to membrane protein sorting signals, J. Cell Biol. 156 (2002) 791–795. [19] A.P. Jackson, A. Flett, C. Smythe, L. Hufton, F.R. Wettey, E. Smythe, Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor l2 kinase, J. Cell Biol. 163 (2003) 231–236. [20] I. Gaidarov, J.H. Keen, Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits, J. Cell Biol. 146 (1999) 755–764. [21] G. Rohde, D. Wenzel, V. Haucke, A phosphatidylinositol (4,5)bisphosphate binding site within l2-adaptin regulates clathrinmediated endocytosis, J. Cell Biol. 158 (2002) 209–214.
Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins A. Alberdi *, T. Sartor, M.A. Sosa Instituto de Histologı´a y Embriologı´a, Facultad de Ciencias Me´dicas, Universidad Nacional de Cuyo, Mendoza (5500), Argentina Received 21 February 2005 Available online 17 March 2005
Abstract Phosphorylation of proteins appears as a key process in early steps of clathrin coated vesicle formation. Here, we report that treatment of post-nuclear fraction with alkaline phosphatase induced redistribution of a subunits of AP-2 adaptor complex to cytosol and this effect was higher in the a2 subunit. A high serine phosphorylation status of a subunits correlated with the higher affinity of AP-2 to membranes. Using a simple binding assay, where membranes were incubated with either purified adaptors or cytosols, we observed an inhibitory effect of tyrphostin, a tyrosine kinase inhibitor, on the binding of AP-2 to membranes, but also an unexpected decrease induced by the phosphatase inhibitor cyclosporine. We also show an inhibitory effect of ATP mediated by cytosolic proteins, although it could not be related to the phosphorylation of AP-2, suggesting an action upstream a cascade of phosphorylations that participate in the regulation of the assembly of AP-2 to membranes. 2005 Elsevier Inc. All rights reserved. Keywords: Vesicular traffic; Clathrin adaptors; Protein phosphorylation; Membranes; ATP; Brain
Clathrin-mediated endocytosis (CME) is the major way of entry into eukaryotic cells and the best-understood mechanism of macromolecule internalization [1]. In brain, CME is required for recycling of membrane proteins after the release of neurotransmitters during synapses [2]. As the first step of CME, coat proteins are recruited to the cytoplasmic side of the plasma membrane, forming clathrin coated pits (CCP), in zones where receptors and their ligands are concentrated. The assembly of the coat in CCP is regulated by specific adaptors through interactions with both cytoplasmic domain of receptors and clathrin. After budding of clathrin coated vesicles (CCV) from the plasma membrane, the coat disassembles, and the clathrin and adaptors are released to the cytoplasm for another round of endocytosis. This process ends when the vesicle
*
Corresponding author. E-mail address: [email protected] (A. Alberdi).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.038
fuses selectively with the proper membrane acceptor [1]. Among the four adaptor complexes described so far, AP-2 is known to play a central role in sorting and packaging of receptors during endocytosis. This complex is a heterotetramer composed by two 100-kDa (named a and b2), one 50-kDa (l2), and one 17-kDa (r2) subunits [3]. Two subtypes of a subunits (named a1/a2 or aA/aC) exist in neurons, and whether they play different roles in the central nervous system is still unknown. Covalent modifications on the coat proteins have been proposed to regulate the assembly–disassembly cycles onto clathrin coated pits. Because l2, b2, and a subunits are phosphoproteins [4], it has been proposed that the mechanisms of AP-2 phosphorylation may also participate in regulating CCV formation [5,6]. For instance, phosphorylation of l2 is crucial for internalization in vitro and in vivo [1]. Despite the two phosphorylation sites has been found on the b2 subunit, only the serine localized in the hinge region has a regulatory role in clathrin binding [4,7]. Although a subunit is also phos-
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phorylated in its hinge region [4,5], little is known about the importance of this covalent modification. In this work, by using an in vitro binding assay, we attempted to know: first, if phosphorylation of adaptor subunits or other cytosolic proteins regulates the assembly–disassembly cycles of AP-2; if so, which types of kinases and/or phosphatases may be involved, and then if the nucleotide ATP exerts a regulatory effect in the AP-2 phosphorylation.
Materials and methods Antibodies and reagents. Monoclonal antibodies against a- and cadaptin, phosphoserine, and phosphotyrosine were from Sigma Chemicals (St. Louis, MO). Kinase- or phosphatase-inhibitors were all from Calbiochem. Brains from freshly slaughtered bovines were obtained from a local slaughterhouse in Mendoza (Argentina). Sprague– Dawley rats were provided by the breeding colony of the Institute de Histologia y Embriologia, Universidad Nacional de Cuyo (Argentina). Distribution of proteins between membranes and cytosol. Adult rat brains were homogenized (1:5 weight/volume) in 0.01 M Tris–HCl, pH 7.2, 0.25 M sucrose, 0.1%(v/v) EDTA, 1 mM PMSF, 1 mM leupeptin, and 1 mM aprotinin (buffer H). The homogenate was centrifuged at 1500g for 10 min, and the post-nuclear supernatant (PNS) was collected and subjected to further centrifugation at 10,000g for 30 min. The supernatant (cytosol) and pellet (membranes) were collected and studied for AP-2 content. In other experiments, PNS were pre-incubated at 37 C for 1 h with 2.5–10 U alkaline phosphatase in buffer H. Pellets or supernatants were solubilized in sample buffer and analysed by immunoblot for the presence of APs, and proteins were phosphorylated in either serine or tyrosine residue. Preparation of coated vesicles and cytosol. Adaptors were prepared from clathrin coated vesicles (CCVs) purified from bovine brain as previously described [8], and fractions enriched in AP-1 and AP-2 were stored in buffer B (1 M Tris–HCl, pH 7.0, containing 0.2 mM EDTA, 0.2%(v/v) b-mercaptoethanol, 0.2 mM PMSF, and 0.02%(v/v) NaN3) at 4 C up to one week. Samples were dialysed against 25 mM Hepes/ KOH buffers, pH 7.0, and supplemented with 125 mM potassium acetate and 5 mM magnesium acetate (buffer C) before using in binding assays. The stripped vesicles were washed sequentially, once with buffer B, three times with buffer C, and then resuspended in the buffer C and stored at 70 C until use. Binding assays. Binding assays were carried out according to Sosa et al. [8]. Briefly, 25 lg of membranes (stripped vesicles) was mixed with either 25 lg of purified APs or 50 lg cytosols in a final volume of 250 ll buffer C. After 15 min incubation at 37 C, 500 ll of buffer C was added, and the tubes were centrifuged at 12,000g for 10 min. The pellets were washed once with 500 ll buffer C, and prepared for SDS/ PAGE and Western blot. On this typical binding assay, phosphatase or kinase inhibitors and/or an ATP-regenerating system (1 mM ATP, 5 mM creatine phosphate, and 10 U/ml creatine phosphokinase) were added to the corresponding experiments. Preparation of cytosol. The post-nuclear supernatants of the bovine brains were subjected to further centrifugation at 1000g for 60 min, supplemented with 1 mM PMSF, 5 lg/ml leupeptin, and 0.1 TIU/ml aprotinin, and stored at 70 C up to use. Samples were dialysed against buffer C before use in binding assays. Fractionation of cytosol. Bovine cytosol was applied to a 70 · 2 cm Sephacryl S-500 column (Pharmacia Biotech) previously equilibrated with buffer B, and 2 ml fractions were collected and analysed by SDS/ PAGE and Western blot. Those fractions devoid of clathrin and APs were pooled into four groups: fractions 1–4, containing proteins ranged between 30–80, 30–60, 20–50, and <30 kDa, respectively. Each
pool was concentrated by ultrafiltration under Nitrogen atmosphere (PM10 membrane, AMICON, Witten, FRG) and stored at 4 C up to use. Before use in binding assays, samples were dialysed against buffer C. In vitro phosphorylation of proteins. Purified APs and/or cytosolic fractions were incubated with 1 mM ATP and 1 ll [c-32P]ATP (3000 Ci/mM) in buffer C for 15 min at room temperature. Reactions were stopped by addition of SDS Laemmli sample buffer [9] and analysed in 7.5% SDS/PAGE gels. The gels were stained with Coomassie blue dye, dried, and the phosphorylated proteins were detected by autoradiography on Kodax X-Omat films. Electrophoresis and Western blotting. Samples were boiled in SDS Laemmli sample buffer [9] and run out on 8% SDS/PAGE gels. The gels were blotted onto nitrocellulose membranes (Sartorius, Go¨ttingen, Germany) and then the membranes were blocked with 5% milk in PBST (10 mM NaH2PO4–Na2HPO4, pH 7.2, 0.05%(v/v) Tween 20), and incubated with the corresponding monoclonal antibody diluted in PBS-T, washed with PBS-T, incubated with a biotin-conjugated antimouse antibody (Sigma, in PBS-T), washed as before, and incubated with peroxidase-conjugated Streptavidin (Sigma, in PBS-T). After washings, the protein bands were detected by enhanced chemiluminescence method (ECL, Amersham), using Kodak X-Omat film. Bands were quantified by densitometric scanning of the films with NIH Image 1.60, and the results were analysed with Graph Pad Prism (Graph Pad Software, San Diego, CA, USA) and KyPlot software (Kyens Lab, Tokyo, Japan).
Results Redistribution of AP-2 by treatment with alkaline phosphatase As a first approach, by immunoblot analysis using anti-a (1 and 2) or b1/b2 adaptins, we measured the distribution of the different AP-2 subunits between membranes and cytosol from a post-nuclear fraction of rat brain, treated or not with alkaline phosphatase. The distribution between the two a subtypes at steady state was different, since a2 was predominantly bound to membranes, whereas a1 was similarly distributed in both, membranes and cytosol (Fig. 1). When the post-nuclear supernatants were treated with alkaline phosphatase, we observed a redistribution of both a-subtypes towards the cytosol, although this was significant for a2. Interestingly, b2 subunit did not follow the redistribution of a2 to the cytosols (Fig. 1), indicating that a2 may alternatively interact with b subunits other than b2, forming hybrid tetrameric complexes. The distribution of c and b1 subunits (AP-1 complex from TGN to late endosomes) was similar to that of a2 subunit in controls. However, they were not redistributed to the cytosol by the treatment with alkaline phosphatase (Fig. 1). Phosphorylation status of a subunit correlates with their distribution to membranes To further explore if redistribution of a subunits to cytosol by treatment with alkaline phosphatase was due to dephosphorylation of these proteins, we analysed
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(PH in Fig. 2). No phosphorylation was detected in tyrosine residues of these proteins (data not shown). ATP as a possible mediator of phosphorylation
Fig. 1. Western blot analysis of AP complex distribution between membranes and cytosol by the treatment with alkaline phosphatase. Post-nuclear fractions from rat brains were incubated or not with alkaline phosphatase (Alk.Ph.) at 37 C for 1 h and centrifuged. The resultant pellets (membranes) or supernatants (cytosol) were solubilized in sample buffer, and analysed by SDS/PAGE and Western blot for the presence of AP subunits (anti-a and -b-AP-2, and anti-b and -cAP1). The graphs show the values of densitometric scanning (OD) of AP-2 and AP-1 bands on the blots.
the phosphorylation status of a1 and a2, before and after the treatment using specific antibodies against either phosphorylated serine or tyrosine residue. As shown in Fig. 2, proteins phosphorylated in serine overlapped with both a subtypes, and underwent partial dephosphorylation by the treatment with alkaline phosphatase. We also observed that the rate of dephosphorylation may be related more to a2 than a1 detachment from the membranes (compare to Fig. 1). When samples were incubated with inhibitors of alkaline phosphatase, both subtypes were overphosphorylated
Fig. 2. Western blot analysis of rat brain membranes and detection of serine phosphorylated proteins and a AP-2 subunits. Post-nuclear supernatants from rat brains were incubated in the presence or absence of alkaline phosphatase and protein kinase inhibitors (DPH), or phosphatase inhibitors (PH). DPH treatment included 10 U of alkaline phosphatase and 30 lM KN93, 1 lM calphostine, staurosporine, and tyrphostin. PH treatment included 100 nM okadaic acid, 1 mM sodium orthovanadate, 100 lM calyculine, and 1 lM cyclosporine. After 1 h incubation at 37 C, samples were centrifuged and the resulting pellets were analysed by Western blot with anti-a-AP-2 (A). The blots were stripped by incubation for 30 min at 50 C in a buffer containing 2%(v/v) SDS, 100 mM 2-mercaptoethanol, and 50 mM Tris (pH 6.7). Blots were then washed extensively with PBS-T and subsequently probed with anti-phosphoserine antibody (B). Bands were quantified by densitometric scanning (OD). CO, control. This figure is representative of three independent experiments (n = 3).
Accumulated evidences showed that the protein phosphorylation mediated by ATP regulates several steps of CCV formation in neuronal cells [10]. The role of ATP in assembly–disassembly cycles of coat proteins is still controversial; while some studies showed that ATP favours the recruitment of AP-2 to membranes [11–13], others consider that ATP is essential for the release of AP-2 from the membranes [14–16]. In a previous report, we demonstrated that ATP inhibits the binding of AP-2 to membranes and this effect was boosted when assayed with cytosols [8]. We here attempted to elucidate whether ATP is an inhibitor of binding, and if it exerts its action via protein phosphorylation. To give a new contribution to the knowledge on building the coat in clathrin coated vesicles, we took advantage of the in vitro binding assay developed by other authors [8]. Purified APs from bovine brain were incubated with membranes in a typical binding assay, either in the presence or the absence of ATP-regeneration system (ATPrs) and in the presence or the absence of cytosol. We confirmed that ATP inhibits the binding of AP-2 to membranes and this effect was enhanced by the presence of cytosol, in a concentration-dependent fashion. Interestingly, the inhibitory effect mediated by ATP-cytosol on a2 was higher than that on a1 (Fig. 3). As a first step towards the identification of ATP-cytosolic factors that interfere with binding of AP-2 to membranes, we carried out a gel filtration from brain cytosols and pooled four fractions depleted of APs (Fig. 4A). One aliquot of each fraction was added to the binding assay of purified APs to membranes, either in the absence or in the presence of ATPrs. As observed in Fig. 4B, the fractions 2 and 3 (at lesser extent) inhibited the binding of AP-2 to membranes in the presence of the nucleotide. As it occurred with the whole cytosols,
Fig. 3. In vitro binding assay of purified APs to vesicle membranes. Stripped vesicles (sv) were incubated with purified APs, in the presence or absence of bovine brain cytosol (Cyt) and in the presence or absence of an ATP-regeneration system at indicated concentrations. Assays were analysed by SDS/PAGE and Western blot, and blots were processed for immunodetection of a subunit of AP-2 and quantified by densitometric scanning of the films (OD). This figure is representative of three independent assays (n = 3).
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Fig. 4. In vitro binding assay of purified APs to stripped vesicles in the presence of cytosolic fractions. (A) Fractionation of cytosol by filtration chromatography. Bovine brain cytosol was applied to a filtration chromatography column and 2 ml fractions were collected. Fractions devoid of clathrin and APs were pooled into four groups: fractions 1–4 and each pool was concentrated by ultrafiltration. Samples of each pool were analysed with SDS/PAGE and gels (7.5%) were stained with Coomassie blue. (B) Stripped vesicles (sv) were incubated with purified APs in the presence of cytosolic fractions (Fr. 1–4) obtained by gel filtration chromatography, in the presence or absence of an ATP-regeneration system at indicated concentrations. Assay was analysed by SDS/PAGE and Western blot, and blots were processed for immunodetection of a subunit of AP-2 and quantified by densitometric scanning of the films (OD). This figure is representative of three independent assays (n = 3). (C) Phosphorylation of APs in vitro. Pure APs (lane 1) or combined with the indicated cytosolic fractions (lanes 2–9) were incubated with [c-32P]ATP and then analysed by SDS/ PAGE. Bands were detected by autoradiography.
the isoform a2 was more sensitive than a1 to the inhibitory effect of ATP. The inhibition mediated by ATP-cytosol is not related to AP-2 phosphorylation If we consider that the inhibitory effect of ATP was due to an increase of AP-2 subunit phosphorylation, cytosolic fractions with inhibitory activity would exhibit higher capability to phosphorylate AP-2 subunits. To assess this possibility, we incubated purified adaptins with aliquots of each cytosolic fraction and [c32P]ATP. We observed that proteins in the range of l subunits were phosphorylated by all the cytosolic fractions and to a similar extent, although with little phosphorylation on proteins ranging between 100 and 110 kDa (Fig. 4C). We observed neither self-phosphorylation of cytosolic proteins or APs. Kinases and/or phosphatases may participate in the complex machinery for the assembly of AP-2 From the results detailed above, we wondered if cytosolic kinases and/or phosphatases were located sequentially and participated in a fairly complex cascade of phosphorylations/dephosphorylations to regulate the AP-2 assembly–disassembly cycle. To explore this, we
pre-incubated cytosols with inhibitors for either phosphatase or kinase prior to the binding assays and evaluated the incidence on the interactions between AP-2 and membranes. Fig. 5A shows that tyrphostin, a tyrosine kinase inhibitor, was able to abolish the binding of AP-2 to membranes, and that this effect was enhanced by ATP. In this case, the effect was more significant for a1 than for a2 subunit. KN93, a well-known inhibitor for calcium-calmodulin-kinase type II (CAMKII), did not significantly affect the binding of AP-2 to membranes (Fig. 5B). According to this, protein phosphorylation favours the binding of AP-2 to membranes, and tyrosine kinases may be involved in some steps of this process. Redistribution of AP-2 to cytosol induced by alkaline phosphatase treatment (Fig. 1) supports this hypothesis. In this way, we may expect that inhibitors of phosphatase could enrich the binding of AP-2 to membranes. Surprisingly, when cytosol was pre-incubated with cyclosporine, an inhibitor of calcineurin, the binding of AP-2 to membranes was reduced (Fig. 5C). Likewise, an additional inhibitory effect was observed when cyclosporine was added in the presence of ATP regeneration system. Although the binding of AP-2 was also reduced by the addition of the ATPase inhibitor sodium orthovanadate, the effect was at lesser extent (Fig. 5D), and no inhibition was observed with okadaic acid (data not shown).
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Fig. 5. In vitro binding assay of cytosolic AP-2 to vesicle membranes after incubation with protein kinase and protein phosphatase inhibitors. Binding assays were performed as previously. Cytosols (50 lg) were incubated with stripped vesicles (sv, 20 lg) in the presence or absence of an ATP-regeneration system at indicated concentrations. Some cytosols were pre-incubated prior to binding with the KN93 (30 mM), tyrphostin (1 mM), sodium orthovanadate (1 mM), or cyclosporine (1 lM) inhibitors. After binding, the samples were prepared, and subjected to SDS/ PAGE and Western blot with antibodies for AP-2. Graphs show the values of densitometric scanning (OD) of a2- and a1-AP-2 bands from blots. Bars represent the mean of independent experiments ± standard error mean (SEM). The number of experiences (n) is indicated. Data were analysed with One-Way ANOVA followed by TukeyÕs multiple comparisons test. Signification level: p < 0.05.
Discussion The results presented in this study, combined with those from others [5,6,17], support that cycles of phosphorylation and dephosphorylation of proteins may regulate rounds of assembly–disassembly of AP-2 in clathrin coated vesicle formation. We also attempted to explain the role of ATP, related to phosphorylation and dephosphorylation of proteins, and to the interaction between AP-2 and membranes. Accumulated evidences suggest that the protein phosphorylation that utilizes ATP regulates several steps of CCV formation in neuronal cells (for review see [10]). However, in vitro and in vivo studies have shown opposite effects of ATP on recruitment and release of AP-2 to and from membranes [11–16]. ATP also participates in clathrin disassembly by a mechanism that involves ATPase activity of Hsc70 and auxilin [15]. In this study, the facts that the treatment with alkaline phosphatase induced a redistribution of AP-2 to cytosol and that tyrphostin inhibited the binding of AP-2 to membranes suggest that phosphorylation of proteins favours that interaction, in agreement with previous reports [5,6,17–19]. However, although a1 and a2 subunits of AP-2 are phosphorylated in serine residues, we consider that this covalent modification is not deter-
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mining for the interaction with membranes, but they may form part of a complex cascade of phosphorylations instead, aimed to enhance that interaction. This was based on the decrease of the binding of AP-2 to membranes by a tyrosine kinase inhibitor (Fig. 5B) and that AP-2 subunits were not phosphorylated in tyrosine. Recently, it was shown that b2 is phosphorylated in tyrosine by EGFR kinase [7]. If phosphorylation of b2 regulates binding of AP-2 to membranes, this may justify the inhibitory effect of tyrphostin. However, the fact that b2 was scarcely redistributed to the cytosol by treatment with alkaline phosphatase indicates that phosphorylation of this subunit does not regulate the binding of AP-2 to membranes. Since ATP favoured phosphorylation of proteins (including l subunits), but it also inhibited the binding of AP-2 to membranes (Fig. 3), we speculate that ATP may be a substrate donor that induces inactivation of certain tyrosine kinases by phosphorylation, in a putative complex cascade of phosphorylations. According to this, a phosphatase (as calcineurin) may activate certain kinases by dephosphorylation and justify the decrease of binding by cyclosporine (Fig. 5D). In support of this, a previous report showed that calcineurin upregulates the CCV assembly in nerve terminals [10]. It is also possible that certain tyrosine kinases (inhibitable by tyrphostin) exert an action downstream the cascade, activating other kinases by phosphorylation (e.g., serine kinases). However, we cannot discard the possibility that, in our model, the proposed tyrosine kinase may phosphorylate other accessory proteins (as dephosphins) that aid in the assembly of AP-2 [10]. On the other hand, we observed that assembly of both a1 and a2 to membranes might be differently regulated. This is supported by the fact that at steady state, a2 is mostly bound to brain membranes meanwhile a1 is distributed to the cytosol. Thus, redistribution of a2 to cytosol by the alkaline phosphatase treatment became more dramatic than for a1. The existence of two a subtypes in neuronal tissue is still poorly understood and from our results we propose that both may belong to different populations of clathrin coated vesicles. Since dephosphorylation by alkaline phosphatase, or incubation with either ATP or inhibitors, did not completely abolish the binding of AP-2, it is also possible that certain AP-2 complexes assemble into clathrin coated pits by a phosphorylation-independent mechanism. One possibility for this alternative binding may be via the phosphoinositide-binding sites of a- [20] and l2 subunits [21]. This could provide an initial targeting of certain AP-2 complexes since the plasma membrane is enriched in phosphatidylinositol 4,5-biphosphate [20]. In conclusion, from the results presented here we suggest that a cascade of protein phosphorylations consist-
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ing in the coordinated action of kinases and phosphatases to activate proteins may be responsible for enhancing the assembly of AP-2 to membranes at the early steps of clathrin coated vesicle formation. Further studies should be done in order to identify the possible kinases involved in this process and the targets for those enzymes.
References [1] S.D. Conner, S.L. Schmid, Differential requirements for AP-2 in clathrin-mediated endocytosis, J. Cell Biol. 162 (2003) 773– 779. [2] J. Hirst, M.S. Robinson, Clathrin and adaptors, Biochim. Biophys. Acta 1404 (1998) 173–193. [3] T. Kirchhausen, Adaptors for clathrin-mediated traffic, Annu. Rev. Cell Dev. Biol. 15 (1999) 705–732. [4] A. Wilde, F.M. Brodsky, In vivo phosphorylation of adaptors regulates their interaction with clathrin, J. Cell Biol. 135 (1996) 635–645. [5] A. Fingerhut, K. von Figura, S. Honing, Binding of AP2 to sorting signals is modulated by AP2 phosphorylation, J. Biol. Chem. 276 (2001) 5476–5482. [6] S.D. Conner, S.L. Schmid, Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis, J. Cell Biol. 156 (2002) 921–929. [7] F. Huang, X. Jiang, A. Sorkin, Tyrosine phosphorylation of the b2 subunit of clathrin adaptor complex AP-2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking, J. Biol. Chem. 278 (2003) 43411–43417. [8] M.A. Sosa, F. Bertini, K. von Figura, A. Hille-Rehfeld, Is an ATPase involved in uncoating of plasma membrane adaptor complex AP-2? FEBS Lett. 407 (1997) 173–176. [9] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685.
[10] M.A. Cousin, P.J. Robinson, The dephosphins: dephosphorylation by calcineurin triggers synaptic vesicle endocytosis, Trends Neurosci. 24 (2001) 659–665. [11] E. Smythe, L.L. Carter, S.L. Schmid, Cytosol- and clathrindependent stimulation of endocytosis in vitro by purified adaptors, J. Cell Biol. 119 (1992) 1163–1171. [12] M.N. Seaman, C.L. Ball, M.S. Robinson, Targeting and mistargeting of plasma membrane adaptors in vitro, J. Cell Biol. 123 (1993) 1093–1105. [13] M.A. West, N.A. Bright, M.S. Robinson, The role of ADPribosylation factor and phospholipase D in adaptor recruitment, J. Cell Biol. 138 (1997) 1239–1254. [14] L.A. Hannan, S.L. Newmyer, S.L. Schmid, ATP- and cytosoldependent release of adaptor proteins from clathrin-coated vesicles: a dual role for Hsc70, Mol. Biol. Cell 9 (1998) 2217–2229. [15] A. Umeda, A. Meyerholz, E. Ungewickell, Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation, Eur. J. Cell Biol. 79 (2000) 336–342. [16] J.R. Morgan, K. Prasad, S. Jin, G.J. Augustine, E.M. Lafer, Uncoating of clathrin-coated vesicles in presynaptic terminals: roles for Hsc70 and auxilin, Neuron 32 (2001) 289–300. [17] P. Ghosh, S. Kornfeld, AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation, J. Cell Biol. 160 (2003) 699–708. [18] D. Ricotta, S.D. Conner, S.L. Schmid, K. von Figura, S. Honing, Phosphorylation of the AP2 l subunit by AAK1 mediates high affinity binding to membrane protein sorting signals, J. Cell Biol. 156 (2002) 791–795. [19] A.P. Jackson, A. Flett, C. Smythe, L. Hufton, F.R. Wettey, E. Smythe, Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor l2 kinase, J. Cell Biol. 163 (2003) 231–236. [20] I. Gaidarov, J.H. Keen, Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits, J. Cell Biol. 146 (1999) 755–764. [21] G. Rohde, D. Wenzel, V. Haucke, A phosphatidylinositol (4,5)bisphosphate binding site within l2-adaptin regulates clathrinmediated endocytosis, J. Cell Biol. 158 (2002) 209–214.