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1 Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine, 65 Tsurumai, Showaku, Nagoya, Aichi, 466-8550, Japan
2 Undergraduate Program for Bioinformatics and Systems Biology, Graduate School of Information Science and Technology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
3 Central Laboratories for Key Technology, Kirin Brewery Co. Ltd, 1-13-5 Fukuura, Kanazawaku, Yokohama, Kanagawa, 236-0004, Japan
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Abstract |
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Introduction |
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Cbl is a multiple-adaptor protein involved in ligand-induced down-regulation of receptor tyrosine kinases. Cbl-mediated ubiquitination of active receptors appears to be essential for receptor degradation and cessation of receptor-induced signal transduction (Joazeiro et al. 1999; Levkowitz et al. 1999; Di Fiore & De Camilli 2001; Thien & Langdon 2001; Waterman & Yarden 2001; Soubeyran et al. 2002). In addition, Cbl regulates EGF receptor endocytosis in a different fashion: Cbl recruits CIN85 (Cbl-interacting protein of 85 kDa) and endophilin (regulatory components of clathrin-mediated endocytosis) to form a complex with activated EGF receptors, thereby controlling receptor internalization (Soubeyran et al. 2002). Endophilin is the regulatory component of clathrin-coated pits (Ringstad et al. 1997; Schmidt et al. 1999; Simpson et al. 1999; Gad et al. 2000), and it can bind to lipid bilayers and induce local changes in membrane curvature (Schmidt et al. 1999). In addition, endophilin associates with other accessory protein such as dynamin, synaptojanin, and amphiphysin, participating in the formation of clathrin-coated vesicle (de Heuvel et al. 1997; Micheva et al. 1997; Ringstad et al. 1997).
The small GTPase Rho regulates various cellular functions, including endocytosis (Etienne-Manneville & Hall 2002; Symons & Rusk 2003). Rho cycles between two conformational states, the GDP-bound (inactive) state and the GTP-bound (active) state (Etienne-Manneville & Hall 2002). Rho is a molecular switch that uses a simple biochemical strategy to control the complex cellular processes of cell migration (Raftopoulou & Hall 2004) and neuronal development (Govek et al. 2005). Rho exerts its biological functions through interaction with specific effectors (Van Aelst & D'Souza-Schorey 1997). We previously identified Rho-associated kinase (Rho-kinase)/ROK
/ROCK II as an effector of Rho (Leung et al. 1995; Ishizaki et al. 1996; Matsui et al. 1996). Rho-kinase is activated by the GTP-bound active form of Rho (Ishizaki et al. 1996; Matsui et al. 1996; Amano et al. 1997). Rho-kinase appears to regulate various cellular responses downstream of Rho (Kaibuchi et al. 1999; Riento & Ridley 2003). Rho regulates transferrin and EGF receptor internalization (Lamaze et al. 1996), and it is involved in the internalization of low-density lipoprotein (LDL) receptor, which requires the clathrin-mediated endocytosis (Hrboticky et al. 2002). Thus, Rho appears to play a critical role in clathrin-mediated receptor endocytosis. However, little is known about how Rho regulates EGF receptor endocytosis.
In the present study, we found that the Rho/Rho-kinase signal regulates EGF receptor endocytosis. We identified endophilin A1 as a novel substrate of Rho-kinase. The phosphomimetic form of endophilin A1 prevented EGF receptor internalization. Furthermore, phosphorylated endophilin A1 decreased its binding activity to CIN85. Thus, Rho/Rho-kinase appears to inhibit the recruitment of endophilin A1 to the activated EGF receptor through the CIN85/Cbl complex, thereby preventing EGF receptor endocytosis.
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Results |
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To examine whether Rho-kinase is involved in EGF receptor endocytosis downstream of RhoA, we measured the internalization of EGF receptors using Alexa 555-conjugated EGF in PC12D cells. When PC12D cells were incubated with Alexa 555-conjugated EGF for 60 min at 4 °C and subsequently incubated for 15 min at 37 °C, some of the labeled EGF was internalized, indicating that the EGF/EGF receptor complex was endocytosed, as previously described (Wilde et al. 1999) (Fig. 1A,B). Compared with nontransfected cells, the cells expressing EGFP did not affect the endocytosis of EGF receptors (Fig. 1A,B).
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Identification of endophilin A1 as a novel substrate of Rho-kinase in bovine brain
To clarify the molecular mechanisms of how the Rho/Rho-kinase signal regulates EGF receptor endocytosis, we sought to identify the responsible substrates of Rho-kinase. To search for the substrates, we separated bovine brain membrane and peripheral membrane proteins by Mono Q column chromatography, and the fractions were subjected to phosphorylation assay using the constitutively active form of Rho-kinase (GST-Rho-kinase CAT), which contains the catalytic domain (6–553 amino acids) of Rho-kinase (Amano et al. 1996, 1997) (Fig. 2A). Among the several proteins detected in this assay, one with a mass of about 40 kDa (p40) was recognized as a major band phosphorylated in a GST-Rho-kinase CAT-dependent manner (Fig. 2B, fractions 14–16). p40 was further purified by S-sepharose column chromatography (Fig. 2A,C), and the molecular identity of p40 was determined by mass spectral analysis. The molecular weight of nine peptide fragments derived from p40 coincided with those from human endophilin A1: KDLREIQHHLK, KLEGRRLDFDYK, KVDVTSRAVMEIMTK, KQNFIDPLQNLHDK, KTIEYLQPNPASRAK, KGPGYPQAEALLAEAMLK, KGGPGYPQAEALLAEAMLK, KFGRELGDDCNFGPALGEVGEAMRELSEVK, and KFGRELGDDCNFGPALGEVGEAMRELSEVK. Furthermore, p40 was recognized by anti-endophilin antibody (Fig. 2D). Thus, we conclude that p40 is endophilin A1. To examine whether Rho-kinase directly phosphorylates endophilin A1, endophilin A1 was produced as a GST-fusion protein and subjected to the phosphorylation assay. GST-endophilin A1 was phosphorylated by GST-Rho-kinase CAT (Fig. 2E). These results indicate that Rho-kinase directly phosphorylates endophilin A1 in vitro.
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Identification of phosphorylation site of endophilin A1 by Rho-kinase
To determine the phosphorylated region of endophilin A1 by Rho-kinase, five fragments of endophilin A1 were produced as GST-fusion proteins (Fig. 3A). The purified GST-endophilin A1 1–352 amino acids, 35–352 amino acids, 1–290 amino acids, 126–290 amino acids, and 291–352 amino acids were phosphorylated by GST-Rho-kinase CAT in the presence of [
-32P]ATP in vitro. GST-endophilin A1 35–352 amino acids, 126–352 amino acids, and 291–352 amino acids were not phosphorylated by Rho-kinase CAT (Fig. 3B). These results suggest that the region containing 1–34 amino acids of endophilin A1 is phosphorylated by Rho-kinase.
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Endophilins A1, A2, and A3 share a sequence similarity in the amino terminus region (Fig. 3F). The sequences around Ser-14 (KAS14) in the amino terminus region of endophilins A2 and A3 were regarded as phosphorylation consensus sequences of Rho-kinase (KXS) (Fig. 3F). It was thus expected that Rho-kinase would phosphorylate Ser-14 of endophilin A2 and A3. To examine whether Rho-kinase phosphorylates endophilin isoforms, GST-endophilins A2 and B were subjected to the phosphorylation assay. Endophilin A2 was slightly phosphorylated by Rho-kinase CAT (Fig. 3G), whereas endophilin B was not (Fig. 3G). Because surrounding amino acid sequences of Ser-14 in endophilin A2 are almost identical to those of Ser-14 in endophilin A3, endophilin A3 may be hardly phosphorylated by Rho-kinase. Thus, endophilin A1 may be the best substrate of Rho-kinase among the endophilin isoforms.
Production and characterization of site- and phosphorylation-state-specific antibody for endophilin A1 at Thr-14
To investigate how the phosphorylation of endophilin A1 by Rho-kinase is regulated in vivo, we prepared the site- and phosphorylation-state-specific antibody for endophilin A1. We prepared rabbit polyclonal antibody (anti-pT14 Ab), raised against the synthetic phosphopeptide GCQFHKA-phosphoT-QKVSEK. The specificity of anti-pT14 Ab was examined by immunoblot analysis. Equal amounts of GST-endophilin A1 with various ratios between phosphorylated and non-phosphorylated forms were loaded on the gel. GST-endophilin A1 phosphorylated by Rho-kinase CAT in vitro was specifically detected by anti-pT14 Ab in a dose-dependent manner (Fig. 4). These results indicate that anti-pT14 Ab specifically recognizes endophilin A1 phosphorylated at Thr-14 by Rho-kinase.
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We examined whether Rho-kinase can phosphorylate endophilin A1 at Thr-14 in vivo. EGFP-endophilin A1 was expressed exogenously with wild-type (WT), dominant negative (RB/PH (TT)), and constitutively active (Rho-kinase CAT) forms of Rho-kinase in COS7 cells. An equivalent amount of EGF-endophilin A1 WT and T14D was expressed in each case (Fig. 5A, lower). The expression of Rho-kinase CAT induced the phosphorylation of endophilin A1, whereas RB/PH (TT) did not. Rho-kinase WT slightly induced the phosphorylation of endophilin A1 (Fig. 5A, upper). EGFP-endophilin T14D co-expressed with Rho-kinase CAT was not recognized by anti-pT14 Ab (Fig. 5A, upper). These results indicate that endophilin A1 is phosphorylated at Thr-14 by Rho-kinase in COS7 cells.
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Rho-kinase is involved in epidermal growth factor receptor internalization through phosphorylation of endophilin A1
To examine whether Rho-kinase regulates the EGF receptor endocytosis through the phosphorylation of endophilin A1, we measured the internalization of EGF receptors using Alexa 555-conjugated EGF. The expression of control EGFP did not affect the internalization of EGF receptor (Fig. 6A,B). The expression of endophilin T14D (substitution of Thr-14 by Asp), which was expected to mimic phosphorylated endophilin A1 (Kamisoyama et al. 1994; Sweeney et al. 1994; Bresnick et al. 1995), inhibited the internalization of EGF receptor, whereas endophilin WT and T14A (substitution of Thr-14 by Ala) did not affect the internalization (Fig. 6A,B). Although the mode of action of endophilin T14D is not well understood, endophilin T14D may substitute for endogenous endophilin A1 to interact with its binding partner, thereby inactivating the complex and inhibiting functions of endogenous endophilin A1.
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Recently, it has been reported that endophilin binds to CIN85 and regulates EGF receptor endocytosis through the endophilin/CIN85/Cbl complex (Soubeyran et al. 2002). We examined whether phosphorylation of endophilin A1 affects its binding activity to CIN85. Purified GST-endophilin A1 was phosphorylated by Rho-kinase CAT in the presence or absence of ATP. Phosphorylated or non-phosphorylated endophilin A1 were immobilized on glutathione-sepharose beads and incubated with porcine brain lysate. CIN85 interacted with non-phosphorylated GST-endophilin A1, whereas CIN85 less effectively interacted with the phosphorylated GST-endophilin A1 (Fig. 7). These results indicate that endophilin A1 phosphorylated by Rho-kinase is prevented from interacting with CIN85.
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Discussion |
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Several groups have shown that RhoA regulates clathrin-mediated endocytosis (Lamaze et al. 1996; Hrboticky et al. 2002). Lamaze et al. (1996) reported that RhoA modulates transferrin and EGF receptor endocytosis; RhoA is also involved in the internalization of LDL receptor, which requires clathrin-mediated endocytosis (Hrboticky et al. 2002). However, the modes of action of Rho in endocytosis have not yet been elucidated.
In the present study, we found that Rho-kinase was involved in the endocytosis of EGF receptor downstream of Rho. We identified endophilin A1 as a substrate of Rho-kinase. Endophilin is a key regulator of clathrin-mediated endocytosis (Ringstad et al. 1997; Schmidt et al. 1999; Simpson et al. 1999; Gad et al. 2000). Endophilin binds to the regulatory components of clathrin-mediated endocytosis, amphiphysin, dynamin, and synaptojanin via the SH3 domain of the C-terminus (de Heuvel et al. 1997; Micheva et al. 1997; Ringstad et al. 1997). Endophilin is involved in membrane bending at the onset of budding during the early stage of endocytosis (Scales & Scheller 1999; Schmidt et al. 1999; Hill et al. 2001; Guichet et al. 2002; Verstreken et al. 2002; Schuske et al. 2003). Other reports suggest a role of endophilin during later stages of endocytosis (Ringstad et al. 1999) during vesicle uncoating (Gad et al. 2000; Fabian-Fine et al. 2003; Schuske et al. 2003) and in the progression of vesicles through the synaptic vesicle cycle (Fabian-Fine et al. 2003). In addition, endophilin appears to be required for stabilizing synaptojanin in synaptic termini and in the recruitment of synaptojanin to endocytotic pits (Schuske et al. 2003; Verstreken et al. 2003). Thus, endophilin participates in multiple stages of clathrin-mediated endocytosis, from early membrane invagination to clathrin-coated vesicle uncoating.
We identified Thr-14 as the phosphorylation site of endophilin A1 by Rho-kinase. We also found that the phospho-mimetic form of endophilin A1 inhibited EGF receptor endocytosis. Taken together, these results suggest that Rho modulates EGF receptor endocytosis through the phosphorylation of endophilin A1 by Rho-kinase.
It may be noted that the phosphorylation of endophilin A1 was not detected in cell lysate without the treatment of phosphatase inhibitor (Calyclin A). Calyclin A-induced phosphorylation of endophilin A1 was strongly inhibited by Y-27632, suggesting that Rho-kinase phosphorylates endophilin A1 in vivo, but this phosphorylation is tightly regulated by phosphatase and rapidly turned over. Rho-kinase phosphorylates MBS, which leads to the inactivation of myosin phosphatase (Kimura et al. 1996). Rho-kinase and myosin phosphatase cooperatively regulate the phosphorylation state of the substrates including myosin light chain (MLC), adducin, and the ezrin/radixin/moesin (ERM) family proteins (Kaibuchi et al. 1999). It is possible that Rho-kinase regulates the phosphorylation of endophilin A1 through the dual regulation, which is the direct phosphorylation by Rho-kinase and inactivation of phosphatase. Thus, it is likely that the spatiotemporal phosphorylation of endophilin A1 occurs near the plasma membranes and plays a critical role in the internalization of EGF receptor.
Molecular mechanisms of EGF receptor endocytosis by Rho/Rho-kinase
Activated EGF receptors are removed from the cell surface via endocytosis and subsequent degradation in the lysosome (Dikic 2003). Ligand-dependent internalization may be a principal process regulating the duration and propagation of signal initiated by tyrosine kinase receptor, thereby preventing overstimulation.
Upon ligand stimulation, the Cbl/CIN85/endophilin complex was shown to promote EGF receptor internalization (Soubeyran et al. 2002). Cbl, which is an ubiquitin ligase, induces the receptor internalization via a pathway that is functionally separable from its ubiquitin ligase activity and is dependent on interactions with adaptor protein CIN85 (Dikic 2003). Several groups independently identified CIN85 (Take et al. 2000) as Ruk (regulator of ubiquitous kinase) (Gout et al. 2000), SETA (SH3 domain-containing gene expressed in tumorigenic astrocytes) (Borinstein et al. 2000), and SH3KBP1 (SH3-domain kinase-binding protein 1) (Narita et al. 2001). CIN85 interacts with two members of the Cbl family, Cbl and Cbl-b, but not Cbl-3 (Szymkiewicz et al. 2002). Binding of CIN85 to Cbl is mediated via its SH3 domains and is enhanced by EGF-induced tyrosine phosphorylation of Cbl, whereas the proline-rich region of CIN85 constitutively interacts with endophilin (Soubeyran et al. 2002). CIN85 appears to rapidly recruit endophilin to activated receptor complex, thus controlling receptor internalization (Petrelli et al. 2002; Soubeyran et al. 2002). Inhibition of the Cbl/CIN85/endophilin interaction is sufficient to block EGF receptor endocytosis and degradation, without perturbing the ability of Cbl to ubiquitinate activated receptors (Soubeyran et al. 2002). This mechanism seems to be common among certain receptor tyrosine kinases; for instance, it has been reported that the Cbl/CIN85/endophilin pathway is involved in the endocytosis of hepatocyte growth factor, platelet-derived growth factor (PDGF), and c-Kit (Petrelli et al. 2002; Szymkiewicz et al. 2002). Thus, endophilin has begun to emerge as a key regulator of receptor tyrosine kinases, and plays a pivotal role in receptor endocytosis.
Here we found that the phosphorylation of endophilin A1 by Rho-kinase inhibited the interaction with CIN85. Thus, endophilin A1 phosphorylated by Rho-kinase may not be recruited to endocytotic sites, resulting in inhibition of EGF-receptor endocytosis (Fig. 8). The present study shows for the first time that the Rho/Rho-kinase signaling pathway mediates EGF receptor endocytosis and reveals the underlying mechanisms.
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EGF stimulation activates p190RhoGAP, which is known as GTPase-activating protein (GAP) for Rho (Burridge & Wennerberg 2004). The activated p190RhoGAP induces GTP hydrolysis of GTP-bound Rho and inactivation of Rho (Burridge & Wennerberg 2004). Following EGF stimulation, both EGF receptor endocytosis and inactivation of Rho are induced rapidly. These observations are reasonable because activated Rho inhibits EGF receptor endocytosis. Thus, to progress the receptor endocytosis, EGF signals may induce the spatiotemporal inactivation of Rho and Rho-kinase. mDia interacting protein (DIP) binds to p190RhoGAP and inactivates Rho following EGF stimulation (Meng et al. 2004). When the expression levels of DIP are suppressed by RNAi, the EGF-induced cell migration is inhibited (Meng et al. 2004). Thus, the inactivation of Rho/Rho-kinase signal upon EGF stimulation may play an important role in the regulation of EGF-receptor endocytosis and cell motility.
EGF receptor endocytosis and trafficking are important for controlling the signaling pathways and cellular responses to EGF. EGF-induced activation of mitogen-activated protein kinase is dependent upon receptor endocytosis (Vieira et al. 1996). Inhibition of Cbl/CIN85/endophilin complex formation was sufficient to block EGF internalization, delay receptor degradation, and enhance EGF-induced gene expression (Soubeyran et al. 2002). It has been reported that only cell surface EGF receptors effectively participate in phospholipase D function (Haugh et al. 1999). A subset of signal transducers requires the normal endocytotic trafficking of EGF receptor for full activation of EGF signals. Thus, endocytotic trafficking of activated EGF receptor plays a critical role not only in attenuating EGF receptor signaling but also in establishing and controlling specific signaling pathways.
Brain-derived growth factor and PDGF induce down-modification of the EGF receptor by increasing the internalization of cell surface receptors (Huang et al. 1988). Elevated intracellular Ca2+ and phorbol esters are thought to inhibit EGF internalization by a mechanism involving activation of protein kinase C (Logsdon & Williams 1984). Several signaling pathways that modify EGF action are mediated through the internalization of EGF receptors. Here we indicate that Rho/Rho-kinase is involved in the EGF receptor internalization. Thus, the Rho/Rho-kinase signal may modulate the EGF signaling through EGF receptor endocytosis downstream of certain extracellular signals.
This study sheds light on the molecular mechanisms underlying EGF receptor endocytosis. We found that Rho regulates EGF receptor endocytosis through the phosphorylation of endophilin A1 by Rho-kinase. Rho/Rho-kinase may regulate EGF signal activity through receptor endocytosis. Understanding how this process is regulated in living cells will be a challenge for further investigations.
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Experimental procedures |
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Anti-endophilin antibody was kindly provided by Dr P. De Camilli (Yale University, New Haven, CT, USA) (Fig. 1D). We also prepared anti-endophilin antibody, which was raised against GST-endophilin A1 (Figs 4 and 5). A rabbit polyclonal antibody against endophilin A1 phosphorylated at Thr-14 was produced against phosphopeptide Gly-Cys-Gln9-Phe-His-Lys-Ala-phosphoThr14-Gln-Lys-Val-Ser-Glu-Lys20 by Biologica Co. (Aichi, Japan). The antisera were then affinity purified against the respective phosphopeptides. cDNA-encoding mouse endophilin A1 was provided by Dr H. D. Söling (Max-Planck Institute, Göttingen, Germany). pCAGGS vector was provided by Dr M. Nakafuku (Children's Medical Research Foundation, Cincinnati). All materials used in the nucleic acid study were purchased from Takara Shuzo Co. (Kyoto, Japan). Other materials and chemicals were obtained from commercial sources.
Plasmid constructs
cDNA fragments encoding endophilin A1 1–352 amino acids (full length), 35–352 amino acids, 1–290 amino acids, 126–290 amino acids, and 291–352 amino acids were amplified by polymerase chain reaction (PCR) using the obtained clone as a template, containing the entire open reading frame of mouse endophilin A1. The endophilin mutants, T14D and T14A, were generated with a site-detected mutagenesis kit (Stratagene, La Jolla, CA, USA) by changing Thr-14 into Asp-14 or Ala-14. cDNAs encoding endophilins A2 and B were cloned from a human fetal library. The cDNA fragments were subcloned into pGEX-4T-1 (Amersham Pharmacia Biotech, Buckinghamshire, UK), pCAGGS-myc, and pEGFP-C1 (Clontech Laboratories, Inc., Palo Alto, CA) vectors.
Phosphorylation assay
The phosphorylation assay of the samples was performed as previously described (Amano et al. 1996). In brief, the kinase reaction for Rho-kinase was carried out in 50 µL of a reaction mixture (50 mM Tris/HCl at pH 7.5, 2 mM EDTA, 1 mM DTT, 7 mM MgCl2, 10 µM[-32P]ATP [1–20 GBq/mmol], and purified GST [glutathione-S-transferase]-constitutively active form of Rho-kinase [GST-Rho-kinase CAT]) for 10–60 min at 30 °C. GST-Rho-kinase CAT was produced in Sf9 cells with a baculovirus system (Matsuura et al. 1987) and purified on glutathione-sepharose 4B beads (Amersham Pharmacia Biotech, Buckinghamshire, UK) (Matsui et al. 1996). Then the reaction mixtures were boiled in SDS sample buffer and subjected to SDS-PAGE. The radiolabelled bands were visualized by an image analyzer (BAS 2000; Fuji, Tokyo, Japan).
Purification of a Rho-kinase substrate, p40
Bovine brain gray matter was homogenized and fractionated with centrifugation at 20 000 g. The proteins contained in the precipitate (Ppt 1) were extracted with 2 M NaCl and centrifuged at 10 000 g. The proteins contained in the precipitate (Ppt 2) were subsequently solubilized with 1% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid (CHAPS) and the solubilized proteins were recovered by centrifugation at 100 000 g in the supernatant (Sup 3). Sup 3 was diluted to 0.5% CHAPS and subjected to Mono Q column chromatography. Proteins were eluted with a linear gradient of NaCl (0.0–0.5 M). The eluates from Mono Q column were phosphorylated with GST-Rho-kinase CAT. The fractions containing phosphorylated proteins were collected and further fractionated by S-sepharose column chromatography with a step-wise gradient of NaCl (0.0–0.5 M). The eluates from S-sepharose were phosphorylated with GST-Rho-kinase CAT.
Mass spectral analysis
The fractions containing p40 were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Problot, Applied Biosystems, Foster, CA). The immobilized protein was reduced, S-carboxymethylated, and digested in situ with Achromobacter protease I (a Lys-C) (Iwamatsu 1992). Molecular mass analyses of Lys-C fragments were performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using a PerSeptive Biosystem Voyager-DE/RP (Foster, CA, USA) (Jensen et al. 1996). We identified protein by comparing the molecular weight determined by v/MS and theoretical peptide masses from the proteins registered in NCBInr.
Culture preparation
PC12D cells were grown in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 5% horse serum at 37 °C in an air with 5% CO2 atmosphere at constant humidity. COS7 cells were cultured in DMEM containing 10% FBS. For transfection experiments, LipofectAMINE 2000 and LipofectAMINE reagents (Invitrogen, Carlsbad, CA, USA) were used.
Pull-down assay
Purified GST-tagged endophilin A1 (GST-endophilin A1) was phosphorylated by GST-Rho-kinase CAT in the presence or absence of ATP. Phosphorylated or non-phosphorylated endophilin A1 was immobilized onto glutathione-sepharose 4B beads for 60 min at 4 °C. The GST-endophilin A1 immobilized beads were incubated with porcine brain lysate for 60 min at 4 °C. After removal of the supernatant, the beads were washed three times with buffer (20 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM DTT, 50 mM NaCl, 10 µg/mL leupeptin, 10 µM APMSF, 0.1% NP-40, 0.1 µM Calyclin A, 25 mM NaF, 2 mM Na-orthovanadate, 10 mMß-glycerophosphate). Then the samples were boiled in SDS sample buffer and subjected to SDS-PAGE and immunoblot analysis with the indicated antibodies.
Internalization assay
Internalization assay with EGF receptor in PC12D cells was performed 24 h after transfection. The cells were washed three times in cold PBS and incubated with 0.5 µg/mL Alexa Fluor 555-conjugated EGF (Molecular Probes, Eugene, OR) in DMEM containing 2 mg/mL BSA and HEPES for 60 min at 4 °C. After the incubation with Alexa Fluor 555-EGF at 4 °C, the cells were transferred to 37 °C for 15 min and incubated with 0.2 M acetic acid (pH 2.5) containing 0.5 M NaCl for 5 min on ice, to remove surface-bound ligand, and washed with cold PBS. The cells were fixed with 3.7% formaldehyde in PBS buffer for 10 min at room temperature, rinsed three times with PBS, and mounted. The cells were observed using a Zeiss Axiophoto microscope (Carl Zeiss, Oberkochen, Germany), and the percentage of the transfected cells displaying significantly reduced levels of endocytosis were counted. Fluorescent images were taken with a confocal laser microscopy system (LSM510; Carl Zeiss).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: kaibuchi{at}med.nagoya-u.ac.jp
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Received: 29 April 2005
Accepted: 5 July 2005
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