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Originally published In Press as doi:10.1074/jbc.M000521200 on April 6, 2000

J. Biol. Chem., Vol. 275, Issue 24, 18399-18406, June 16, 2000
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Regulation of Complex Formation of POB1/Epsin/Adaptor Protein Complex 2 by Mitotic Phosphorylation*

Kenji Kariya, Shinya Koyama, Shintaro Nakashima, Takafumi Oshiro, Kenji Morinaka, and Akira KikuchiDagger

From the Department of Biochemistry, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan

Received for publication, January 23, 2000, and in revised form, April 2, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RalBP1 and POB1, the downstream molecules of small GTP-binding protein Ral, are involved in receptor-mediated endocytosis together with Epsin and Eps15. The regulation of assembly of the complex of these proteins was examined. RalBP1, POB1, Epsin, and Eps15 formed a complex with alpha -adaptin of AP-2 in Chinese hamster ovary cells, but the formation was reduced in mitotic phase. RalBP1, POB1, Epsin, and Eps15 were all phosphorylated in mitotic phase. The phosphorylated forms of POB1 and Epsin were recognized by the antibody MPM2, which is known to detect mitotic phosphoproteins. POB1 and Epsin were phosphorylated by p34cdc2 kinase in vitro. Their phosphorylation sites (Ser411 of POB1 and Ser357 of Epsin) were determined. Phosphorylated Epsin and EpsinS357D formed a complex with alpha -adaptin less efficiently than wild type Epsin. Although the EH domain of POB1 bound directly to Epsin, phosphorylation of Epsin inhibited the binding. Furthermore, EpsinS357D but not EpsinS357A lost the effect of Epsin on the insulin-dependent endocytosis. These results suggest that phosphorylation of Epsin in mitotic phase inhibits receptor-mediated endocytosis by disassembly of its complex with POB1 and alpha -adaptin.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The uptake of a variety of hormones and nutrients by eukaryotic cells occurs by receptor-mediated endocytosis. Occupied cell surface receptors are internalized via clathrin-coated vesicles and are transported to endosomes, where ligands dissociate during the gradual acidification of the endosomal lumen (1). The main structural component on clathrin-coated vesicles is clathrin, a trimeric scaffold protein, which organizes itself into cage-like lattices (2). Clathrin has the shape of a triskelion, where each of the three legs is made of a heavy and a light chain. The assembly of the clathrin lattice on the cytosolic side of the plasma membrane occurs during the formation of a coated pit, leading to the production of a coated vesicle. Clathrin is thus an organizing framework for the proteins that carry out receptor sorting and several steps in the cycle of vesicle assembly, uncoating, and fusion. The major proteins that drive clathrin coat formation are APs1 (3, 4). APs can promote clathrin cage assembly, linking clathrin to the membranes and interacting with membrane proteins that contain appropriate signals for sorting into clathrin-coated vesicles. Each AP contains four subunits. Different APs are associated with specific clathrin-coated vesicle populations and confer distinct sorting properties onto these vesicles (5). AP-1 complexes are associated with clathrin-coated vesicles derived from the trans-Golgi network and contain gamma , beta 1, µ1, and sigma 1 subunits. AP-2 complexes are associated with endocytic clathrin-coated vesicles and contain alpha , beta 2, µ2, and sigma 2 subunits. AP-3 and AP-4 have been identified but have not yet been well characterized (6, 7). The beta  subunits of AP-1 and AP-2 interact with clathrin and drive the formation of coats.

Membrane proteins (receptors) that are internalized in clathrin-coated vesicles contain at least one signal sequence that direct the proteins into clathrin-coated pits (8). Tyrosine-based signals of the form of YXXPhi and dileucine (LL)-containing signals in the cytoplasmic domains of the receptors interact with AP-2 complexes. YXXPhi motif binds directly to the µ2 subunit, but the binding site on AP-2 for LL signals is not yet clearly established. Although the mechanism by which the receptor is captured by clathrin-coated pits is not clear, the AP-2/receptor complex may recruit cytosolic clathrin to form a coated pit or be captured by available clathrin already located at the edge of a coated pit. The crystal structure of the appendage domain of the alpha  subunit (alpha -adaptin) of AP-2 has been solved (9). The N-terminal beta -sandwich subdomain appears to function as a scaffold and spacer that supports a C-terminal platform subdomain to which various proteins implicated in endocytosis including Eps15, Epsin, and AP180 bind (10-12).

Eps15 was originally identified as a substrate of EGF receptor kinase and is constitutively associated with the plasma membrane and AP-2 (13, 14). Membrane-bound Eps15 is mainly associated with clathrin-coated pits and vesicles. Furthermore, expression of the AP-2-binding region of Eps15 in CV-1, COS, and HeLa cells inhibits internalization of the EGF and transferrin receptors (15, 16). Thus, Eps15 is involved in receptor-mediated endocytosis. Eps15 has three copies of the EH domain in the N terminus. The EH domain has been found in several proteins of mammals, yeast, insects, and nematodes, thus establishing its evolutionary conservation (14). We have identified an EH domain-containing protein that binds to RalBP1 (Ral-binding protein 1), which is an effector protein of small G protein Ral (17), and named it POB1 (partner of RalBP1) (18). POB1 has a single EH domain in its N-terminal region and two proline-rich motifs and a coiled-coil structure in its C-terminal region. The activated Ral forms a complex with POB1 through RalBP1. POB1 binds directly to Grb2 but not to Nck or Crk. Furthermore, EGF stimulates tyrosine phosphorylation of POB1, which leads to the formation of a complex between EGF receptor and POB1 (18). Expression of the EH domain and the C-terminal region of POB1 inhibits the internalization of EGF and insulin (19). Other proteins with the EH domain include Eps15R (for Eps15-related), Reps1 (for RalBP1-associated Eps homology domain protein 1), intersectin, Pan1, and End3. Eps15R has 47% amino acid identity with Eps15 and exhibits characteristics similar to those of Eps15 (20, 21). The POB1-related protein Reps1 has been identified as a RalBP1-binding protein (22). Intersectin (Ese) has five Src homology 3 domains in addition to two EH domains and is involved in the regulation of internalization of the transferrin receptor (23, 24). Pan1 and End3 are Saccharomyces cerevisiae dimeric partners that are necessary for endocytosis of the alpha -mating factor receptor and for normal organization of the actin cytoskeleton (25, 26). Thus, the EH domain containing proteins are likely to regulate endocytosis.

Epsin has been identified as a binding protein of the EH domains of Eps15, POB1, and intersectin (11, 19, 23, 24, 27). The N-terminal region of Epsin has the evolutionarily conserved domain called ENTH (for Epsin N-terminal homology) (28), the central region is characterized by the presence of eight repeats of Asp-Pro-Trp (DPW) motif, and the C-terminal region of Epsin contains three Asn-Pro-Phe (NPF) motifs, which are known to constitute the binding sequence of the EH domain (14). The central region of Epsin binds to AP-2, and overexpression of this region inhibits the clathrin-dependent internalization of the receptors for EGF and transferrin (11). Epsin has a Leu-Val-Asp-Leu-Asp (LVDLD) sequence. Similar amino acid sequences are found in beta  subunits of clathrin adaptors (AP-1, AP-2, and AP-3), arrestin 3, and amphiphysin and are shown to bind clathrin directly (29). Therefore, Epsin would regulate endocytosis by interacting with AP-2 and clathrin.

We have demonstrated that the small G protein Ral and its downstream molecules, RalBP1 and POB1, are involved in receptor-mediated endocytosis for EGF and insulin (19). Furthermore, we have found that Eps15 and Epsin bind directly to the EH domain of POB1 (19, 27). These results suggest that the signal from Ral to Eps15 and Epsin through RalBP1 and POB1 regulates clathrin-mediated endocytosis. However, how assembly and disassembly of this complex are regulated is not known. It has been shown that the complex formation of proteins involved in the endocytosis of synaptic vesicles is regulated by their phosphorylation (30). Therefore, we examined whether phosphorylation affects receptor-mediated endocytosis in the Ral signaling pathway.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Chemicals-- CHO-IR cells (insulin receptor-overexpressing Chinese hamster ovary cells) and the anti-GST and anti-MBP antibodies were kindly supplied by Drs. Y. Ebina (Tokushima University, Tokushima, Japan), and M. Nakata (Sumitomo Electric Industries, Yokohama, Japan), respectively. Histone H1 and pACT2/alpha -adaptin were generous gifts from Drs. H. Usui (Hiroshima University, Hiroshima, Japan) and H. Ohno (Kanazawa University, Kanazawa, Japan), respectively. Hygromycin-resistant CHO-IR cells that stably express RalBP1, POB1, human Epsin (hEpsin), or their mutants were propagated as described (19). The anti-POB1 and anti-hEpsin antibodies were prepared by immunizing rabbits with GST-POB1-(126-227) and hEpsin-(1-205), respectively. GST and MBP fusion proteins were purified from Escherichia coli. [125I]Insulin was purchased from Amersham Pharmacia Biotech, Inc. (Buckinghamshire, United Kingdom). The anti-Eps15 and anti-alpha -adaptin antibodies were from Transduction Laboratories, Inc. (Lexington, KY). The anti-RalBP1 antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody MPM2 and the GST-p13suc1 agarose were from Upstate Biotechnology, Inc. (Lake Placid, NY). Other materials and chemicals were from commercial sources.

Plasmid Constructions-- pEF-BOS-Myc/hEpsin (full length), pMAL-c2/hEpsin-(204-458), pCGN/POB1 (full length), pCGN/POB1-(1-374), pGEX-2T/POB1-(126-227) (EH domain), pGEX-2T/POB1-(375-521), and pGEX-2T/Eps15-(1-330) (EH domain) were constructed as described (18, 19, 27). pEF-BOS-Myc/hEpsinS357D and pEF-BOS-Myc/hEpsinS357A were constructed as follows. The 0.53-kb fragment encoding hEpsin-(297-474), in which Ser357 was mutated to Asp or Ala, was synthesized by polymerase chain reaction, digested with StuI, and inserted into StuI-cut pBSKS/hEpsin (full length). To construct pEF-BOS-Myc/hEpsinS357D and pEF-BOS-Myc/hEpsinS357A, pBSKS/hEpsinS357D and pBSKS/hEpsinS357A were digested with XbaI and then inserted into XbaI-cut pEF-BOS-Myc. pCGN/POB1S411D and pCGN/POB1S411A were constructed as follows. The 0.57-kb fragment encoding POB1-(321-512) in which Ser411 was mutated to Asp or Ala was synthesized by polymerase chain reaction, digested with BglII and MunI, and inserted into BglII- and MunI-cut pCGN/POB1 (full length). To construct pGEX-2T/alpha -adaptin appendage domain (app), the 0.7-kb fragment encoding alpha c-adaptin (701-938) with BamHI and EcoRI sites was synthesized by polymerase chain reaction, digested with BamHI and EcoRI, and inserted into BamHI- and EcoRI-cut pGEX-2T. To construct pGEX-2T/RalBP1-(364-647), pMAL-c2/RalBP1-(364-647) was digested with BamHI, and the fragment encoding RalBP1-(364-647) was inserted into BamHI-cut pGEX-2T. To construct pGEX-2T/POB1- (1-125) and pGEX-2T/POB1-(228-406), pBSKS/POB1-(1-125) and pBSKS/POB1-(228-406) were digested with PmaCI and EcoRI, and the 0.4- and 0.55-kb fragments were inserted into SmaI- and EcoRI-cut pGEX-2T. To construct pGEX-2T/POB1-(322-521), pUC19/POB1 (full length) was digested with BglII and blunted with Klenow fragment, and the 0.6-kb fragment encoding POB1-(322-521) was inserted into pGEX-2T that had been digested with BamHI and blunted with Klenow fragment. To construct pCGN/POB1-(322-521), pUC19/POB1 (full length) was digested with BglII and inserted into BamHI-cut pMAL-c2, then digested with EcoRI, blunted with Klenow fragment, and digested with XbaI. The fragment encoding POB1-(322-521) was inserted into pCGN that had been digested with XbaI and SmaI. To construct pEF-BOS-Myc/hEpsin (204-551), pBSKS/hEpsin (full length) was digested with BamHI and XbaI and then blunted with Klenow fragment. The 1-kb fragment encoding hEpsin-(204-551) was inserted into pEF-BOS-Myc that had been digested with XbaI and blunted with Klenow fragment.

Synchronization of CHO Cells-- Mitotic CHO cells were isolated as described (31, 32). Briefly, cells were subjected to 16-h thymidine block, followed by 2.5-h incubation in the absence of thymidine. 40 ng/ml of nocodazole was added to the medium to arrest cells in mitotic phase. Mitotic cells were collected 5 h later by mechanical shake off, washed with phosphate-buffered saline, and lysed as described (31). Cells without thymidine block remaining on flasks after nocodazole treatment were lysed and used as interphase cells.

Complex Formation of RalBP1, POB1, Epsin, and Eps15 with alpha - Adaptin in Vitro-- The lysates of CHO-IR cells were prepared as described (19, 27). The lysates (1 mg of protein) of CHO-IR cells were incubated with 1 µM GST fused appendage domain of alpha -adaptin (GST-app) or GST for 1 h at 4 °C. GST-app or GST was precipitated with glutathione-Sepharose 4B, and the precipitates were probed with the anti-Eps15, anti-Epsin, anti-POB1, and anti-RalBP1 antibodies.

Complex Formation of POB1 and Epsin with alpha -Adaptin in Intact Cells-- To show the interaction of POB1 with alpha -adaptin, CHO-IR cells expressing HA-POB1 or its mutants were lysed. The lysates (1 mg of protein) were immunoprecipitated with the anti-HA antibody, and the immunoprecipitates were probed with the anti-alpha -adaptin and anti-HA antibodies. To determine which region of POB1 associates with alpha -adaptin, deletion mutants of HA-POB1 were expressed in COS cells. The complex formation of Epsin with alpha -adaptin in intact cells was examined in the same way.

Overlay Assay-- The overlay assay was performed as described (19, 27, 33). The lysates (100 µg of protein) of mitotic phase and interphase of CHO-IR cells expressing Myc-RalBP1, HA-POB1, or Myc-hEpsin were subjected to SDS-PAGE and transferred to nitrocellulose membranes. GST-app, GST-POB1-(322-521), GST-RalBP1-(364-647), GST-POB1-(126-227), GST-Eps15-(1-330), or GST was incubated with the nitrocellulose membranes in overlay buffer (10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 3% bovine serum albumin, 1 mM dithiothreitol, 0.1% Tween-20) at a final concentration of 2.2-50 nM for 12 h at 4 °C. The membranes were then washed and probed with the anti-GST antibody.

Treatment of the Immunoprecipitates with Alkaline Phosphatase-- The Myc-RalBP1, HA-POB1, and Myc-hEpsin immune complexes were incubated with 3 units of calf intestinal alkaline phosphatase in 20 µl of buffer (50 mM Tris/HCl, pH 8.0, 10 mM MgCl2, 0.1 mg/ml bovine serum albumin) for 30 min at 37 °C.

Kinase Assay-- MBP-hEpsin deletion mutants, GST-POB1 deletion mutants, and histone H1 (1 µM of protein each) were incubated with p34cdc2 kinase precipitated with GST-p13suc1 from synchronized cells in 25 µl of kinase assay mixture (50 mM Hepes/NaOH, pH 7.2, 50 mM KCl, 10 mM EGTA, 5 mM MgCl2, 50 µM [gamma -32P]ATP (500-1500 cpm/pmol), 50 mM NaF, 100 mM beta -glycerophosphate) for 15 min at 37 °C. The phosphorylation of each protein was detected by autoradiography.

Insulin Binding and Internalization Assay-- The activities of the binding and the internalization of insulin in CHO-IR cells were determined as described previously (19, 27, 34, 35). Briefly, confluent wild type CHO-IR cells and CHO-IR cells expressing POB1, hEpsin or their mutants (35-mm-diameter dishes) were incubated with 100 pM [125I]insulin (4-5 × 103 cpm/pmol) for 5 h at 4 °C. Internalization was initiated by adding warm binding medium (Ham's F-12 medium containing 1 mg/ml bovine serum albumin, 50 mM Hepes/NaOH, pH 7.4) at 37 °C after the cells had been washed with cold phosphate-buffered saline three times. At various times the medium was removed, and the cells were washed with the acidic washing buffer (0.2 M acetic acid, 0.5 M NaCl). The acid-stripped cells were lysed with phosphate-buffered saline containing 1% Triton X-100 and 0.1% SDS. The rate of the internalization of insulin was expressed as the percentage of internalized [125I]insulin relative to the sum of surface-bound and internalized [125I]insulin.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Complex Formation of alpha -Adaptin with POB1-- The structures of RalBP1, POB1, hEpsin, Eps15, alpha -adaptin, and their deletion mutants used in this study are summarized in Fig. 1. The appendage domain of alpha -adaptin of AP-2 binds directly to Eps15 and Epsin (10, 11). Because we have shown that RalBP1 binds to POB1 and that POB1 binds to Eps15 and Epsin (18, 19, 27), we examined whether alpha -adaptin forms a complex with RalBP1 and POB1. The appendage domain of alpha -adaptin was purified as a GST fusion protein (GST-app). GST-app but not GST precipitated Eps15, RalBP1, Epsin, and POB1 from the lysates of CHO-IR cells (Fig. 2A), suggesting that RalBP1 and POB1 form a complex with alpha -adaptin. To determine which region of POB1 forms a complex with alpha -adaptin, various deletion mutants of GST-POB1 were incubated with the lysates of CHO-IR cells. GST-POB1-(126-227) (EH domain), but not GST-POB1-(1-125), GST-POB1-(228-406), or GST-POB1-(375-521), precipitated the alpha -adaptin of AP-2 (Fig. 2B). Next, we examined whether POB1 forms a complex with AP-2 in intact cells. Consistent with the previous observations (11), Myc-hEpsin formed a complex with alpha -adaptin in CHO-IR cells (Fig. 2C). When the lysates of CHO-IR cells expressing HA-POB1 were immunoprecipitated with the anti-HA antibody, alpha -adaptin was observed in the POB1 immune complexes (Fig. 2C). In COS cells, the N-terminal half of POB1 (POB1-(1-374)), which contains the EH domain, but not the C-terminal half (POB1-(322-521)) was immunoprecipitated with alpha -adaptin (Fig. 2D). These results suggest that the EH domain of POB1 is responsible for forming a complex with alpha -adaptin of AP-2 in intact cells. However, GST-app did not bind directly to POB1 in the overlay assay under the conditions in which it interacted with Epsin (Fig. 2E). Therefore, POB1 would associate indirectly with AP-2 through other proteins, probably Eps15 or Epsin.


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  		Fig. 1.   Schematic representations of RalBP1, POB1, Epsin, Eps15, alpha -adaptin, and their deletion mutant constructs used in this study.


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  		Fig. 2.   Complex formation of alpha -adaptin with POB1. A, complex formation of Eps15, RalBP1, Epsin, and POB1 with alpha -adaptin. The lysates of CHO-IR cells were precipitated with GST or GST-app, and the precipitates were probed with the anti-Eps15, anti-RalBP1, anti-Epsin, and anti-POB1 antibodies. B, interaction of POB1-EH with alpha -adaptin. The lysates of CHO-IR cells were probed directly with the anti-alpha -adaptin antibody (lane 1) or precipitated with GST (lane 2), GST-POB1-(1-125) (lane 3), GST-POB1-(126-227) (EH domain) (lane 4), GST-POB1-(228-406) (lane 5), or GST-POB1-(375-521) (lane 6). The precipitates were probed with the anti-alpha -adaptin antibody. C, complex formation of POB1 and Epsin with alpha -adaptin in CHO-IR cells. The lysates of wild type (WT) CHO-IR cells (lanes 1, 4, and 6), CHO-IR cells expressing Myc-hEpsin (lanes 2 and 5) or HA-POB1 (lanes 3 and 7) were probed directly with the anti-alpha -adaptin antibody and anti-Myc or anti-HA antibody (lanes 1-3). The lysates were immunoprecipitated (IP) with the anti-Myc (lanes 4 and 5) or anti-HA antibody (lanes 6 and 7). The immunoprecipitates were probed with the anti-alpha -adaptin antibody and the anti-Myc or anti-HA antibody. Ab, antibody; Ig, immunoglobulin. D, complex formation of POB1 deletion mutants with alpha -adaptin in COS cells. The lysates of COS cells expressing HA-POB1-(1-374) (lanes 1 and 3) or HA-POB1-(322-521) (lanes 2 and 4) were probed with the anti-alpha -adaptin and anti-HA antibodies (lanes 1 and 2) or immunoprecipitated with the anti-HA antibody (lanes 3 and 4). The immunoprecipitates were probed with the anti-alpha -adaptin and anti-HA antibodies. E, direct interaction of alpha -adaptin with Epsin but not with POB1. The lysates of CHO-IR cells expressing Myc-hEpsin (lanes 1 and 3) or HA-POB1 (lanes 2 and 4) were probed with the anti-Myc (lane 1) or anti-HA antibody (lane 2). The same lysates were subjected to SDS-PAGE and transferred to nitrocellulose membrane. The membrane was overlaid with GST-app, and the bound proteins were detected using the anti-GST antibody (lanes 3 and 4).

Mitotic Phosphorylation of RalBP1, POB1, and Epsin-- It has been shown that fluid phase and receptor-mediated endocytosis are inhibited during mitosis (31, 36, 37) and that Epsin and Eps15 are phosphorylated in mitotic phase but not in interphase (38). We examined whether the complex formation of RalBP1, POB1, Epsin, and Eps15 with AP-2 is affected in mitotic phase. Nocodazole-arrested CHO-IR cells were released from flasks by shaking and used as mitotic cells. The cells remaining on the flasks after nocodazole treatment were used as interphase cells. When precipitated with GST-app, RalBP1, POB1, Epsin, and Eps15 complexed with GST-app were all reduced in the lysates of mitotic cells in comparison with those of interphase cells (Fig. 3A). In this experiment, we found that in addition to Eps15 and Epsin, RalBP1 and POB1 also show a slow migration on SDS-PAGE in mitotic phase. Therefore, we examined whether RalBP1 and POB1 are indeed phosphorylated in mitotic phase. Consistent with the previous observations (38), Epsin from mitotic phase of CHO-IR cells exhibited a slower migration on SDS-PAGE than that from interphase (Fig. 3B). Similarly, RalBP1 and POB1 from mitotic phase showed a gel band shift (Fig. 3B). To confirm that the electrophoretic shifts are due to direct phosphorylation, these proteins from mitotic phase were incubated with alkaline phosphatase. Treatment of Epsin in mitotic phase with phosphatase reversed the mobility shift (Fig. 3B), indicating that Epsin is phosphorylated in mitotic phase. Treatment of RalBP1 and POB1 with phosphatase exhibited a faster migration than those from interphase without treatment (Fig. 3B), indicating that RalBP1 and POB1 are phosphorylated in interphase and further phosphorylated in mitotic phase. The antibody MPM2 recognizes mitotic phosphoproteins, and the recognized sequence is the phosphorylated form of Leu-Thr-Pro-Leu-Lys (LTPLK) or Phe-Thr-Pro-Leu-Gln (FTPLQ) (39, 40). MPM2 reacted with Epsin in mitotic phase but not in interphase. MPM2 also reacted with POB1 from mitotic phase and weakly with that from interphase, but it did not detect RalBP1 (Fig. 3C). These results indicate that POB1 is phosphorylated in both mitotic phase and interphase and that RalBP1 is subjected to phosphorylation that is not recognized by the antibody MPM2.


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  		Fig. 3.   Mitotic phosphorylation of RalBP1, POB1, and Epsin. A, complex formation of RalBP1, POB1, Epsin, and Eps15 with alpha -adaptin in mitotic phase and interphase. The lysates from interphase (I, lanes 1 and 3) and mitotic phase (M, lanes 2 and 4) of CHO-IR cells were probed with the anti-Eps15, anti-RalBP1, anti-Epsin, and anti-POB1 antibodies (lanes 1 and 2) or precipitated with GST-app (lanes 3 and 4). The precipitates were probed with each antibody. B, mitotic phosphorylation of RalBP1, POB1, and Epsin in CHO-IR cells. Myc-RalBP1, HA-POB1, and Myc-hEpsin immunoprecipitated from the lysates of CHO-IR cells from interphase (I) or mitotic phase (M) were incubated with (+) or without (-) alkaline phosphatase. C, recognition by the antibody MPM2. The same lysates of CHO-IR cells expressing Myc-RalBP1, HA-POB1, and Myc-hEpsin from interphase (I) or mitotic phase (M) prepared in B were immunoprecipitated with the anti-Myc or anti-HA antibody. The immunoprecipitates were probed with the antibody MPM2. The arrow and arrowhead indicate the positions of Myc-hEpsin and HA-POB1, respectively.

The antibody MPM2 detects the proteins phosphorylated by p34cdc2 kinase activated in mitotic phase (41). Therefore, we examined whether p34cdc2 kinase phosphorylates Epsin and POB1 directly. hEpsin-(204-551) and POB1-(322-521) showed a gel mobility shift in the mitotic phase of CHO-IR cells (Fig. 4A), suggesting that these regions contain the direct phosphorylation site for p34cdc2 kinase. p34cdc2 kinase precipitated from CHO cells in the mitotic phase contained kinase activity toward histone H1, whereas that from the interphase cells exhibited weak histone H1 kinase activity (Fig. 4B). MBP-hEpsin-(204-458) was phosphorylated more by p34cdc2 kinase from mitotic phase than interphase (Fig. 4B). Neither MBP nor MBP-hEpsin-(1-205) was phosphorylated by p34cdc2 kinase (Fig. 4B and data not shown). GST-POB1-(375-521) was phosphorylated more by p34cdc2 kinase from mitotic phase than interphase (Fig. 4B). The phosphorylation of GST-POB1-(1-125) or GST-POB1-(228-406) was not enhanced by this kinase (data not shown). These results demonstrate that the C-terminal regions of Epsin and POB1 have direct phosphorylation site(s) for p34cdc2 kinase.


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  		Fig. 4.   Phosphorylation of POB1 and Epsin by p34cdc2 kinase. A, mitotic phosphorylation of the C-terminal region of Epsin and POB1. Lysates of CHO-IR cells stably expressing Myc-hEpsin-(204-551) or HA-POB1-(322-521) from interphase (I) and mitotic phase (M) were probed with the anti-Myc or anti-HA antibody, respectively. B, phosphorylation of POB1 and Epsin by p34cdc2 kinase in vitro. MBP, MBP-hEpsin-(204-458), GST-POB1-(375-521), and histone H1 were incubated with p34cdc2 kinase precipitated from interphase (I) or mitotic phase (M) of CHO cells. Left panel, Coomassie staining; Right panel, autoradiography. The arrowhead indicates the position of GST-POB1-(375-521). C, expression of Epsin and its mutants in CHO-IR cells. The lysates of CHO-IR cells expressing Myc-hEpsin, Myc-hEpsinS357D, and Myc-hEpsinS357A were probed with the anti-Myc antibody. WT, wild type. D, expression of POB1 and its mutants in CHO-IR cells. The lysates of CHO-IR cells expressing HA-POB1, HA-POB1S411D, and HA-POB1S411A from interphase (I) or mitotic phase (M) were probed with the anti-HA antibody.

The consensus sequence for the phosphorylation by p34cdc2 kinase is (S/T)PX(K/R) (where X is a polar amino acid)) (42). hEpsin has a single putative phosphorylation site, 357SPAK. We made hEpsinS357D and hEpsinS357A, in which Ser357 is mutated to Asp and Ala, respectively. When expressed in CHO-IR cells, hEpsinS357D exhibited a slower migration on SDS-PAGE than wild type hEpsin and hEpsinS357A (Fig. 4C). Neither hEpsinS357D nor hEpsinS357A was phosphorylated by p34cdc2 kinase in vitro (data not shown), indicating that Ser357 of hEpsin is a major phosphorylation site for the kinase. POB1 also has a single putative phosphorylation site for p34cdc2 kinase, 411SPAK. We made POB1S411D and POB1S411A, in which Ser411 is mutated to Asp and Ala, respectively. Unlike Epsin, POB1S411D did not exhibit a gel mobility shift, but neither POB1S411D nor POB1S411A showed a mobility shift in mitotic phase (Fig. 4D). Further, these mutants were not phosphorylated by p34cdc2 kinase in vitro (data not shown), suggesting that Ser411 is a major phosphorylation site for the kinase.

Effect of the Phosphorylation of RalBP1, POB1, and Epsin on Their Mutual Binding-- We next examined whether mitotic phosphorylation of RalBP1, POB1, and Epsin affects their mutual binding. To this end, GST-POB1-(322-521), GST-RalBP1-(364-647), and GST-POB1-(126-227) (EH domain) were purified to investigate their binding to RalBP1, POB1, and Epsin in mitotic phase and interphase by overlay assay. GST-POB1-(322-521) bound to Myc-RalBP1 in mitotic phase as efficiently as in interphase (Fig. 5A, lanes 1-6). GST-RalBP1-(364-647) also bound to HA-POB1 in both mitotic phase and interphase with similar efficiency (Fig. 5A, lanes 7-12). GST-POB1-(126-227) bound to Myc-hEpsin less efficiently in mitotic phase than interphase (Fig. 5B). Further, consistent with the results, hEpsinS357D hardly formed a complex with GST-POB1-(126-227) under the conditions that wild type hEpsin did (Fig. 5C). In contrast, Eps15-(1-330) (EH domain) bound to hEpsin in mitotic phase and interphase with similar efficiency (data not shown). These results indicate that mitotic phosphorylation of POB1 and RalBP1 does not affect their interaction but that mitotic phosphorylation of Epsin inhibits its binding to the EH domain of POB1.


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  		Fig. 5.   Effect of the phosphorylation of RalBP1, POB1, and Epsin on their mutual binding. A, interaction of POB1 with RalBP1. The lysates of CHO-IR cells from interphase (I) or mitotic phase (M) expressing Myc-RalBP1 (lanes 1-6) or HA-POB1 (lanes 7-12) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The membranes were overlaid with GST (lanes 1, 2, 7, and 8), GST-POB1-(322-521) (lanes 3-6), or GST-RalBP1-(364-647) (lanes 9-12), and the bound proteins were detected using the anti-GST antibody. Upper panel, overlay assay; lower panel, expression levels of Myc-RalBP1 and HA-POB1. B, interaction of Epsin with POB1. The lysates of CHO-IR cells from interphase (I) or mitotic phase (M) expressing Myc-hEpsin were overlaid with GST (lanes 1 and 2) or GST-POB1-(126-227) (lanes 3-6). The bound proteins were detected using the anti-GST antibody. Upper panel, overlay assay; lower panel, expression level of Myc-hEpsin. C, interaction of Epsin and its mutant with POB1-EH. The lysates of COS cells expressing Myc-hEpsin or Myc-hEpsinS357D were probed with the anti-Myc antibody (lanes 1 and 2) or precipitated with GST (lanes 3 and 4) or GST-POB1-(126-227) (lanes 5 and 6). The precipitates were probed with the anti-Myc antibody. WT, wild type.

Effect of the Phosphorylation of POB1 and Epsin on Their Complex Formation with AP-2-- To examine the effect of mitotic phosphorylation of POB1 and Epsin on endocytosis, we investigated the binding of their mutants to AP-2. Epsin did not form a complex with alpha -adaptin in mitotic phase of intact cells, and hEpsinS357D bound to alpha -adaptin less efficiently than wild type hEpsin and hEpsinS357A (Fig. 6A). Although POB1 did not form a complex with alpha -adaptin in mitotic phase, POB1S411D formed a complex with alpha -adaptin as efficiently as wild type POB1 and POB1S411A (Fig. 6B). These results suggest that the phosphorylation of Epsin in mitotic phase inhibits its binding to AP-2 but that the mitotic phosphorylation itself of POB1 is not involved in the disassembly of its complex with AP-2.


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  		Fig. 6.   Effect of the phosphorylation of POB1 and Epsin on their complex formation with AP-2. A, interaction of Epsin and its mutants with alpha -adaptin. The lysates of CHO-IR cells expressing Myc-hEpsin from interphase (I, lanes 1 and 3) or mitotic phase (M, lanes 2 and 4) were probed directly with the anti-alpha -adaptin and anti-Myc antibodies (lanes 1 and 2) or immunoprecipitated (IP) with the anti-Myc antibody (lanes 3 and 4). The same lysates of CHO-IR cells prepared in Fig. 4C were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-alpha -adaptin and anti-Myc antibodies (lanes 5-7). WT, wild type. B, interaction of POB1 and its mutants with alpha -adaptin. The lysates of CHO-IR cells expressing HA-POB1 from interphase (I, lanes 1 and 3) or mitotic phase (M, lanes 2 and 4) were probed directly with the anti-alpha -adaptin and anti-HA antibodies (lanes 1 and 2) or immunoprecipitated with the anti-HA antibody (lanes 3 and 4). The lysates of CHO-IR cells expressing HA-POB1, HA-POB1S411D, and HA-POB1S411A were immunoprecipitated with the anti-HA antibody, and the immunoprecipitates were probed with the anti-alpha -adaptin and anti-HA antibodies (lanes 5-7).

Effect of the Phosphorylation of Epsin and POB1 on the Endocytosis-- We have previously shown that Epsin and POB1 are involved in receptor-mediated endocytosis for insulin (19, 27). To examine the effect of mitotic phosphorylation of Epsin and POB1 on this endocytosis, we established CHO-IR cells expressing hEpsinS357D, hEpsinS357A, POB1S411D, or POB1S411A. Expression of these proteins did not affect the insulin binding activity (data not shown). As reported previously (27), expression of wild type hEpsin resulted in a reduction of the internalization of insulin. This could reflect the fact that expression of hEpsin blocks endocytosis perhaps through sequestration of AP-2 or other Epsin binding proteins into nonfunctioning complexes during endocytosis. hEpsinS357D did not affect the insulin internalization, whereas hEpsinS357A inhibited the internalization with similar efficiency to wild type hEpsin (Fig. 7A). These results suggest that phosphorylated hEpsin loses its function. Expression of wild type POB1 did not affect the internalization of insulin, and neither did POB1S411D or POB1S411A (Fig. 7B).


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  		Fig. 7.   Effects of Epsin, POB1, and their mutants on internalization of insulin. A, hEpsin. The insulin internalization activities of wild type CHO-IR cells (open circle ) and CHO-IR cells expressing Myc-hEpsin (), Myc-hEpsinS357D (black-triangle), or Myc-hEpsinS357A () were measured. B, POB1. The insulin internalization activities of wild type CHO-IR cells (open circle ) and CHO-IR cells expressing HA-POB1 (), HA-POB1S411D (black-triangle), or HA-POB1S411A () were measured. The results shown are means of five independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we have shown that RalBP1 and POB1, downstream molecules of small G protein Ral, form a complex with AP-2 as well as Eps15 and Epsin. Although the EH domain of POB1 is involved in the complex formation with AP-2, they do not bind directly to each other. Eps15 and Epsin interact directly with AP-2 (10, 11, 15, 16, 43). Because the EH domain of POB1 binds directly to Epsin and Eps15, the results suggest that POB1 forms a complex with AP-2 probably through Epsin or Eps15. Eps15 interacts directly with AP-2 and Epsin (11, 14). Another EH domain containing protein Ese (intersectin) binds to dynamin and Eps15 (24). Further, in yeast, Pan1 interacts with yAP180 and Epsin-like protein that bind to clathrin (44, 45). Thus, it is likely that the EH domain containing proteins binds directly or indirectly to molecules that regulate endocytosis including AP-2, clathrin, and dynamin. Taken together with the observations that the deletion mutants of POB1 inhibit the internalization of insulin and EGF (19), these findings support that POB1 is involved in the regulation of receptor-mediated endocytosis.

It has been reported that Epsin and Eps15 are phosphorylated in mitotic phase and that their mitotic phosphorylation inhibits the binding to the appendage domain of alpha -adaptin in vitro (38). We have shown that RalBP1 and POB1 are also phosphorylated in mitotic phase. Forty or more of the mitotic phosphoproteins are reactive with the monoclonal antibody MPM2 (41, 42). The sequence of the MPM2-reactive phosphorylated site overlaps with the consensus for phosphorylation by p34cdc2 kinase, (S/T)PX(K/R), suggesting that proteins containing the sequence are recognized by the antibody MPM2 in mitotic phase (42). The antibody reacts with POB1 and Epsin, which are indeed phosphorylated by p34cdc2 kinase in vitro. Based on these observations, we have examined the effects of the mitotic phosphorylation of RalBP1, POB1, and Epsin on their mutual binding. Neither the phosphorylation of RalBP1 nor POB1 affects their binding. The phosphorylation of Epsin inhibits the binding to POB1. The complex formation of POB1 and Epsin with AP-2 is also reduced in mitotic cells. Consistent with these observations, a mutation of hEpsin (hEpsinS357D), which mimics the phosphorylated state, inhibits the complex formation with POB1 and AP-2. These are the first demonstration showing that mitotic phosphorylation of Epsin inhibits its complex formation with the binding partners in intact cells. Thus, the phosphorylation of Epsin in mitotic phase controls the assembly of the complex formation with POB1 and AP-2, and this may dissociate it from the clathrin coated pits. However, POB1S411D forms a complex with AP-2 as well as wild type POB1. The reduction of the complex formation of POB1 with AP-2 may be due to the dissociation of POB1 from Epsin or that of Epsin from AP-2 by the phosphorylation of Epsin.

It is known that receptor-mediated endocytosis is completely arrested during mitosis (37). The invagination of coated pits is inhibited by mitotic cytosol in vitro, and p34cdc2 kinase can substitute for mitotic cytosol (31). These results suggest that some proteins that are involved in mitotic regulation of endocytosis may be targets of p34cdc2 kinase. As reported previously (27), expression of wild type hEpsin inhibits the internalization of insulin. This is not surprising, because expression of Ese, which contains two EH domains and five Src homology 3 domains, also blocks endocytosis of transferrin (24). Expressed hEpsin would function as a dominant inhibitory protein through recruitment of partners into nonfunctional complexes that do not contain all of the components necessary for endocytosis. Therefore, the observation that expression of hEpsinS357D does not affect the internalization of insulin indicates that Epsin is a target protein of p34cdc2 kinase to suppress endocytosis in mitotic phase and that the Epsin phosphorylated in mitotic phase loses its function. It has been demonstrated that depolarization of synaptosomes results in dephosphorylation of Epsin and that the reduced state of phosphorylation of Epsin increases its binding to AP-2 (38). Therefore, the phosphorylation of Epsin may be critical for the regulation of endocytosis generally. The physiological significance of the phosphorylation of POB1 remains to be clarified.

RalBP1 is an effector protein of small G protein Ral (17). It has been reported that cytocentrin is the same gene product as RalBP1 (46, 47). Cytocentrin is a cytosolic protein that transiently associates with the mitotic spindle poles in early prophase and regulates diplosome separation and assembly of the mitotic spindle. RalBP1 is phosphorylated in mitotic phase, although which kind of protein kinase phosphorylates RalBP1 is not known. The phosphorylated form of RalBP1 may associate with centrosome. Because the phosphorylation of RalBP1 does not affect its binding to POB1, RalBP1/POB1 complex may recognize different binding partners when they are phosphorylated in mitotic phase. It is intriguing to speculate that RalBP1 and POB1 have different functions in interphase and mitotic phase. In interphase, they are involved in the regulation of assembly of the receptor and endocytic proteins, whereas in mitotic phase, they may regulate the assembly and function of the mitotic apparatus.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Y. Ebina, M. Nakata, H. Usui, and H. Ohno for gifts of cell lines, antibodies, proteins, and plasmids, respectively.

    FOOTNOTES

* This work was supported by Grants-in-Aid for Scientific Research and for Exploratory Research from the Ministry of Education, Science, and Culture, Japan (1998 and 1999) and by grants from the Yamanouchi Foundation for Research on Metabolic Disorders (1998 and 1999) and the Uehara Memorial Foundation (1998).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-82-257-5130; Fax: 81-82-257-5134; E-mail: akikuchi@mcai.med.hiroshima-u.ac.jp.

Published, JBC Papers in Press, April 6, 2000, DOI 10.1074/jbc.M000521200

    ABBREVIATIONS

The abbreviations used are: AP, adaptor protein complex; EGF, epidermal growth factor; EH, Eps15 homology; G protein, GTP-binding protein; GST, glutathione S-transferase; MBP, maltose-binding protein; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; kb, kilobase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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