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J. Biol. Chem., Vol. 280, Issue 24, 23048-23056, June 17, 2005

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Convergence of Cell Cycle Regulation and Growth Factor Signals on GRASP65^*

Shin-ichiro Yoshimura, Katsuji Yoshioka¶, Francis A. Barr||**, Martin Lowe, Kazuhisa Nakayama, Shoji Ohkuma, and Nobuhiro Nakamura¶¶

From the Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan, the Japan Society for the Promotion of Science, 6 Ichiban-cho, Chiyoda, Tokyo 102-8471, Japan, the ^¶Division of Cell Cycle Regulation, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan, the ^||Department of Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, Martinsried D-82152, Germany, the School of Biological Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom, and the Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan

Received for publication, March 4, 2005 , and in revised form, April 13, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. During mitosis, GRASP65 is heavily phosphorylated, and in turn, cisternal stacking is inhibited leading to the breakdown of the Golgi apparatus. Here we show that GRASP65 is phosphorylated on serine 277 in interphase cells, and this is strongly enhanced in response to the addition of serum or epidermal growth factor. This is directly mediated by ERK suggesting that GRASP65 has some role in growth factor signal transduction. Phosphorylation of Ser-277 is also dramatically increased during mitosis, however this is mediated by Cdk1 and not by ERK. The microinjection of recombinant GRASP65 without N-terminal myristoylation or a peptide fragment containing Ser-277 into the cytosol of normal rat kidney cells inhibits passage through mitosis. This effect is abolished when Ser-277 is replaced with alanine suggesting the phosphorylation of Ser-277 plays an important role in cell cycle regulation. The convergence of cell cycle regulation and growth factor signals on GRASP65 Ser-277 suggests that GRASP65 may function as a signal integrator controlling the cell growth.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Golgi apparatus is composed of flattened cisternal membranes connected and further organized to produce a juxtanuclear ribbon-like structure in most mammalian cells. During mitosis, the Golgi apparatus is fragmented to produce clusters of vesicles that disperse throughout the cytoplasm. These mitotic Golgi fragments are equally partitioned into the daughter cells during mitosis, and then the perinuclear Golgi apparatus is reconstituted simultaneously with the reformation of the nuclear envelope upon mitotic exit (for a review, see Ref. 1). Cdk1-cyclin B promotes the events of mitosis and was shown to be necessary for mitotic Golgi fragmentation by in vitro reconstitution experiments using purified Golgi stacks (2–4). On the other hand, MEK1,¹ and not Cdk1-cyclin B, was shown to be responsible for the disassembly of the Golgi apparatus by in vitro reconstitution experiments using a permeabilized cell system (5). Furthermore, there is also a report that both Cdk1-cyclin B and MEK1 take part in different stages of the mitotic disassembly of the Golgi apparatus using a similar permeabilized cell system (6). Whether either of these kinases or both are responsible for the mitotic disassembly of the Golgi apparatus remains to be confirmed.

GRASP65 (golgi reassembly stacking protein 65) is a peripheral membrane protein enriched on the cis-face of the Golgi apparatus. It was identified as an N-ethylmaleimide-sensitive factor required for the stacking of Golgi cisternae from mitotically disassembled Golgi fragments in a cell-free system (7). GRASP65 is myristoylated at the N terminus and has two PDZ-like domains. The second PDZ-like domain binds to GM130, a protein thought to function in vesicle tethering with p115 and giantin (8–10). The myristoylation and the GM130 binding were shown to be necessary for the targeting of GRASP65 to the Golgi apparatus (11). We have recently shown that newly synthesized GRASP65 is directly targeted to the Golgi apparatus soon after the synthesis in the cytoplasm. The specific binding of GRASP65 to the Golgi membrane is partly supported by pre-existing GM130 and by Golgi membrane components (12, 13). GRASP65 is heavily phosphorylated on multiple site during mitosis, and this is thought to promote cisternal unstacking (7, 14). Of the mitotically activated kinases, only Cdk1-cyclin B and polo-like kinase (Plk) have been proposed to phosphorylate GRASP65 (14–16). However, the one or more exact phosphorylation sites responsible for the regulation of GRASP65 function remain to be elucidated.

Two major models have been proposed to explain how the structure of the Golgi relates to its function (for a review, see Ref. 17). First is the vesicular transport model. In this model, only exocytic materials are transported to downstream cisternae by transport vesicles. Each Golgi cisterna forms a stable compartment, and Golgi resident proteins stay in certain cisternae and are excluded from transport vesicles. Therefore, the stacked cisternae are stable and do not have to be unstacked for exocytic transport. Second is the cisternal maturation model. In this model, Golgi resident proteins are continuously transported back to an upstream cisterna by transport vesicles, so that cis-cisterna eventually "matures" to become medial- and then trans-cisterna. Exocytic cargo is thus transported in these maturing cisternae from cis to trans. The Golgi cisternae have to be unstacked and fragmented after reaching the trans position, and their content delivered to the plasma membrane, endosomes, or regulated secretory granules. It is still under debate which of these models or a mixed intermediate model is correct.

Importantly, the function and dynamics of GRASP65 are predicted to be completely different in the above two models. In the vesicular transport model, GRASP65 stays at the cis-cisternae and constantly functions for cisternal stacking. In the cisternal maturation model, the function of GRASP65 has to be switched off when a cis-cisterna reaches the medial position and reactivated upon retrieval back to the cis position. In this latter scenario, the function and the dynamics of GRASP65 may be regulated by some biochemical modification, for example, a post-translational modification such as phosphorylation. Interestingly, we noticed during the course of our previous work that GRASP65 shows a mobility shift on SDS-PAGE after synthesis and targeting to the Golgi apparatus in interphase cells (12, 18). We, therefore, sought to identify the cause of this mobility shift to gain insight into the dynamics of the stacked Golgi cisternae in interphase cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression Vector Constructions—GRASP65 mutant fragments were produced by PCR with pairs of appropriate primers or site-specific single primer mutagenesis using GRASP65 cDNA and cloned into pcDNA3.1 (Invitrogen) (7). pFLAG-CMV2-ERK2, pFLAG-CMV2-JNK1, pFLAG-CMV2-p38, and pcDNA3-myc-Raf-C (C-terminal fragment of Raf-1) were used to express FLAG- or myc-tagged proteins as described previously (19, 20).

Cell Culture and Enrichment of Mitotic Cell—HeLa, COS7, NRK, and NRK-E52 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% fetal bovine serum (FCS) (Equitec-Bio, Inc., Ingram, TX). For epidermal growth factor (EGF) stimulation, NRK cells were first incubated with DMEM containing 0.05% FCS for 48 h. After that, 50 ng/ml EGF (Sigma) was added, and the mixture was further incubated for the indicated times. To enrich mitotic NRK cells, the cells were incubated with DMEM (10% FCS) containing 2.5 µg/ml aphidicolin for 12 h, then washed three times by DMEM (10% FCS) and incubated for 7 h. The mitotic cells were then shaken off from the culture dish with vigorous tapping. To inactivate MEK and ERK, 20 µM U0126 (Promega Corp., Madison, WI) was added during the incubation after the removal of aphidicolin. The cells were finally collected and lysed by SDS-PAGE sample buffer.

Peptides and Antibodies—Oligonucleotide-peptide (>90% purity) and anti-phosphorylated peptide antibody were supplied from Asahi Techno Glass Corp. (Tokyo, Japan). Briefly, a peptide corresponding to the GRASP65 sequence from 270 to 284 residues and phosphorylated at serine 277 was chemically synthesized and used for immunizing rabbits. The antiserum was affinity-purified on a phosphorylated peptide column and adsorbed with a corresponding nonphosphorylated peptide column. Rabbit anti-rat GRASP65 antibody (12), mouse anti-GRASP65 antibody 7E10 (21), and rabbit anti-PS25 (22) were described previously. Rabbit anti-active MAPK polyclonal antibody ERK (pERK) (Promega), rabbit anti-ERK2 antibody (BD Biosciences), mouse anti-GM130 (BD Biosciences), Alexa-488-conjugated goat anti-mouse (Molecular Probes, Inc., Eugene, OR), Cy3-conjugated goat anti-rabbit (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and horseradish peroxidase-conjugated goat anti-rabbit antibodies (Jackson) were purchased.

Pulse-chase Labeling and Immunoprecipitation—1 x 10⁵ of HeLa cells were grown in 3.5-cm dishes and transfected with GRASP65 mutants. After 24 h, cells were incubated with methionine/cysteine-free DMEM (Invitrogen) (10% FCS, dialyzed) for 60 min. The cells were then labeled with 7.4 MBq/ml ³⁵S-Express protein labeling mix (PerkinElmer Life Sciences) for 5 min, washed, and chased with DMEM with 10% FCS for 30 min. The cells were washed with ice-cold phosphate-buffered saline, and lysed by 500 µl of radioimmune precipitation assay buffer (1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0)) containing protease inhibitors mixture (1 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, 20 µM leupeptin, 15 µM pepstatin, and 15 µM chymostatin). The lysate was immunoprecipitated with rabbit anti-rat GRASP65 antibody. The immunoprecipitated materials were washed three times with radioimmune precipitation assay buffer and once with NEB buffer 3 (50 mM Tris-HCl (pH 7.9), 10 mM MgCl₂, 100 mM NaCl, 1 mM dithiothreitol), and treated by 50 units/ml of calf intestine alkali phosphatase (CIP) (New England Biolabs Inc., Beverly, MA) for 60 min at 37 °C. After the treatment, the materials were subjected to SDS-PAGE and analyzed by autoradiography.

Indirect Immunofluorescence and Confocal Microscopy—This was performed as described previously (18). Briefly, cells were fixed with paraformaldehyde and incubated by first antibodies (anti-PS277 antibody and 7E10), washed, treated with secondary antibodies, and finally stained with 1 µg/ml Hoechst 33258. Antibody was preincubated with 1 mg/ml synthetic oligonucleotide-peptide for 60 min at room temperature where indicated. The cells were post-fixed with 4% paraformaldehyde and observed with an LSM510 confocal microscope (Carl Zeiss, Jena, Germany).

In Vitro Kinase Assay—In vitro kinase assays were performed as described previously with slight modifications (19). MAPKs were immunoisolated as follows. For ERK2, pFLAG-CMV2-ERK2 was transfected solely (control) or co-transfected with pcDNA3-myc-Raf-C (activated) in COS7 cells and incubated for 30 h. For JNK1, pFLAG-CMV2-JNK1 was transfected in COS7 cells and incubated for 30 h (control), and culture medium was removed and irradiated by UV (10⁵ µJ/cm²) using a Spectrolinker XL-1500 UV Crosslinker (Spectronics Corp., Westbury, NY) (activated). For p38, pFLAG-CMV2-p38 was transfected in COS7 cells and incubated for 30 h (control) and treated with OPTI-MEM I (Invitrogen) containing 0.6 M sorbitol for 15 min (activated) (23). The cells were collected and lysed by a lysis buffer (25 mM HEPES-NaOH (pH 7.5), 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 0.5 M NaCl, 50 mM NaF, 1 mM sodium orthovanadate, 5 mM EDTA, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). The kinases were immunoprecipitated with anti-FLAG-M2 monoclonal antibody (Sigma) from the lysate, washed three times with the lysis buffer, and incubated with 3 µg of substrates in 30 µl of kinase buffer (25 mM HEPES-NaOH (pH 7.6), 20 mM MgCl₂, 20 mM -glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM sodium orthovanadate, 2 mM dithiothreitol) at 30 °C for 30 min in the presence of 20 µM ATP and 5 µCi of [-³²P]ATP (Amersham Biosciences). Cold reactions were performed in the same condition without radioactive ATP. Myelin basic protein was purchased from Sigma. GST-c-Jun and Trx-ATF2 were prepared as previously described (20, 24). His-GRASP65 was produced with pET-28a(+) vector and BL21(DE3) Rossetta-pLysS (Novagen, Merck KGaA, Darmstadt, Germany) and purified by using TALON metal affinity resin (BD Biosciences) following the manufacturer's protocol. The reactions were terminated with SDS-PAGE sample buffer, and the products were resolved by SDS-PAGE and analyzed by autoradiography.

Rat liver Golgi membranes and mitotic HeLa-S3 cytosol were prepared as previously described (8, 9). To inhibit ERK activation, HeLa-S3 cells synchronized in mitosis were treated with 20 µM U0126 for 30 min before homogenization. 50 µl of cytosol (8 mg/ml) was desalted and incubated with 10 µg Golgi membranes for 30 min at 30 °C. The reaction was centrifuged at 100,000 x g for 30 min to precipitate the membranes. The membranes were lysed with SDS-PAGE sample buffer and analyzed by Western blotting using anti-GM130, anti-PS25, anti-GRASP65, and anti-PS277 antibodies.

Microinjection—NRK cells were seeded at 1.5 x 10⁴/cm², grown on glass coverslips for 24 h, and incubated with DMEM (10% FCS) containing 2.5 µg/ml aphidicolin for 12 h. The cells were then washed with DMEM (10% FCS) and incubated for 2 h. The cells were microinjected with 10 mg/ml recombinant His₆-tagged GRASP65 or 0.3 mg/ml peptide over 30 min and further incubated for indicated time. 10 mg/ml Alexa-488-conjugated dextran (Molecular Probes) was co-injected as an injection marker. The cells were fixed and stained by mouse anti-GM130 antibody followed by Cy3-anti-mouse antibody and finally with Hoechst 33258. For all the cells showing Alexa-488 fluorescence (more than 200 cells, typically 300 cells), the number of the cells showing chromosomal condensation was counted and the ratio (%) was shown as a mitotic index.

View larger version (32K): [in this window] [in a new window]   FIG. 1. Phosphorylation of newly synthesized GRASP65. NRK cells were pulse-labeled with [35S]Met/Cys for 5 min and chased for 30 min in the presence or absence of Brefeldin A (BFA). The cells were then solubilized with SDS-containing buffer and immunoprecipitated by anti-rat GRASP65 antibody. Some of the immunoprecipitated materials were treated with alkaline phosphatase (CIP) and analyzed by SDS-PAGE followed by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The targeting of GRASP65 to the Golgi membrane is very rapid, and >80% of GRASP65 is bound to the Golgi membrane just after the 5-min pulse labeling (12). Therefore, GRASP65 is likely to be phosphorylated after binding to the Golgi membranes. Interestingly, the lower mobility band was not seen when the cells were treated with brefeldin A (BFA) before the pulse labeling and chase (lane 3). In the presence of BFA, the Golgi apparatus is disassembled and the anterograde transport is blocked. Most of the medial- and trans-Golgi proteins are transported back to the ER, whereas many cis-Golgi proteins, including syntaxin 5 and GRASP65, remain in distinct cytoplasmic punctate structures with ERGIC53 (8, 25). Therefore, it is probable that the one or more kinases responsible for the GRASP65 phosphorylation localize to the Golgi apparatus in untreated interphase cells, and this is segregated together with other Golgi proteins from GRASP65 after BFA treatment such that it can no longer efficiently phosphorylate GRASP65.

Determination of the Phosphorylation Site Responsible for the Mobility Shift—We have found a similar mobility shift of GRASP65 in HeLa cells.² Therefore, we took advantage of the higher transfection efficiency of HeLa cells to determine the phosphorylation site of GRASP65 responsible for the mobility shift. A further advantage of this approach is that only the transfected rat GRASP65 and its mutants were precipitated using the anti-rat GRASP65 antiserum, thus simplifying the analysis. HeLa cells were transfected with various mutants of rat GRASP65 (Fig. 2, A and B), and the cells were pulse-labeled, chased, and analyzed as before. The mobility shift was observed for the transfected full-length GRASP65 and for C372 (C-terminal residues from 373 onward were deleted) and C292 mutants (C-terminal residues from 293 onward were deleted) but not for C202 (C-terminal residues from 203 onward were deleted) (Fig. 2C, lane 3). The shift was abolished by the treatment of immunoprecipitated material with alkaline phosphatase (Fig. 2C, lane 4). Therefore, this suggests that residues between 203 and 292 are responsible for the mobility shift. Correspondingly, an internal deletion mutant (203–292), lacking residues between 203 and 292, did not show a mobility shift (Fig. 2D, lane 3). On the other hand, an internal deletion mutant (203–240), lacking residues between 203 and 240, showed a mobility shift suggesting that residues between 241 and 292 are responsible for the mobility shift (Fig. 2D, lane 7).

There are 9 residues that are potentially phosphorylated between residues 241 and 292 (Fig. 2B; 6 serines, 1 threonine, and 2 tyrosines). To pinpoint the residues responsible for the mobility shift, we mutated some of these residues to alanine to prevent the phosphorylation. Mutants where both serines 244 and 245 were replaced with alanine (S244A, S245A) or both serines 251 and 254 were replaced with alanine (S251A, S254A) still showed the mobility shift (Fig. 2E, lanes 3 and 7). In contrast, a mutant where serine 277 was replaced with alanine (S277A) did not show a mobility shift (Fig. 2 E, lane 11). These results indicate that the phosphorylation of serine 277 (Ser-277) is responsible for the mobility shift of GRASP65.

Phosphorylation of Ser-277 in Interphase Cells—To further characterize the phosphorylation of Ser-277, we produced an anti-phospho-Ser-277 antibody using a chemically synthesized phosphorylated peptide shown in Fig. 3A (PS277). When purified rat liver Golgi membranes were probed with the affinity-purified anti-PS277 antibody, a 65-kDa band was detected (Fig. 3B, lane 1). This corresponded to the lower mobility band detected by anti-GRASP65 antibody (lane 4). The reaction with the anti-PS277 antibody was abolished after the treatment of Golgi membranes with alkaline phosphatase (lane 3). Consistently, the lower mobility band detected by anti-GRASP65 antibody also disappeared after phosphatase treatment (lane 6). The reactivity with the antibodies was preserved after the mock incubation confirming that disappearance of the signal was caused specifically by the added phosphatase activity (lanes 2 and 5). These results indicate that the anti-PS277 antibody specifically recognizes GRASP65 phosphorylated at Ser-277 and reinforce that the mobility shift is indeed caused by phosphorylation of Ser-277.

The anti-PS277 antibody was then used to stain NRK cells by indirect immunofluorescence. A GRASP65 monoclonal antibody was also used for double staining the cells (Fig. 3C). The anti-PS277 antibody clearly stained a juxtanuclear ribbon-like structure, and the staining pattern was almost completely coincident with that of anti-GRASP65 monoclonal antibody (top panels). The staining with anti-PS277 antibody was abolished after the pretreatment of the antibody with PS277 peptide (bottom panels) but not with non-phosphorylated Ser-277 control peptide (middle panels) confirming the specificity of the staining. These results confirmed that GRASP65 is phosphorylated at Ser-277 in interphase cells.

The surrounding sequence of Ser-277 (see Fig. 3A) matches the consensus sequence for MAPKs (especially for ERKs) (PX(S/T)P) and cyclin-dependent kinases (Cdks) ((S/T)PX(K/R)) (26, 27). We therefore, decided to find out whether Ser-277 is actually phosphorylated by these kinases.

Ser-277 Is Phosphorylated by ERK after EGF Stimulation— It is well known that ERK is activated by EGF stimulation (28). Therefore, we first analyzed whether the phosphorylation of Ser-277 is increased after stimulation with EGF. NRK cells were cultured in low serum conditions for 48 h. EGF was then added to the medium, and the cells were further incubated up to 180 min. After the incubation, the cells were either fixed and processed for immunofluorescence (Fig. 4A) or lysed and analyzed by Western blotting (Fig. 4C) using the anti-PS277 antibody. The PS277 signal decreased after serum starvation but persisted at a low level (Fig. 4, A and C, 0 min). The signal greatly increased in the 5 min after addition of EGF and remained at a similar level for a further 5 min. The signal was then reduced by 30 min to a level lower than that before EGF addition and persisted at this level for up to 180 min. As shown in Fig. 4C, the change in PS277 signal correlates well with that of activated ERK monitored by ERK phosphorylation (pERK). When cells were pretreated with U0126, a specific MEK inhibitor, ERK was not activated by EGF treatment (Fig. 4D, pERK). Correspondingly, the PS277 signal was greatly reduced and did not increase in response to EGF (Fig. 4, B and D). These results suggest that Ser-277 is phosphorylated by ERK in response to the EGF treatment.

View larger version (45K): [in this window] [in a new window]   FIG. 2. Determination of the phosphorylation site responsible for the mobility shift. The constructs shown were transfected in HeLa cells and analyzed as described in Fig. 1. A, schematic representation of the full-length and deletion constructs of GRASP65 used in this study. N-terminal myristoylation (myr), two PDZ domains (PDZ), and serine/proline rich region (Ser/Pro) are indicated. B, amino acid sequence of the region responsible for the mobility shift and residues changed in point mutants are shown. C, full-length and C-terminal deletion mutants. D, internal deletion mutants. E, point mutants. Arrowheads indicate higher mobility bands, and arrows indicate lower mobility bands. The asterisk in D indicates a nonspecific precipitate.

View larger version (65K): [in this window] [in a new window]   FIG. 3. Production of antibody to GRASP65 phosphorylated at Ser-277. A, a 15-residue region used for immunization is shown. B, rat liver Golgi membrane was untreated (input), mock treated (CIP –), or treated with alkaline phosphatase (CIP +) and then subjected to Western blotting using anti-PS277 antibody (left panel) or anti-GRASP65 antibody (right panel). C, NRK cells were fixed and double stained with anti-GRASP65 antibody (mouse monoclonal: left panels) and anti-PS277 antibody (light panels). The antibody was preincubated with either Ser-277 oligonucleotide-peptide (Ser-277, middle panels) or phosphorylated Ser-277 oligonucleotide-peptide (PS277, bottom panels) where indicated. Bar = 20 µm.

Phosphorylation of Ser-277 in Mitotic Cells—We noticed that some of the NRK cells showed a brighter signal with the anti-PS277 antibody in immunofluorescence experiments. Because GRASP65 is known to be phosphorylated during mitosis (7), we investigated whether these cells were in mitosis. We triple stained asynchronous cultures of several cell lines with anti-GRASP65 for total GRASP65, anti-PS277 antibody for Ser-277 phosphorylation, and Hoechst 33258 for chromosomes. Here we only show the staining results of NRK-E52, an epithelioid NRK line, in which the cells in mitosis are more strongly attached and spread on glass coverslips and more suitable for the morphological observation of the cells in mitosis. Similar results were obtained with parental fibroblastic NRK cells and with HeLa cells. Almost all of the cells showing brighter staining for anti-PS277 antibody (>97%) had condensed chromosomes and vice versa (Fig. 6, M-phase). None of the cells with relaxed chromosome staining showed brighter PS277 staining (Fig. 6, interphase).

We tried to confirm the increased phosphorylation of Ser-277 in mitotic cells biochemically. The parental fibroblastic NRK cells were arrested in S-phase by aphidicolin treatment and released to enter into mitosis for 7 h. The cells loosely attached to the culture dish were collected to enrich mitotic cells (mitotic index = 85 ± 3%, n = 3), then lysed and analyzed by Western blotting (Fig. 7A). The phosphorylation of Ser-277 was greatly increased in mitotic cells (lane 2) compared with randomly growing cells (lane 1). As reported previously, phosphorylation of serine 25 of GM130 (PS25) was also increased in mitotic cells (22).

View larger version (69K): [in this window] [in a new window]   FIG. 4. Ser-277 was phosphorylated by EGF stimulation via an ERK signaling pathway. NRK cells were cultured under the low serum condition. After 48 h, epidermal growth factor (EGF, 50 ng/ml) was added and the mixture was incubated for the indicated time (minutes). To inhibit MEK kinase activity, U0126 (20 µM) was pretreated before adding EGF (B and D). At each time point, the cells were either fixed and double-stained (A and B) by anti-PS277 (PS277) and anti-GRASP65 antibodies (GRASP65) or lysed and analyzed by Western blotting (C and D) using anti-PS277 (PS277), anti-GRASP65 antibodies (GRASP65), anti-phosphorylated active form of ERK (pERK), or anti-ERK2 (ERK2). To compare the total amount of GRASP65, band shift was suppressed using a thick gel (10%). Bar = 20 µm.

View larger version (66K): [in this window] [in a new window]   FIG. 5. Ser-277 is directly phosphorylated by ERK. In vitro kinase assays were performed with purified activated or control MAPKs (ERK2, JNK1, and p38) using recombinant His-tagged GRASP65 or control substrates (myelin basic protein, c-Jun, and ATF2). A, an assay was performed in the presence of [-32P]ATP, and phosphorylated substrates were detected by SDS-PAGE followed by autoradiography. B, an assay was performed in the presence of cold ATP, and the samples were analyzed by Western blotting using anti-PS277 or anti-GRASP65 antibodies.

Ser-277 Is Phosphorylated by Cdk but Not by ERK in Mitotic Cells—The surrounding sequence of Ser-277 matches the consensus of Cdks suggesting the phosphorylation of Ser-277 during mitosis is mediated by the mitotic kinase, Cdk1-cyclin B. We, therefore, tried to confirm Ser-277 is indeed phosphorylated by Cdk using an in vitro experimental system that was shown to reproduce the mitotic fragmentation of the Golgi apparatus (2, 3, 9). Purified rat liver Golgi membranes were incubated with interphase or mitotic HeLa cytosol in the presence of an ATP regenerating system and the membranes were recovered and analyzed by Western blotting (Fig. 7B). The phosphorylation of Ser-277 was greatly increased after the incubation with mitotic cytosol (lane 3). When the mitotic cytosol was pretreated with roscovitine, a Cdk-specific inhibitor, the phosphorylation of Ser-277 was completely abolished (lane 4). This result was in good accordance with the phosphorylation status of serine 25 of GM130 (PS25). It was previously shown that the serine 25 of GM130 is phosphorylated by Cdk1-cyclin B under similar experimental conditions (3). We have also confirmed that purified Cdk1-cyclin B can phosphorylate GRASP65.² These results strongly suggested that the Ser-277 is phosphorylated by Cdk1-cyclin B under mitotic condition.

View larger version (61K): [in this window] [in a new window]   FIG. 6. Ser-277 is phosphorylated during mitosis. Randomly growing NRK cells were fixed and stained with anti-PS277 (PS277), anti-GRASP65 (GRASP65), and Hoechst 33258 (Hoechst). The stage in cell cycle was judged from the cell and the chromosome morphology. Pictures represent a typical staining pattern for each stage of the cell cycle.

View larger version (56K): [in this window] [in a new window]   FIG. 7. Ser-277 is phosphorylated by Cdk but not by ERK during mitosis. A, NRK cells were synchronized in S phase by 12-h aphidicolin treatment. The cells were then released to enter mitosis for 7 h in the presence (M+U) or absence (M) of U0126. Asynchronous cell cultures were used as control (I). The cells were collected, lysed, and subjected to Western blotting with anti-PS277, GRASP65, PS25, GM130, pERK, or ERK2 antibodies. B, purified rat liver Golgi membranes were incubated with interphase (I) or mitotic (M) cytosol prepared from HeLa cells in the presence or absence of an ERK inhibitor (U0126) and/or Cdk inhibitor (roscovitine) as indicated. The membranes were precipitated by centrifugation and subjected to Western blotting using anti-PS277, GRASP65, PS25, and GM130 antibodies. C, cytosol used in B was Western blotted with anti-pERK and ERK2 antibodies.

Inhibition of Mitotic Entry by GRASP65 Fragment Containing S277—Sütterlin et al. (30) have recently reported that introduction of an antibody to GRASP65 or a C-terminal fragment of GRASP65 inhibited NRK cell to enter mitosis. This was suggested to be caused by the inhibition of mitotic disassembly of the Golgi apparatus, because the inhibition of mitotic entry was abolished by the forced disassembly of the Golgi apparatus using brefeldin A or nocodazole. To explore the significance of Ser-277 phosphorylation for mitotic entry, we employed their experimental system. Initially, we reproduced their results using purified C-terminal fragment of GRASP65.² We then used purified N-terminally His-tagged full-length GRASP65 to evaluate the importance of Ser-277 in the context of entire molecule. NRK cells were arrested in S-phase by aphidicolin treatment and then released to enter mitosis. The cells started to enter mitosis 5 h after the release, and >35% of the cells were found to be in mitosis after 7 h (Fig. 8A, solid bars). Most of these cells then synchronously exited mitosis following an additional 2 h of incubation. When His-tagged GRASP65 was microinjected into the cytoplasm, only 15% of the cells were in mitosis after 7 h (white bars). No effect was seen after the injection of the buffer alone (hatched bars). In the majority of the cells injected with His-tagged GRASP65, mitotic entry is prevented rather than delayed, because the peak time in the number of cells in mitosis was not delayed (7 h after the incubation). As previously reported (30), the inhibition of mitotic entry was abolished by the disassembly of the Golgi apparatus by brefeldin A treatment (Fig. 8B, GR).

View larger version (30K): [in this window] [in a new window]   FIG. 8. Inhibition of mitotic entry by Ser-277. A, NRK cells were incubated for 12 h in the presence of aphidicolin to synchronize in S phase. The cells were washed and incubated for 2 h and then microinjected with buffer (mock) or His-GRASP65 (GR65) with injection marker. Cells were then incubated for indicated time after the removal of the drug. Cells were fixed and stained by Hoechst 33258, and the mitotic index was analyzed as described under "Materials and Methods." B, cells were synchronized and microinjected with buffer (mock) or His-GRASP65 (GR) as in A and then incubated for 7 h after the removal of aphidicolin. Brefeldin A (BFA) was added just after the removal of aphidicolin and was present for the rest of the incubation. C, cells were synchronized and microinjected with buffer (mock), His-GRASP65 (GR), S277A mutant of His-GRASP65 (SA), and analyzed as in A. D, the oligonucleotide-peptide sequences used for the microinjection in E. E, cells were synchronized and microinjected with oligonucleotide-peptide shown in D and analyzed as in A.

To evaluate the specificity of the inhibitory effect, we performed similar microinjection experiments using synthetic peptides. Four serine residues known to be phosphorylated by Cdk1-cyclin B (31) were selected, and corresponding peptides were synthesized (Fig. 8D, Ser-277, Ser-217, Ser-376, and Ser-400). Remarkably, a similar inhibitory effect was observed when the Ser-277 peptide was injected at the same molar concentration as His-GRASP65 (Fig. 8, D and E, 1: Ser-277). Similar to His-GRASP65 injection, the mutation of the Ser-277 to alanine abolished the inhibition (3: S277A). The inhibition was not observed with Ser-217/Thr-220, Ser-376, and Ser-400 peptides (4: Ser-217/Thr-220, 5: Ser-376, and 6: Ser-400). These results strongly suggest that the inhibition is specifically caused by the region of GRASP65 surrounding Ser-277 and not by simple competition for the catalytic site of Cdk1-cyclin B. Interestingly, more efficient inhibition was observed when the peptide phosphorylated at Ser-277 was microinjected (2: PS277). By immunofluorescence using anti-PS277, microinjected His-GRASP65 was found to be phosphorylated at the Ser-277.² Therefore, it is probable that the microinjected His-GRASP65 and Ser-277 peptide needed to be phosphorylated to exert an inhibitory effect on cell cycle progression.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mitotic phosphorylation of GRASP65 and its role in cisternal unstacking have been analyzed extensively (31, 32), but interphase phosphorylation has been poorly explored. Here, we have shown that serine 277 (Ser-277) of GRASP65 is phosphorylated in interphase cells. The phosphorylation is markedly enhanced by growth factor stimulation (EGF) of interphase cells (Fig. 4), and this is mediated by ERK (Figs. 4 and 5). The phosphorylation of Ser-277 of GRASP65 causes a mobility shift on SDS-PAGE, and the mutation of Ser-277 to alanine abolishes the shift (Figs. 1 and 2). Therefore, the shift is specifically caused by the phosphorylation of Ser-277. A similar mobility shift was observed in in vitro phosphorylation experiments using recombinant GRASP65 and interphase or mitotic cytosol (Fig. 7), and this was abolished when a S277A mutant was used.² On the other hand, the S277A mutant was labeled with ³²P to a comparable level with wild type GRASP65 when in vitro phosphorylation experiment was carried out in the presence of [-³²P]ATP.² Taken together, our results strongly suggest that GRASP65 is phosphorylated on many sites in interphase and mitotic condition but only the phosphorylation of Ser-277 causes a mobility shift.

Our results suggest that Ser-277 is phosphorylated on the Golgi apparatus in interphase cells and that disassembly of the Golgi apparatus by BFA treatment abolishes the phosphorylation (Fig. 1). An intriguing interpretation is that a kinase localized on the Golgi apparatus is responsible for Ser-277 phosphorylation, and the relocation of this kinase after BFA treatment abolishes the phosphorylation. However, we cannot exclude the possibility that BFA inhibited the kinase activity directly or indirectly without affecting its localization. We are now examining these possibilities. Because ERK is reported to be localized on the Golgi apparatus (5, 33–35), this is a prime candidate for the interphase kinase of Ser-277 at the Golgi apparatus. Actually, we have shown that Ser-277 can be directly phosphorylated by ERK in vitro (Fig. 5). Furthermore, the phosphorylation of Ser-277 was reduced by serum starvation and was strongly enhanced by serum² or EGF treatment in correspondence with the increase of activated ERK1 and ERK2 (Fig. 4). The Ras-Raf-MEK-ERK pathway is activated by signals controlling cell proliferation, differentiation, or cell migration (for a review, see Ref. 28). It was recently reported that the activation of Ras occurs on the Golgi apparatus and plays an important role for nerve growth in PC12 cells (36). Correspondingly, we have found that the phosphorylation of the Ser-277 occurs in PC12 cells after the treatment of nerve growth factor.² Therefore, it is probable that GRASP65 is a general downstream target of the ERK signaling pathway. Interestingly, our preliminary experiments showed that high salt-washed Golgi membrane, in which most of ERK was removed, retained a kinase activity for GRASP65. We also noted that weak but significant phosphorylation of Ser-277 was always observed when serum-starved interphase cells were treated with U0126, an MEK inhibitor (Fig. 4). Therefore, it is possible that there is another kinase on the Golgi apparatus, and this is also responsible for the interphase phosphorylation of Ser-277.

The downstream events after the phosphorylation of Ser-277 by ERK remain obscure. GRASP65 Ser-277 phosphorylation may have some role in the maintenance of the Golgi morphology as we initially presumed. However, we have only found subtle morphological changes in the Golgi apparatus even when the phosphorylation of Ser-277 was enhanced by EGF treatment at least by the resolution of immunofluorescence observation (Fig. 4A). Detailed morphological analyses by electron microscopy and analyses of the effects on the rate or quality of the vesicular traffic through the Golgi apparatus may provide some evidence of the change. Alternatively, GRASP65 may function as a scaffold in one or more signal transduction pathways, and the phosphorylation of Ser-277 may affect some downstream signaling molecules without affecting Golgi morphology. Indirect evidence in support of this idea comes from the observation that GM130 is a scaffold for Ste20 kinases in interphase cells (37). Isolation of the one or more proteins that specifically bind to the phosphorylated or non-phosphorylated Ser-277 peptide will allow us to examine this possibility.

GRASP65 is known to be heavily phosphorylated during mitosis (7). We have now identified Ser-277 as one of the phosphorylation sites and Cdk1-cyclin B as the relevant kinase (Figs. 6 and 7). A mass-spectrum fingerprinting analysis of mitotically phosphorylated GRASP65 also confirmed that Ser-277 is one of the phosphorylation sites in mitosis (31). During the course of our work, Wang et al. (14) have reported that the mitotic phosphorylation of GRASP65 can inhibit the higher order oligomer formation. We have tried to find whether the mutation of Ser-277 can cause any change in biochemical character of GRASP65. However, we have not been able to find any difference with wild type GRASP65 and the S277A mutant by several methods, including an in vitro binding experiment similar to that employed by Wang et al. Therefore, the phosphorylation of Ser-277 alone seems to be not enough to cause the disassembly of GRASP65 oligomer. This is consistent with the recent findings of Wang et al. (32), who reported that phosphorylation of GRASP65 at multiple sites is necessary for the inhibition of the higher order oligomer formation. Preisinger et al. (31) have also shown that at least 5 other serine and 1 threonine residues in the C terminus that are mitotically phosphorylated. These results argue against Ser-277 phosphorylation alone causing the dissolution of GRASP65 oligomers and cisternal unstacking, although it may make a significant contribution to these events. It is possible that in vivo, several kinases, including Cdk1-cyclin B (3), collaborate to phosphorylate GRASP65 on multiple sites and cause the disassembly of GRASP65 oligomer during mitosis. Actually, GRASP65 was shown to be phosphorylated by Plk1 directly (14, 15), although the exact phosphorylation sites were not identified. Whether other kinases are responsible for Ser-277 phosphorylation and how they are involved in Golgi disassembly has to be examined in future.

Consistent with the former report by Sütterlin et al. (30), the microinjection of His-GRASP65 into the cytoplasm of G₂-phase cells blocked the cells to enter mitosis (Fig. 8A). In this report, we have found that Ser-277 plays a key role in causing the inhibitory effect (Fig. 8, D and E). The inhibition was enhanced by Ser-277 phosphorylation (Fig. 8, D and E, 3: PS277) and abolished by the substitution of Ser-277 for alanine to prevent the phosphorylation (Fig. 8C, SA). These results suggest that the Ser-277 region is phosphorylated to exert the inhibitory effect. Consistent with this, we found that microinjected His-GRASP65 was efficiently phosphorylated at Ser-277 by immunostaining the cells with anti-PS277 antibody² providing an explanation for its inhibition of mitotic entry. An alternative explanation is that unphosphorylated Ser-277 has a basic inhibitory activity and that its phosphorylation merely enhances this activity.

How then does Ser-277 inhibit mitotic entry? Immunofluorescence observation revealed that microinjected His-GRASP65 or Ser-277 peptide distributed diffusely in the cytosol and did not affect the localization of endogenous GRASP65 or the structure of the Golgi apparatus.² Therefore, it is unlikely that microinjected His-GRASP65 or Ser-277 peptide need to be localized to the Golgi apparatus to inhibit mitotic entry. It is plausible that the Ser-277 region of His-GRASP65 and Ser-277 peptide binds to some cytosolic factor(s) required for regulating mitotic entry and inhibits its function. In this regard, Cdk1-cyclin B is a prime candidate for a target of the Ser-277 region. However, it is unlikely that the Ser-277 region directly affects the activity of Cdk1-cyclin B by sequestering active enzyme, because the microinjection of peptides containing other phosphorylation site of Cdk1-cyclin B (Ser-217/Thr-220, Ser-376, or Ser-400) did not affect mitotic entry (Fig. 8, D and E). This raises the possibility that the Ser-277 region affects activation or localization of Cdk1-cyclin B. Detailed in vitro and in vivo experiments are necessary to evaluate these possibilities. Other candidates are factor(s) that indirectly affect Cdk1-cyclin B activity. One such candidate is the kinase Plk1, because Plk1 was shown to bind mitotically phosphorylated GRASP65 (31), and Plk1 is implicated in the regulation of Cdk1 activity (38). Our preliminary experiments have shown that less Plk1 binds to mitotically phosphorylated GRASP65 with S277A mutation suggesting that Plk1 preferentially binds to the phosphorylated Ser-277 region.² If so, the introduction of excess phosphorylated Ser-277 fragments would perturb Plk1 function.

This still leaves the question of how the phosphorylation of Ser-277 of GRASP65 contributes to the regulation of mitotic entry. As we discussed above, the phosphorylated Ser-277 region most probably interacts with one or more regulators of mitosis. Therefore, it is reasonable to think that GRASP65 has a role in controlling mitotic entry. The inhibition of mitotic entry induced by the GRASP65 Ser-277 region was overridden by disrupting the Golgi apparatus (Fig. 8B) indicating that the existence of an intact Golgi apparatus is necessary to inhibit the mitotic entry. This suggests that the intact Golgi apparatus can inhibit the mitotic entry in G₂ phase, and the inhibitory activity is lost after Golgi disassembly during mitosis. This is not the only evidence that the Golgi apparatus has an inhibitory role in mitotic entry. Myt1, an inhibitor kinase for Cdk1, localizes on the Golgi apparatus (39). Upon mitotic transition, the kinase activity of Myt1 is inactivated and the resulting dephosphorylation of Cdk1 by Cdc25, a Cdk1-specific phosphatase, results in the activation of Cdk1-cyclin B (see Liu et al. (39) and references therein). It was recently shown that Myt1 is phosphorylated by Plk1 (38) and inactivated by Plx1, a Xenopus homologue of Plk1 (40). Because Plk1 is shown to bind phosphorylated GRASP65 (31), phosphorylated GRASP65 may recruit Plk1 to the Golgi apparatus and promote the inactivation of neighboring Myt1 leading to the activation of Cdk1-cyclin B. The activated Cdk1-cyclin B then phosphorylates GRASP65 producing a positive feedback loop. The intact Golgi apparatus may serve as a scaffold for Myt1 to phosphorylate Cdk1-cyclin B and promote its inactivation. If so, the scaffold for Myt1 to inactivate Cdk1-cyclin B will be lost after the disassembly of the Golgi apparatus. This scenario fits well with the loss of the inhibition of mitotic entry after the disassembly of the Golgi apparatus. We are now examining this possibility with further experiments.

The fact that Ser-277 of GRASP65 is phosphorylated by multiple kinases (ERKs and Cdk1) raises the possibility that GRASP65 may function as a signal integrator controlling cell growth. For example, the inhibition of ERKs might affect mitotic entry through phosphorylation of Ser-277. However, it is unlikely that this simple idea is true, because we did not observe inhibition of mitotic entry and activation of Cdk1 even when the activation of ERKs was prohibited by the presence of U0126 (Fig. 7A). Nevertheless, it is still possible that there are as yet unidentified kinases that phosphorylate Ser-277 or proteins binding to the Ser-277 region, and these factors link different signaling cascades to control cell growth.

Finally we emphasize that our results presented here strongly argue that the phosphorylation of GRASP65 and the disassembly of the Golgi apparatus are not only the results of mitosis or the mitotic activation of Cdk1-cyclin B but are also actively involved in regulating mitotic entry and Cdk1-cyclin B activation themselves. Our finding, that the phosphorylation of Ser-277 of GRASP65 has an important role in mitotic entry, provides an important clue to understand this novel regulatory pathway for cell cycle control.

	FOOTNOTES

 
^* This work was supported in part by a Grant-in-Aid for Scientific Research (15570156), Grants-in-Aid for Scientific Research on Priority Areas (15032216 and 16044218) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a special research project grant from Kanazawa University (2001, 2002, and 2003) (to N. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** Supported by the Max-Planck Society.

^¶¶ To whom correspondence should be addressed: Tel./Fax: 81-76-234-4466; E-mail: osaru3{at}kenroku.kanazawa-u.ac.jp.

¹ The abbreviations used are: MEK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1; EGF, epithelial growth factor; NRK, normal rat kidney; DMEM, Dulbecco's modified Eagles medium; FCS, fetal calf serum; CIP, calf intestinal alkali phosphatase; Plk, polo-like kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; pERK, phosphorylated active form of ERK; JNK1, c-Jun N-terminal kinase 1; GST, glutathione S-transferase; BFA, brefeldin A; ER, endoplasmic reticulum; Cdk, cyclin-dependent kinase.

² S. Yoshimura and N. Nakamura, unpublished observation.

	ACKNOWLEDGMENTS

 
We thank Drs. M. Takano-Maruyama (Kanazawa Medical University); H. Nishitani, H. Kobayashi, and K. Mihara (Kyushu University); K. Yamashita, S. Uchida, and S. Takano (Kanazawa University); Y. Misumi (Fukuoka University); H. Nakajima and E. Nishida (Kyoto University); C. Preisinger (Max-Planck-Institute of Biochemistry); and all the member of the Nakayama and Ohkuma Laboratories for helpful comments and discussions.

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