M Phase Phosphorylation of Cytoplasmic Dynein Intermediate Chain and p150Glued*

Chi-Ying F. HuangDagger §, Chao-Pei Betty ChangDagger , Chia-Lin Huang§, and James E. Ferrell Jr.Dagger parallel

From the Dagger  Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332 and the § National Health Research Institutes, Division of Molecular and Genomic Medicine, Taipei 115, Taiwan, Republic of China

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To understand how the dramatic cell biological changes of oocyte maturation are brought about, we have begun to identify proteins whose phosphorylation state changes during Xenopus oocyte maturation. Here we have focused on one such protein, p83. We partially purified p83, obtained peptide sequence, and identified it as the intermediate chain of cytoplasmic dynein. During oocyte maturation, dynein intermediate chain became hyperphosphorylated at the time of germinal vesicle breakdown and remained hyperphosphorylated throughout the rest of meiosis and early embryogenesis. p150Glued, a subunit of dynactin that has been shown to bind to dynein intermediate chain, underwent similar changes in its phosphorylation. Both dynein intermediate chain and p150Glued also became hyperphosphorylated during M phase in XTC-2 cells and HeLa cells. Thus, two components of the dynein-dynactin complex undergo coordinated phosphorylation changes at two G2/M transitions (maturation in oocytes and mitosis in cells in culture) but remain constitutively in their M phase forms during early embryogenesis. Dynein intermediate chain and p150Glued phosphorylation may positively regulate mitotic processes, such as spindle assembly or orientation, or negatively regulate interphase processes such as minus-end-directed organelle trafficking.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fully grown (stage VI) Xenopus oocytes are arrested in a G2-like state. Exposure to progesterone releases oocytes from this arrest and causes meiotic maturation. The maturing oocyte undergoes germinal vesicle breakdown (GVBD),1 forms a meiotic spindle, segregates its homologous chromosomes, completes the first meiotic division, enters meiosis 2, and then arrests in metaphase of meiosis 2.

Oocyte maturation depends upon the activation of Cdc2-cyclin B complexes (1), just as entry into mitotic M phase does (reviewed in Refs. 2-4). In the Xenopus oocyte, Cdc2 activation is tightly linked to activation of p42 mitogen-activated protein kinase (MAPK) and its upstream activators Mek-1 (a MAPK kinase) and Mos (a MAPK kinase kinase) (5-7). Interfering with the activation of Mos (8), Mek-1 (9), or p42 MAPK (10) inhibits progesterone-induced Cdc2 activation, and overexpression of Mos (11, 12) or introduction of active forms of Mek-1 (13) or p42 MAPK (14) can cause activation in the absence of progesterone. p42 MAPK is activated in an all-or-none fashion (15), ensuring that the oocyte's decision to carry out maturation is decisive and irrevocable.

Relatively little is known about how the activation of MAPK and Cdc2 brings about the dramatic cell biological changes of maturation. Some of the relevant substrates of these kinases are undoubtedly involved in entry into mitosis as well; others must be specific for meiosis or maturation. Expression cloning studies have identified a number of proteins that become phosphorylated at the onset of mitosis (16-18), and work is under way to determine whether and how their mitotic phosphorylation affects their function.

Here we have approached the identification of meiotic phosphoproteins by antiphosphotyrosine immunoblotting, the strategy that implicated p42 MAPK in Xenopus oocyte maturation (19-22). We looked for immunoreactive bands that increased in intensity during oocyte maturation (as does p42 MAPK), decreased in intensity (as does Cdc2), or changed in their electrophoretic mobility.

This paper focuses on the first of these proteins, p83, a band that shifts up in its apparent molecular weight during oocyte maturation but does not change substantially in intensity. Through purification and peptide sequencing, we have identified p83 as a component of the minus-end-directed microtubule motor dynein, the cytoplasmic dynein intermediate chain (dynein IC). We found that dynein IC comigrates with p83 and undergoes mobility shifts that exactly parallel those of p83; that the mobility shift of dynein IC is due to phosphorylation; and that dynein IC cross-reacts with various phosphotyrosine antisera but is phosphorylated mainly at serine in vivo. Dynein IC was found to undergo hyperphosphorylation just prior to germinal vesicle breakdown and to remain hyperphosphorylated throughout maturation and early embryogenesis.

In addition, we examined the phosphorylation of the p150Glued subunit of dynactin, because of the physical and functional association between dynein and dynactin (23-28). We found that p150Glued also becomes hyperphosphorylated during oocyte maturation and, like dynein IC, remains in a shifted form during early embryogenesis. Finally, we found that both dynein IC and p150Glued exhibit mobility shifts in nocodazole-treated XTC-2 cells and HeLa cells. These findings demonstrate that multiple components of the dynein/dynactin system undergo coordinated phosphorylation changes at the G2/M transitions of both meiosis and mitosis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Isolation of Xenopus Oocytes, Eggs, and Embryos-- Xenopus ovarian tissue was obtained surgically and defolliculated with 2 mg/ml collagenase as described (29). Stage VI oocytes were sorted manually and incubated at 16 °C for at least 8 h. Eggs were obtained by dorsal lymph sac injection of female frogs with human chorionic gonadotropin. Eggs were fertilized and dejellied as described (30).

Oocyte Maturation-- Immature stage VI oocytes were treated with progesterone (5 µg/ml) in modified Barth's saline containing Ca2+ and bovine serum albumin at room temperature for various lengths of time to induce maturation. GVBD was inferred from the appearance of a distinct white dot at the oocyte's animal pole. Groups of oocytes were quick-frozen on dry ice.

Preparation of Oocyte, Egg, and Embryo Lysates-- Ten oocytes, eggs, or embryos were added to 100 µl of ice-cold extract buffer (250 mM sucrose, 100 mM NaCl, 2.5 mM MgCl2, 20 mM HEPES, pH 7.2) containing 0.5 mM Na3VO4 and protease inhibitors (10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml chymostatin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) and lysed by pipetting through a 200-µl pipette tip. Lysates were centrifuged for 5 min in a microcentrifuge with a right angle rotor. Samples of clarified cytoplasm were removed from the overlying lipid and underlying yolk protein. Cytoplasm was frozen on dry ice and stored at -80 °C.

Preparation of Concentrated Oocyte and Egg Extracts-- Concentrated cell-free Xenopus oocyte extracts were prepared essentially as described previously (29). Cycling egg extracts were prepared as described (32).

Protein Purification-- G2 phase oocyte lysates containing approximately 600 mg of protein were subjected to a 20-35% ammonium sulfate cut. The precipitate was resuspended in 60 ml of buffer A (50 mM MES, pH 6.5, 20 mM NaCl, 0.1 mM EGTA, 1 mM dithiothreitol, and 10% glycerol) and centrifuged at 10,000 rpm for 10 min at 4 °C to remove insoluble proteins. The supernatant was loaded on an S-Sepharose Fast Flow column (Amersham Pharmacia Biotech) equilibrated with buffer A. The column was washed extensively with buffer A, and proteins were eluted with buffer B (50 mM MES, pH 6.5, 1 M NaCl, 0.1 mM EGTA, 1 mM dithiothreitol, and 10% glycerol). The eluent was diluted with a 10× volume of buffer C (20 mM Tris, pH 7.6, 1 mM dithiothreitol, and 10% glycerol) and then spun at 10,000 rpm for 15 min at 4 °C. The supernatant was filtered through a 0.22-µm filter. The filtrate was applied to a 5-ml FPLC HiTrap Blue column (Amersham Pharmacia Biotech) equilibrated with buffer D (buffer C plus 10 mM NaCl). Protein was eluted with a 35-ml linear gradient from buffer C to buffer E (buffer C plus 1.5 M NaCl and 2% ethylene glycol). Fractions were collected and assayed for p83 by antiphosphotyrosine immunoblotting. The 83-kDa doublet bands were excised from the SDS-PAGE and sent to the W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University) for sequencing.

32P Labeling in Vivo-- For in vivo 32P labeling, groups of 50 oocytes were placed in a 12-well plate containing 2 mCi of [32P]orthophosphate/ml in modified Barth's saline containing Ca2+ and bovine serum albumin as described above and incubated with or without progesterone (5 µg/ml). Once the progesterone-treated oocytes reached GVBD, both groups of oocytes were transferred to nonradioactive modified Barth's saline solution, washed four times, and frozen on dry ice. Lysates were prepared as described above.

Immunoblotting-- Samples were separated on 7.5 or 10% low-bis polyacrylamide SDS gels (acrylamide:bisacrylamide ratio 100:1) and transferred to Immobilon P (Millipore Corp.) blotting membranes. Proteins were detected with polyclonal antiphosphotyrosine antiserum (21), dynein IC monoclonal antibody (clone 70.1; Sigma), or p150Glued monoclonal antibody (Transduction Laboratories), followed by 125I-protein A for phosphotyrosine detection or alkaline phosphatase-conjugated secondary antibodies (Sigma) for dynein IC and p150Glued detection.

Immunoprecipitation-- Oocyte lysates were diluted with 1 volume of IP buffer (25 mM Tris, pH 8.0, 10 mM MgCl2, 15 mM EGTA, 0.1% Triton X-100, 0.5 mM NaF, 0.5 mM Na3VO4, 60 mM 2-glycerolphosphate, and 0.1% bovine serum albumin). The solution was incubated with dynein IC antibody 70.1 or p150Glued antiserum at 4 °C for 2-4 h. Anti-mouse IgM agarose (for dynein IC) (Sigma) or anti-mouse IgG agarose (for p150Glued) (Sigma) was added and incubated for another 2 h at 4 °C. Immune complexes were washed four times with immunoprecipitation buffer lacking NaF, Na3VO4, and 2-glycerolphosphate. The resulting pellets were resuspended either in SDS sample buffer for SDS-PAGE analysis or lambda -phosphatase buffer (50 mM Tris, pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, and 2 mM MnCl2).

lambda -Phosphatase Treatment-- Dynein IC or p150Glued immunoprecipitates were incubated with 400 units of lambda -phosphatase (New England BioLabs) in lambda -phosphatase buffer at 30 °C for 30 min. This was followed by adding an additional 400 units of lambda -phosphatase for a further 60 min at 30 °C.

Phosphoamino Acid Analysis-- 32P-Labeled lysates were subjected to immunoprecipitation with dynein or p150Glued antibodies followed by SDS-PAGE and transfer to Immobilon P membranes as described above. Dynein IC and p150Glued bands were excised and subjected to partial acid hydrolysis (33). The hydrolysates were mixed with phosphoamino acid standards and subjected to two-dimensional (pH 1.9 followed by pH 3.5) electrophoresis on thin layer cellulose plates (34).

Cell Culture and Synchronization-- XTC-2 cells were grown in 70% L-15 medium supplemented with 10% fetal calf serum. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were arrested in interphase (S phase) by double thymidine block. Cells were treated with 2 mM thymidine for 24 h, grown in regular medium for 12 h, and then grown again for 24 h in medium containing 2 mM thymidine. Cells were arrested in M phase by release from double thymidine block followed by treatment with nocodazole (100 ng/ml) for 12-16 h. Mitotic cells were recovered by gentle shake off. Mitotic index was determined by staining an aliquot of the cells with Hoechst 33342 dye followed by epifluorescence microscopy. Samples for immunoblotting were prepared by suspending washed pelleted cells in phosphate-buffered saline and lysing with SDS sample buffer.

Recombinant Proteins and Other Chemicals-- Bacterially expressed wild-type malE-Mos protein was purified as described (12). Okadaic acid was purchased from Life Technologies, Inc.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hyperphosphorylation of p83 during Oocyte Maturation-- We used antiphosphotyrosine immunoblotting to search for proteins that undergo phosphorylation changes during Xenopus oocyte maturation, with the aim of identifying new regulators or effectors of meiosis. In agreement with previous reports (19, 21, 22), the most prominent antiphosphotyrosine-reactive bands were a ~42-kDa band corresponding to p42 MAP kinase, which appeared just prior to GVBD, and a ~33-kDa band corresponding to Cdc2, which disappeared just prior to GVBD (Fig. 1A). In addition, we identified five other bands recognized by at least one antiphosphotyrosine antiserum that changed in intensity or mobility during maturation, with apparent molecular masses of 83, 95, 100, 116, and 140 kDa (Fig. 1A and data not shown). Early attempts to identify these proteins by testing plausible candidates proved to be unsuccessful. We therefore purified each of the proteins from Xenopus oocytes or eggs, using antiphosphotyrosine immunoblotting as an assay.


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Fig. 1.   Tyrosine phosphorylation of p83. A, antiphosphotyrosine immunoblot of G2 and M phase oocyte lysates. Stage VI oocytes were treated with progesterone to induce maturation. G2 and M phase oocyte lysates were subjected to SDS-PAGE, transferred to a blotting membrane, and probed with antiphosphotyrosine antiserum. Migration of molecular weight standards is indicated on the left. B, antiphosphotyrosine immunoblot of Xenopus extracts treated with okadaic acid or Mos. Cell-free G2 phase oocyte extracts were treated with okadaic acid (5 µM) or Mos (40 nM). At the indicated times (top), samples were removed and analyzed by antiphosphotyrosine immunoblotting.

Here we shall focus on p83, which migrated as a doublet in G2 phase with the upper band becoming more prominent during maturation (Fig. 1A). Both p83 bands were recognized by several antiphosphotyrosine antisera (PY20, 4G10, and the polyclonal serum used in the blots shown herein), and their recognition was blocked by phenylphosphate (40 mM) or phosphotyrosine (1 mM) but not by phosphothreonine (1 mM) or phosphoserine (1 mM) (data not shown).

Both p83 bands were detected in G2 phase Xenopus extracts and shifted to the upper band in extracts treated with okadaic acid, a phosphatase inhibitor, and Mos, an activator of the MAP kinase cascade (Fig. 1B). p83 also shifted in response to added active Cdc2-cyclin B (Fig. 4 and data not shown).

Purification of p83 and Identification as Dynein Intermediate Chain-- Initial attempts to immunopurify p83 with antiphosphotyrosine antibodies were unsuccessful; we therefore used classical protein purification techniques. G2 phase oocyte lysates were subjected to ammonium sulfate precipitation (taking a 20-35% cut) followed by S-Sepharose cation exchange chromatography and HiTrap Blue dye affinity chromatography. The HiTrap Blue column profile is shown in Fig. 2. p83 eluted as a doublet in fractions 31-35 as judged by antiphosphotyrosine immunoblotting (Fig. 2C). Corresponding bands were seen by Coomassie staining (Fig. 2B).


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Fig. 2.   HiTrap Blue column fractionation of p83 from G2 phase Xenopus oocyte lysates. G2 phase lysates were fractionated by ammonium sulfate precipitation and S-Sepharose cation exchange chromatography as described under "Experimental Procedures." Fractions containing p83 were diluted, applied to a HiTrap Blue column, and eluted with a linear salt gradient. Fractions were collected and analyzed by SDS-PAGE (B) and immunoblotting with antiphosphotyrosine antiserum (C). A, elution profile from the HiTrap Blue column. Fractions 31-35 contained p83. Migration of an 85-kDa molecular mass standard is indicated on the left.

To address whether the two p83 bands were derived from the same protein, both bands were cut out from SDS-polyacrylamide gels and subjected to tryptic digestion and reversed phase microbore HPLC analysis. The patterns of tryptic peptides from the two bands were nearly identical (Fig. 3A). All of the major peaks were present in both samples, indicating that these two proteins were probably closely related. To further test this conclusion, four peptide peaks from each of the samples were subjected to matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The masses of the upper and lower band peptides were very similar (Fig. 3B), again supporting the idea that the upper and lower p83 bands were derived from a single protein. These findings also suggested that both of the two p83 bands were essentially pure (or, possibly, that both bands were contaminated by the same non-p83 protein).


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Fig. 3.   Tryptic analysis of the upper and lower p83 bands. Purified p83 was eluted from an SDS-polyacrylamide gel, digested with trypsin, and subjected to microbore reversed phase HPLC. A, elution profiles of tryptic peptides from the upper and lower p83 bands. B, MALDI-MS analysis of peaks 1-4 from A. The mass/charge ratio (M/Z) of each peak is shown. Peak 4 contained two peptides. C, sequence alignments. The protein sequence of tryptic peptide 5 from the p83 lower band (A) and a similar tryptic peptide from rat cytosolic dynein intermediate chain are shown.

The MALDI-MS analysis was used to identify peptide peaks that would be suitable for gas phase microsequencing. One such peak (peak 5, from the p83 lower band) was sequenced and found to comprise VTQVDFAPR. This peptide was similar (8 of 9 identities) to a predicted tryptic peptide from rat cytoplasmic dynein intermediate chain (dynein IC) (Fig. 3C). The predicted molecular mass of rat dynein IC is 74 kDa (31), close to the apparent molecular weight of Xenopus p83. We therefore hypothesized that p83 was Xenopus dynein IC.

To test this hypothesis, we made use of a monoclonal dynein IC antibody that cross-reacts with the Xenopus protein. G2 phase oocyte extracts were obtained and treated with buffer, Mos, okadaic acid, or Cdc2-cyclin B. Aliquots of the extracts were subjected to immunoblotting with the dynein IC antibody 70.1. Dynein IC was found to migrate as a closely spaced 83-kDa doublet in G2 phase extracts (Fig. 4A), and dynein IC shifted to a single upper band in response to Mos, okadaic acid, or Cdc2-cyclin B (Fig. 4A). Thus, dynein IC (Fig. 4A) and p83 (Fig. 1B) migrate similarly and respond similarly to various stimuli. Immunoprecipitated dynein IC was recognized on immunoblots by antiphosphotyrosine antibodies (Fig. 4B). Taken together, the common properties of the two proteins identify p83 as dynein IC.


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Fig. 4.   Effects of Mos, Cdc2-cyclin B, and okadaic acid on dynein intermediate chain phosphorylation. Cell-free Xenopus extracts were treated with extract buffer, Mos (40 nM), okadaic acid (5 µM), or Cdc2-cyclin B (50 units/µl). Samples were taken at various times and analyzed by immunoblotting with dynein antibody 70.1 (A) or by immunoprecipitation with dynein antibody 70.1 followed by immunoblotting with antiphosphotyrosine antiserum (B).

The Dynein IC Upper Band Is Hyperphosphorylated-- Next, we addressed whether the upper dynein IC band represented a hyperphosphorylated form of the lower band. Dynein IC was immunoprecipitated from G2 and M phase lysates. Immunoprecipitates were then treated with extract buffer, lambda -phosphatase (a dual specificity phosphatase), or lambda -phosphatase plus vanadate (an inhibitor of lambda -phosphatase), and the products were subjected to electrophoresis and dynein IC immunoblotting. G2 phase dynein IC migrated as a doublet (Fig. 5A, lane 1), and the upper band was eliminated by treatment with lambda -phosphatase (lane 2) but not by treatment with lambda -phosphatase plus vanadate (lane 3). Dynein IC isolated from M phase lysates migrated as a single upper band (lane 4) and was shifted to a lower band by treatment with lambda -phosphatase (lane 5) but not by treatment with lambda -phosphatase plus vanadate (lane 6). These findings establish the dynein IC upper band to be a hyperphosphorylated form of the lower band.


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Fig. 5.   The phosphorylation state of the upper and lower dynein intermediate chain bands. A, effect of phosphatase treatment on dynein IC electrophoretic mobility. G2 or M phase lysates were immunoprecipitated by using dynein IC antibody 70.1. The immunoprecipitates were treated with extract buffer (lanes 1 and 4), lambda -phosphatase (lanes 2 and 5), or lambda -phosphatase plus Na3VO4 (lanes 3 and 6). Samples were then analyzed by the SDS-PAGE and immunoblotting with dynein antibody 70.1. B, phosphoamino acid analysis of in vivo labeled dynein intermediate chain. Stage VI oocytes, treated with or without progesterone, were metabolically labeled in vivo with [32P]orthophosphate. Labeled oocytes were lysed and immunoprecipitated with dynein antibody 70.1. Two-dimensional phosphoamino acid analyses of 32P-dynein intermediate chain immunoprecipitates from G2 (lower and upper bands) and M phases are shown. The positions of nonradioactive phosphoamino acid standards are shown on the right. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine. C, lack of effect of phosphatase treatment on dynein IC antiphosphotyrosine immunoreactivity. Dynein IC was immunoprecipitated from M phase lysates and treated with lambda -phosphatase with or without Na3VO4.

Phosphoamino Acid Analysis of in Vivo Labeled Dynein IC-- The fact that both the upper and lower bands of dynein IC were recognized by antiphosphotyrosine antibodies (Fig. 1) suggested that both forms were phosphorylated at tyrosine. However, previous phosphoamino acid analysis of axonemal dynein reportedly yielded only phosphoserine (35). Consequently, we examined further whether dynein IC is phosphorylated on tyrosine.

Dynein IC was labeled with [32P]orthophosphate in vivo, subjected to immunoprecipitation, and transferred to a blotting membrane. Radiolabel was found to be incorporated into G2 phase dynein IC (both bands) and M phase dynein IC, and the overall level of labeling of G2 and M phase dynein IC was similar (data not shown). The dynein IC bands were excised from the blot and subjected to partial acid hydrolysis and thin layer electrophoresis. The two G2 phase dynein IC bands and the M phase dynein IC band all yielded primarily phosphoserine (Fig. 5B), consistent with a previous report (35), and contrary to the antiphosphotyrosine immunoblotting results (Figs. 1, 2, and 4), no phosphotyrosine was detected. This finding suggests that the recognition of dynein IC by antiphosphotyrosine antibodies might have been spurious. We also immunoprecipitated dynein IC from M phase oocytes and treated the immunoprecipitates with lambda -phosphatase. The lambda -phosphatase caused dynein IC to shift completely to the lower band without decreasing its antiphosphotyrosine immunoreactivity (Fig. 5C). The simplest interpretation of these data is that the antiphosphotyrosine antibodies recognize dynein IC in a phosphorylation state-independent fashion.

Dynein IC Phosphorylation during Maturation, Mitosis in Extracts, and Embryogenesis-- To determine whether dynein IC hyperphosphorylation was specific to meiosis or occurred in other M phases, we examined in detail the timing of dynein IC hyperphosphorylation during maturation, mitosis in cycling extracts, and the mitotic cycles of fertilized eggs and early embryos. Dynein IC shifted to its hyperphosphorylated form just prior to GVBD (Fig. 6A), at about the time when Cdc2 and p42 MAPK become activated (data not shown). Dynein IC remained hyperphosphorylated throughout the rest of meiosis 1 and into meiosis 2. There was no detectable decrease in dynein IC phosphorylation during the period before meiosis 2 when Cdc2 activity drops (Fig. 6A and data not shown; see also Ref. (62).


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Fig. 6.   Dynein intermediate chain phosphorylation during Xenopus oocyte maturation, in cycling egg extracts, and during early embryogenesis. A, dynein IC phosphorylation during progesterone-induced oocyte maturation. Stage VI oocytes were treated with progesterone for the times indicated. Cell cycle progression was monitored by the appearance of a white dot and by Cdc2-cyclin B activity (not shown). Dynein IC was detected with antibody 70.1. B, dynein IC phosphorylation in cycling egg extracts. Extracts from electrically activated eggs were warmed to room temperature to initiate cycling, and samples were taken at various times. Demembranated sperm chromatin (~500 nuclei/µl) was added to the cycling extract to allow monitoring of cell cycle progression. C, dynein phosphorylation in fertilized Xenopus eggs. Eggs were fertilized and dejellied, and samples were taken at various times after fertilization. Unfertilized eggs (t = 0), and stage X embryos are also shown. Cleavages occurred at 90, 125, and 155 min.

Next, we assessed dynein IC phosphorylation in cycling egg extracts, with cell cycle progression assessed by observation of nuclei formed from added sperm chromatin. As shown in Fig. 6B, the extracts were initially in interphase and then entered mitosis at about 60 min and exited mitosis at about 80 min. During all of these cell cycle phases, dynein IC remained in its hyperphosphorylated form (Fig. 6B).

Finally, we examined dynein IC phosphorylation during the completion of meiosis 2 and the first three mitotic cycles of fertilized eggs. Once again, dynein IC remained constitutively in its hyperphosphorylated form (Fig. 6C). Dynein IC was also found to be hyperphosphorylated in stage X (gastrula) embryos. Taken together, these results show that dynein IC goes from partially hyperphosphorylated to fully hyperphosphorylated at the G2/meiosis 1 transition during oocyte maturation, and then remains constitutively hyperphosphorylated throughout the remainder of meiosis, mitosis, and early embryogenesis.

p150Glued Undergoes Cell Cycle-dependent Phosphorylation Changes-- Dynein IC physically interacts with the p150Glued component of dynactin complex (27, 28). Since dynein IC underwent cell cycle-dependent hyperphosphorylation during oocyte maturation, we set out to determine whether p150Glued did as well. As shown in Fig. 8A, p150Glued progressively shifted from a lower apparent molecular weight band to a higher one during oocyte maturation. The mobility shift was due to hyperphosphorylation, since lambda -phosphatase treatment caused M phase p150Glued to shift from the higher band to the lower one (Fig. 7A). In vivo 32P labeling and phosphoamino acid analysis of p150Glued showed that G2 phase p150Glued was essentially nonphosphorylated, and M phase p150Glued was phosphorylated predominantly at serine (Fig. 7B). Like dynein IC, p150Glued hyperphosphorylation persisted throughout meiosis and early embryogenesis (Fig. 8, A, B, and D), and p150Glued became hyperphosphorylated in Mos-, okadaic acid-, and Cdc2-cyclin B-treated G2 phase oocyte extracts (although less rapidly and less completely than did dynein IC; Fig. 8C versus Fig. 4A). Okadaic acid also caused p150Glued to shift to a third more highly shifted band that we have not detected during the normal cell cycles of oocytes and eggs (Fig. 8C).


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Fig. 7.   Phosphorylation of p150Glued in G2 and M phase oocytes. A, effect of phosphatase treatment on p150Glued electrophoretic mobility. G2 or M phase lysates were immunoprecipitated with p150Glued antiserum. The immunoprecipitates were treated with extract buffer (lanes 1 and 4), lambda -phosphatase (lanes 2 and 5), or lambda -phosphatase plus Na3VO4 (lanes 3 and 6). Samples were then analyzed by the SDS-PAGE followed by immunoblotting with p150Glued antiserum. B, phosphoamino acid analysis of in vivo labeled p150Glued. Stage VI oocytes, treated with or without progesterone, were metabolically labeled in vivo with [32P]orthophosphate. Labeled oocytes were lysed and immunoprecipitated with p150Glued antiserum. Two-dimensional phosphoamino acid analyses of 32P-dynein intermediate chain immunoprecipitates from G2 and M phases are shown. The positions of nonradioactive phosphoamino acid standards are shown on the right. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine.


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Fig. 8.   p150Glued phosphorylation during Xenopus oocyte maturation, in cycling egg extracts, in G2 phase oocyte extracts, and during early embryogenesis. A, p150Glued phosphorylation during progesterone-induced oocyte maturation. Stage VI oocytes were treated with progesterone for the times indicated. Lysates were subjected to SDS-PAGE and immunoblotting with p150Glued antiserum. The percentage of oocytes with a white dot is shown below the blot. B, p150Glued phosphorylation in cycling egg extracts. Extracts from electrically activated eggs were warmed to room temperature to initiate cycling, and samples were taken at various times. Demembranated sperm chromatin (~500 nuclei/µl) was added to the cycling extract to allow monitoring of cell cycle progression. G2 phase and M phase lysates are included for comparison. C, effects of Mos, Cdc2-cyclin B, and okadaic acid on p150Glued phosphorylation. Cell-free Xenopus extracts were treated with extract buffer, Mos (40 nM), okadaic acid (5 µM), or Cdc2-cyclin B (50 units/µl). Samples were taken at the indicated times and subjected to immunoblotting with p150Glued antiserum. D, p150Glued phosphorylation in fertilized Xenopus eggs. Eggs were fertilized and dejellied, and samples were taken at various times after fertilization. Unfertilized eggs (t = 0) and stage X embryos are also shown. Cleavages occurred at 90, 120, and 140 min.

Mitotic Phosphorylation of Dynein IC and p150Glued in XTC-2 Cells and HeLa Cells-- Finally, we assessed whether dynein IC and p150Glued were hypophosphorylated in interphase (as was the case for oocytes) or hyperphosphorylated in interphase (as was the case for early embryos) in two somatic cell lines, Xenopus tadpole XTC-2 cells and human HeLa cells, and whether there was any change in their phosphorylation when the cells entered mitosis. Interphase cells were obtained after double thymidine block; mitotic cells were obtained by releasing the double-thymidine blocked cells, arresting them in M phase by nocodazole treatment, and harvesting the mitotic cells by gentle shake off. This treatment yielded approximately 80% cells in mitosis as judged by chromosome staining.

In XTC-2 cells, dynein IC migrated as three bands in interphase (Fig. 9). In mitosis, the bottom band disappeared, and the proportion of the dynein IC in the highest band increased (Fig. 9). In HeLa cells, a single interphase band was detected, and most of the dynein IC shifted to a higher band in M phase. Thus dynein IC undergoes hyperphosphorylation both in meiosis in oocytes and in mitosis in these two cell lines. Similar results were found when unsynchronized XTC-2 cells were compared with nocodazole-treated cells (data not shown).


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Fig. 9.   p150Glued and dynein intermediate chain phosphorylation in S phase and M phase XTC-2 cells and HeLa cells. S phase cells were obtained by double thymidine block. M phase cells were obtained by releasing the S phase cells from the double thymidine block in the presence of nocodazole (100 ng/ml). Cell lysates were blotted with p150Glued (top) and dynein IC (bottom) antibodies.

p150Glued migrated as two bands in interphase XTC-2 cells, with most of the p150Glued found in the lower band (Fig. 9). Most of the p150Glued shifted to the higher band in mitosis (Fig. 9). Similar results were found with HeLa cells (Fig. 9). Likewise, similar results were found when unsynchronized XTC-2 cells were compared with nocodazole-treated cells (data not shown). Thus, both p150Glued and dynein IC become hyperphosphorylated during mitosis in XTC-2 and HeLa cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Initially, we identified p83 as a band on an antiphosphotyrosine immunoblot that shifts from a lower to a higher apparent molecular mass during oocyte maturation (Fig. 1). We purified both the upper and lower p83 bands (Fig. 2) and demonstrated by peptide sequencing and mass spectroscopy that they most likely represent the intermediate chain of cytoplasmic dynein (Fig. 3). In support of this identification, we have shown that dynein IC runs as two bands that co-migrate with the two p83 bands (Fig. 4 and data not shown) and that immunoprecipitated dynein IC is recognized by antiphosphotyrosine antibodies on immunoblots (Fig. 4). Moreover, stimuli that cause p83 to shift to the upper band also cause dynein IC to shift to the upper band (Fig. 4). We conclude that p83 is dynein IC.

Both the upper and lower dynein IC bands are phosphorylated in vivo (Fig. 5B). The upper band is converted to the lower band by lambda -phosphatase treatment (Fig. 5A), indicating that it represents a hyperphosphorylated form. Both bands are phosphorylated predominantly at serine; no phosphotyrosine is detected by in vivo labeling and phosphoamino acid analysis (Fig. 5B). Moreover, the antiphosphotyrosine reactivity of dynein IC is not diminished by lambda -phosphatase treatment (Fig. 5C). Thus, although the antiphosphotyrosine reactivity of p83/dynein IC is what originally allowed us to identify and purify the protein, we believe that dynein IC is not phosphorylated at tyrosine. However, it is the serine phosphorylation of dynein IC, not the (apparent) tyrosine phosphorylation, that changes at the G2/M transition, so it is the serine phosphorylation that is of particular interest. Thus, we examined under what circumstances the serine hyperphosphorylation occurs.

Dynein IC becomes hyperphosphorylated during oocyte maturation at about the time of germinal vesicle breakdown and remains hyperphosphorylated throughout maturation and early embryogenesis (Fig. 6). Dynein IC undergoes a shift from hypo- to hyperphosphorylated forms in nocodazole-treated M phase XTC cells and HeLa cells (Fig. 9). Thus, dynein IC represents a novel M phase phosphoprotein that remains constitutively in its M phase form during the rapid cell cycles of early embryogenesis.

This is an unusual phosphorylation pattern; most M phase phosphoproteins and M phase kinases cycle in their phosphorylation/activity during early embryogenesis. However, one of the kinases responsible for the phosphorylation of cyclin B2 (a kinase of uncertain identity that phosphorylates N-terminal site(s) different from serine 90) is activated during oocyte maturation and remains active throughout early embryogenesis (36). It will be of interest to determine whether this cyclin B2 kinase is also responsible for dynein IC phosphorylation.

Because dynein IC physically and functionally interacts with the p150Glued component of dynactin, we examined p150Glued phosphorylation as well. We found that p150Glued, like dynein IC, becomes phosphorylated during oocyte maturation and remains hyperphosphorylated throughout maturation and early embryogenesis (Fig. 8). It also shifts to a hyperphosphorylated form in M phase in XTC cells and is less subtle in this shift than is dynein IC (Fig. 9). Thus, p150Glued is another novel M phase phosphoprotein and undergoes phosphorylation changes similar to those of its binding partner dynein IC.

Phosphorylation of Dynein IC and p150Glued in Other Systems-- Pfister and co-workers have previously demonstrated that dynein IC is a phosphoprotein, and that different cellular pools of dynein exhibit differences in the relative phosphorylation of various dynein subunits (35, 37, 38). p150Glued has also been previously shown to be a phosphoprotein, and its phosphorylation has been shown to be affected by activators of protein kinases A and C (39). The present work extends these observations by showing that dynein IC and p150Glued undergo marked changes in their phosphorylation during meiotic and mitotic M phases. This suggests the hypothesis that dynein IC and p150Glued phosphorylation contribute to the dramatic changes in microtubule function that occur at the G2/M transition. The demonstration that dynein and p150Glued phosphorylation can be manipulated in Xenopus oocyte extracts (Figs. 1, 4, 8) establishes a powerful system for future work aimed at testing this hypothesis.

Mitotic Phosphorylation of Other Dynein Components-- Niclas and co-workers (40) previously examined dynein phosphorylation in cycling Xenopus egg extracts. They found a ~12-fold increase in the phosphorylation of dynein light intermediate chain during M phase. Thus, there are two different temporal patterns for the M phase phosphorylation of dynein and dynactin: that shown by dynein IC and p150Glued, with the proteins attaining their M phase form during oocyte maturation and remaining locked in that state throughout early embryogenesis and in cycling extracts, and that shown by dynein light intermediate change, with the protein cycling between M phase and interphase forms in cycling extracts.

M Phase Functions of Cytoplasmic Dynein and Dynactin-- Cytoplasmic dynein is a multisubunit complex consisting of heavy chains, intermediate chains, light intermediate chains, and light chains (23, 41-43). Dynein can transport cargo along microtubules toward the minus-end. Dynein IC can directly interact with the p150Glued component of dynactin, which connects dynein to organelles and other structures that are to be transported.

Dynein and dynactin have been implicated in a number of the dramatic changes in cellular organization that occur at the G2/M transition. During mitosis, cytoplasmic dynein is associated with kinetochores, spindle, and centrosomes in mammalian cells in culture (44, 45), placing it in an appropriate location for control of chromosomes or the spindle. Dynactin localization appears to be very similar to that of dynein (46, 47).

Moreover, genetic studies and immunoneutralization studies have functionally implicated dynein and dynactin in spindle assembly and positioning. Budding yeasts with mutations in dynein show defects in spindle orientation (48, 49). Immunodepletion or microinjection of dynein antibodies interferes with spindle pole formation and positioning (50-52), as does immunodepletion with antibodies to the dynein-associated protein NuMA (53). Overexpression of p50 dynamitin, a subunit of dynactin, results in distortion of the spindle and dissociation of the dynactin complex from kinetochores (54).

Finally, biochemical studies have shown that phosphorylation has the potential of regulating kinetochore associated plus- and minus-end-directed microtubule motors (55) and that phosphorylation has important effects on a variety of dynein- and kinesin-associated spindle components (56-60). It will clearly be of interest to determine whether and how the M phase phosphorylation of dynein IC and p150Glued described here contributes to these events. In addition, minus-end-directed organelle transport decreases during M phase in cycling extracts (61), apparently as a result of a phosphorylation-associated decrease in the binding of dynein to its membranous cargo (40). It is plausible that M phase phosphorylation of dynein IC and p150Glued might be involved in shutting down interphase processes that depend upon dynein and dynactin.

    ACKNOWLEDGEMENTS

We thank Xiao Min Wang for help with the XTC-2 cell experiments; Ramesh Bhatt, Tom Guadagno, and Sarah Walter for providing egg extracts; and Xiao Min Wang, Joanne Westendorf, and other members of the Ferrell laboratory for critical reading of this manuscript. We thank David Gard, Tim Mitchison, Tim Stearns, and Ron Vale for helpful discussions; Monica Murakami and George Vande Woude for providing malE-Mos plasmids; and the W. M. Keck Foundation Biotechnology Resource Laboratory for sequencing and MALDI-MS analysis.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM46383 and a Leukemia Society of America fellowship (to C.-Y. F. H.).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.

Present address: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021.

parallel To whom correspondence should be addressed. Tel.: 650-725-0765; Fax: 650-725-2952; Email: ferrell{at}cmgm.stanford.edu.

    ABBREVIATIONS

The abbreviations used are: GVBD, germinal vesicle breakdown; dynein IC, cytoplasmic dynein intermediate chain; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; MAPK, mitogen-activated protein kinase; XTC-2, Xenopus tadpole cell line 2; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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