M Phase Phosphorylation of Cytoplasmic Dynein Intermediate Chain and p150Glued *

To understand how the dramatic cell biological changes of oocyte maturation are brought about, we have begun to identify proteins whose phosphorylation state changes duringXenopus 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.

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. p150 Glued

, 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 p150 Glued 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 G 2 /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 p150
Glued phosphorylation may positively regulate mitotic processes, such as spindle assembly or orientation, or negatively regulate interphase processes such as minus-end-directed organelle trafficking.
Fully grown (stage VI) Xenopus oocytes are arrested in a G 2 -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.
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 p150 Glued subunit of dynactin, because of the physical and functional association between dynein and dynactin (23)(24)(25)(26)(27)(28). We found that p150 Glued 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 p150 Glued 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 G 2 /M transitions of both meiosis and mitosis.

EXPERIMENTAL PROCEDURES
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 g/ml) in modified Barth's saline containing Ca 2ϩ 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 l of ice-cold extract buffer (250 mM sucrose, 100 mM NaCl, 2.5 mM MgCl 2 , 20 mM HEPES, pH 7.2) containing 0.5 mM Na 3 VO 4 and protease inhibitors (10 g/ml leupeptin, 10 g/ml pepstatin, 10 g/ml chymostatin, 10 g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) and lysed by pipetting through a 200-l 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 Ϫ80°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-G 2 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 10ϫ 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-m 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. 32 P Labeling in Vivo-For in vivo 32 P labeling, groups of 50 oocytes were placed in a 12-well plate containing 2 mCi of [ 32 P]orthophosphate/ ml in modified Barth's saline containing Ca 2ϩ and bovine serum albumin as described above and incubated with or without progesterone (5 g/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.
-Phosphatase Treatment-Dynein IC or p150 Glued immunoprecipi-tates were incubated with 400 units of -phosphatase (New England BioLabs) in -phosphatase buffer at 30°C for 30 min. This was followed by adding an additional 400 units of -phosphatase for a further 60 min at 30°C. Phosphoamino Acid Analysis-32 P-Labeled lysates were subjected to immunoprecipitation with dynein or p150 Glued antibodies followed by SDS-PAGE and transfer to Immobilon P membranes as described above. Dynein IC and p150 Glued 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.

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 ϳ42-kDa band corresponding to p42 MAP kinase, which appeared just prior to GVBD, and a ϳ33-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. Here we shall focus on p83, which migrated as a doublet in G 2 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 G 2 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. G 2 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).
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).
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. G 2 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 G 2 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.
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 G 2 and M phase lysates. Immunoprecipitates were then treated with extract buffer, -phosphatase (a dual specificity phosphatase), or -phosphatase plus vanadate (an inhibitor of -phosphatase), and the products were subjected to electrophoresis and dynein IC immunoblotting. G 2 phase dynein IC migrated as a doublet (Fig. 5A 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 [ 32 P]orthophosphate in vivo, subjected to immunoprecipitation, and transferred to a blotting membrane. Radiolabel was found to be incorporated into G 2 phase dynein IC (both bands) and M phase dynein IC, and the overall level of labeling of G 2 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 G 2 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 -phosphatase. The -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).
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  (lanes 1 and 4), -phosphatase (lanes 2 and 5), or -phosphatase plus Na 3 VO 4 (lanes 3 and 6). the G 2 /meiosis 1 transition during oocyte maturation, and then remains constitutively hyperphosphorylated throughout the remainder of meiosis, mitosis, and early embryogenesis.
p150 Glued Undergoes Cell Cycle-dependent Phosphorylation Changes-Dynein IC physically interacts with the p150 Glued component of dynactin complex (27,28). Since dynein IC underwent cell cycle-dependent hyperphosphorylation during oocyte maturation, we set out to determine whether p150 Glued did as well. As shown in Fig. 8A, p150 Glued progressively shifted from a lower apparent molecular weight band to a higher one during oocyte maturation. The mobility shift was due to hyperphosphorylation, since -phosphatase treatment caused M phase p150 Glued to shift from the higher band to the lower one (Fig. 7A). In vivo 32 P labeling and phosphoamino acid analysis of p150 Glued showed that G 2 phase p150 Glued was essentially nonphosphorylated, and M phase p150 Glued was phosphorylated predominantly at serine (Fig. 7B). Like dynein IC, p150 Glued hyperphosphorylation persisted throughout meiosis and early embryogenesis (Fig. 8, A, B, and D), and p150 Glued became hyperphosphorylated in Mos-, okadaic acid-, and Cdc2cyclin B-treated G 2 phase oocyte extracts (although less rapidly and less completely than did dynein IC; Fig. 8C versus Fig. 4A). Okadaic acid also caused p150 Glued 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).
Mitotic Phosphorylation of Dynein IC and p150 Glued in XTC-2 Cells and HeLa Cells-Finally, we assessed whether dynein IC and p150 Glued 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).
p150 Glued migrated as two bands in interphase XTC-2 cells, with most of the p150 Glued found in the lower band (Fig. 9).  1 and 4), -phosphatase (lanes 2 and 5), or -phosphatase plus Na 3 VO 4 (lanes 3 and 6). Samples were then analyzed by the SDS-PAGE followed by immunoblotting with p150 Glued antiserum. B, phosphoamino acid analysis of in vivo labeled p150 Glued . Stage VI oocytes, treated with or without progesterone, were metabolically labeled in vivo with [ 32 P]orthophosphate. Labeled oocytes were lysed and immunoprecipitated with p150 Glued antiserum. Two-dimensional phosphoamino acid analyses of 32  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 (ϳ500 nuclei/l) 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 ϭ 0), and stage X embryos are also shown. Cleavages occurred at 90, 125, and 155 min.
Most of the p150 Glued 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 p150 Glued and dynein IC become hyperphosphorylated during mitosis in XTC-2 and HeLa cells. DISCUSSION 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 -phosphatase treatment (Fig. 5A), indicating that it represents a hyperphosphorylated form. Both bands are phosphorylated predominantly at serine; no phosphotyrosine is de-tected by in vivo labeling and phosphoamino acid analysis (Fig.  5B). Moreover, the antiphosphotyrosine reactivity of dynein IC is not diminished by -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 G 2 /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 p150 Glued component of dynactin, we examined p150 Glued phosphorylation as well. We found that p150 Glued , 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, p150 Glued is another novel M phase phosphoprotein and undergoes phosphorylation changes similar to those of its binding partner dynein IC. FIG. 8. p150 Glued phosphorylation during Xenopus oocyte maturation, in cycling egg extracts, in G 2 phase oocyte extracts, and during early embryogenesis. A, p150 Glued 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 p150 Glued antiserum. The percentage of oocytes with a white dot is shown below the blot. B, p150 Glued 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 (ϳ500 nuclei/l) was added to the cycling extract to allow monitoring of cell cycle progression. G 2 phase and M phase lysates are included for comparison. C, effects of Mos, Cdc2-cyclin B, and okadaic acid on p150 Glued phosphorylation. Cell-free Xenopus extracts were treated with extract buffer, Mos (40 nM), okadaic acid (5 M), or Cdc2-cyclin B (50 units/l). Samples were taken at the indicated times and subjected to immunoblotting with p150 Glued antiserum. D, p150 Glued phosphorylation in fertilized Xenopus eggs. Eggs were fertilized and dejellied, and samples were taken at various times after fertilization. Unfertilized eggs (t ϭ 0) and stage X embryos are also shown. Cleavages occurred at 90, 120, and 140 min. Phosphorylation of Dynein IC and p150 Glued 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). p150 Glued 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 p150 Glued undergo marked changes in their phosphorylation during meiotic and mitotic M phases. This suggests the hypothesis that dynein IC and p150 Glued phosphorylation contribute to the dramatic changes in microtubule function that occur at the G 2 /M transition. The demonstration that dynein and p150 Glued (23,(41)(42)(43). Dynein can transport cargo along microtubules toward the minus-end. Dynein IC can directly interact with the p150 Glued 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 G 2 /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 dyneinand 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 p150 Glued 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 p150 Glued might be involved in shutting down interphase processes that depend upon dynein and dynactin.