©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G2/M Transition Requires Multisite Phosphorylation of Oncoprotein 18 by Two Distinct Protein Kinase Systems (*)

Niklas Larsson , Helena Melander , Ulrica Marklund , rjan Osterman , Martin Gullberg (§)

From the (1) Department of Cell and Molecular Biology, University of Umeå, S-901 87 Umeå, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Oncoprotein 18 (Op18) is a conserved cytosolic protein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. The four residues Ser, Ser, Ser, and Ser are all subject to cell cycle-regulated phosphorylation. Ser and Ser are targets for cyclin dependent kinases (CDKs), while Ser and Ser are phosphorylated by an unidentified protein kinase. We have recently shown that induced expression of a CDK target site-deficient mutant, Op18-S25A,S38A, blocks human cell lines during G2/M transition. In the present report we show that mitosis is associated with complete phosphorylation of the two Op18 CDK target sites Ser and Ser and that Ser and Ser are also phosphorylated to a high stoichiometry. To evaluate the function of multisite phosphorylation of Op18, we expressed and analyzed the cell cycle phenotype of different kinase target site-deficient mutants. The data showed that induced expression of the S16A,S63A, S25A,S38A, and S16A,S25A,S38A,S63A mutants all resulted in an indistinguishable phenotype, i.e. immediate G2/M block and subsequent endoreduplication, a given fraction of G2 versus M-phase blocked cells, and a characteristic nuclear morphology of M-blocked cells. This result was unexpected; however, a likely explanation was provided by analysis of Op18 phosphoisomers, which revealed that mutations of the CDK sites interfere with phosphorylation of Ser and Ser. The simplest interpretation of our results is that phosphorylation of Ser and Ser is essential during G2/M transition and that the phenotype of the S25A,S38A mutant is mediated by the observed block of Ser/Ser phosphorylation.


INTRODUCTION

Progression through the cell cycle and cell division is regulated by cyclin-dependent kinases (CDKs)() in all eukaryotes (for review, see Ref. 1). The enzymatic activity of these kinases requires association with a family of regulatory subunits called cyclins (for review, see Ref. 2). In higher eukaryotes, the G1 and S-phase functions are controlled by the CDK2, CDK4, and CDK5 kinases, while the prototypic member of the CDK family, p34-cdc2, controls entry into mitosis (for review, see Refs. 3 and 4). In addition to CDKs, other protein kinases have also been implicated in cell cycle control, such as mitogen-activated protein kinases, MPM-2 epitope kinases, calcium/calmodulin kinase II, or NimA (for review, see Refs. 4 and 5). However, the specific role of these protein kinases during the cell cycle is not yet as well understood as the role of CDKs. Cell cycle control by protein kinases clearly depends on phosphorylation of specific substrates, but only a limited number of cell cycle-regulated physiological substrates have been definitively identified (for review, see Ref. 6). Moreover, even fewer protein substrates have been identified whose phosphorylation is known to be important during cell division.

Oncoprotein 18 (Op18) is a conserved cytosolic protein that has been identified in several cellular systems and studied under different names such as p19, 19K, p18, prosolin, stathmin, and Op18 (7, 8, 9, 10, 11) . This protein has evoked interest due to its up-regulated expression in various neoplasms, prompting the designation Op18 (12, 13) , and its complex pattern of phosphorylation. Many investigators have proposed a regulatory role of Op18 based on the following: (i) phosphorylation of Op18 in response to diverse extracellular signals, (ii) developmental control and differentiation-specific regulation of Op18 expression levels, and (iii) profound up-regulation of Op18 in various malignancies (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .

Previous studies have identified all four sites of Op18 that are phosphorylated in intact cells, namely Ser, Ser, Ser, and Ser(22, 23, 24, 25, 26, 27, 28) . During our search for the function of Op18, we have identified three protein kinase systems that specifically phosphorylate these residues (23, 24, 27, 29). Analysis of T-cell antigen receptor-induced phosphorylation of Op18 revealed two distinct protein kinase families that phosphorylate Ser and Ser. Our site-mapping studies performed in vivo and in vitro have shown Ser is a major target for members of the mitogen-activated protein kinase family and that Ser is a major target for the Ca/calmodulin-dependent kinase-Gr(23, 24, 27). We have also observed cell cycle-regulated fluctuations of Op18 phosphorylation on all four Ser residues that are phosphorylated in intact cells. Site-mapping studies performed in vivo and in vitro identified CDKs as the kinase system involved in cell cycle-regulated phosphorylation of Ser and Ser, but the cell cycle-regulated kinase system(s) involved in Ser and Ser phosphorylation remains to be identified (23, 29) . The analysis of Op18 phosphorylation outlined above suggests that Op18 resides at a junction where receptor and cell cycle-regulated kinase families interact with the same substrate.

To address the function of Op18 and the potential importance of its cell cycle-regulated phosphorylation, we have recently expressed a CDK target site-deficient mutant of Op18 and searched for defects on the level of cell cycle regulation (30) . The result demonstrated that Ala substitution of the two CDK sites of Op18 results in rapid accumulation of cells in the G2/M phase of the cell cycle. The block in G2 was transient, and prolonged incubation resulted in a large fraction of the transfected cells entering S-phase in the absence of mitosis, i.e. endoreduplication. In addition, a fraction of the transfected cells was blocked in mitosis. These cells appeared to be blocked in early M-phase, since they lacked the mitotic spindle, and also exhibited a serious defect during mitotic chromosome segregation. Analyses of the mechanism behind the phenotype of the mutants containing Ala substitutions of Ser and Ser suggested an essential CDK-regulated role for Op18 during cell division and that the mutant interfered with the function of the endogenous gene product (30) .

In the present study we investigated the stoichiometry of phosphorylation of the four targets of Op18 and the cell cycle phenotypes of mutants containing Ala substitutions of the phosphorylated residues. The data demonstrate that a cascade of phosphorylation on all four Ser residues results in multisite-phosphorylated Op18 and that Op18 may play a more central role in G2/M transition than previously anticipated.


MATERIALS AND METHODS

Reagents

Rabbit anti-Op18 was raised against Escherichia coli produced Op18. Anti-Op18 specific for the COOH-terminal part (amino acid residue 34-149, anti-Op18:34-149) was affinity purified on recombinant Op18 as described in detail elsewhere (19). The PC10 monoclonal anti-proliferating cell nuclear antigen was purchased from Santa Cruz Biotechnology Inc. Protein A and Protein A bound to Sepharose was purchased from Pharmacia Biotech Inc.

DNA Transfection, Suppression of the Human Metallothionein IIa (hMTIIa) Promotor, and Cell Culture Condition

Construction of mutant Op18 cDNA, where the codons for Ser (S16A), Ser (S25A), Ser (S38A), and Ser (S63A) are changed to Ala, have previously been described (23, 24, 27) . Using standard DNA manipulation techniques (31), we have combined these single point mutations to generate constructs expressing di-, tri-, and tetra- combinations of the amino acid substitutions. Construction of Op18 cDNA with the sequence encoding amino acids 4-55 deleted, designated Op18-4-55, has also been described (30) . Op18 cDNA derivatives were excised from pBluescript SK(+) (Strategene) as BamHI to HindIII fragments and cloned into the corresponding sites in the poly-linker of the episomal Epstein-Barr virus (EBV) expression vector pMEP4 (Invitrogen) (32) . The resulting plasmids allow expression of Op18 derivatives under control of the Cd-induced hMTIIa promotor. The pMEP4 shuttle vector contains the EBV origin of replication and the EBNA-1 gene to allow high copy episomal replication, and the hph gene, which confers hygromycin B resistance in mammalian cells. pCMV-EBNA (Invitrogen) contains the EBNA-1 gene under the control of the cytomegalovirus promotor, which directs transient expression of EBNA-1. To maximize the frequency of hygromycin B-resistant transfectants of the EBV-negative K562 erythroleukemia cell line, pCMV-EBNA was co-transfected with pMEP-Op18 derivatives. A detailed description of the transfection procedure has been presented elsewhere (30) . In brief, K562 cells were transfected by electroporation in the presence of 25 µg of pMEP4-Op18 derivatives, 10 µg of pCMV-EBNA, and 25 µg of pBluescript SK(+) as carrier DNA. Cells were thereafter recultured in a medium containing EDTA (50 µM), which has been specifically designed to support cell growth under conditions that minimize expression from the hMTIIa promotor (30) . Hygromycin (0.5 mg/ml; Boehringer Mannheim) was used to select for transfected cells as described (30) , and about 50-70% of all pMEP4-transfected cells surviving electroporation were resistant to the drug, while essentially all mock-transfected cells were killed within 3 or 4 days. Flow cytometric analysis of Op18 expression of transfected cells revealed that Cd (0.1 µM) induced a 5-20-fold increased ectopic expression of Op18 within 12 h. To avoid selection for fast growing clones, cells were used between 5 and 10 days post-electroporation. Flow Cytometric Analysis and [H]Thymidine Incorporation-Single parameter DNA stainings were performed with propidium iodide as described (30) . The BrdU labeling and detection kit I (Boehringer Mannheim) was used to for the detection of BrdU incorporation into cellular DNA according to the manufacturer's instructions. In brief, cells (0.5-1 10) were cultured for 24 h in the presence of BrdU (1 µM) and thereafter fixed in 70% ethanol buffered with 50 mM glycine buffer, pH 2 (-20 °C). Cells were thereafter washed and incubated with a monoclonal anti-BrdU, which was revealed by fluorescein-conjugated sheep anti-mouse immunoglobulin. Prior to analysis, cells were resuspended in phosphate-buffered saline containing 10 µg/ml propidium iodide, 0.1% Triton X-100, and 10 µg/ml RNase. DNA and BrdU dual parameter analysis was performed using a FACScan (Becton Dickinson). Evaluation of flow cytometry data was performed using the Consort 30 or FACScan software. Cellular proliferation was analyzed by culturing cells (0.2 ml/culture, triplicate cultures) in flat bottom microtiter plates in the presence of [H]thymidine (1 µCi/well) for 2 h. Cultures were thereafter precipitated on glass filter and incorporated [H]thymidine determined by liquid scintillation.

Western Blot, SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and Immunoprecipitation

Preparation of cellular proteins by cell lysis in Triton X-100 and separation of proteins by 10-20% gradient SDS-PAGE has been described (30) . Affinity-purified anti-Op18, specific for the COOH-terminal part (anti-Op18:34-149), was used for Western blot analysis and immunoprecipitation as described (30) . I-Protein A was used to reveal bound antibodies in Western blot analysis and PhosphorImager analysis of radioactive bands was used for quantification. As a control for equal loading, the relevant parts of filters were routinely probed with a monoclonal anti-proliferating cell nuclear antigen (PC10) or anti-triose-phosphate isomerase.

Separation of Op18 Phosphoisomers by Native PAGE and Tryptic P-Phosphopeptide Analysis

To separate Op18 phosphoisomers, we employed a native PAGE system that separates Op18 according to the charge differences introduced by phosphorylation (23). Unlabeled Op18 was purified prior to analysis by a small scale purification protocol (23) , and P-labeled Op18 was purified by immunoprecipitation. Cells were P-labeled by incubating HeLa cells for the indicated time in phosphate-free Dulbecco's minimum Eagle's medium (10 cells/ml, 1 ml) containing P (100 µCi/ml). Thereafter, cells were solubilized in Triton X-100 containing lysis buffer, and Op18 was immunoprecipitated using anti-Op18:34-149 as described (23) . Bound Op18 was eluted by heating the beads to 70 °C (5 min) in 40 mM Tris-glycine, pH 8.8, 10% glycerol, and 1% 2-mercaptoethanol. Eluted proteins were separated by native PAGE and electrotransferred to nitrocellulose filters. Radioactive bands were localized by autoradiography and excised. Immobilized proteins were digested with tosylphenylalanyl chloromethyl ketone-treated trypsin (Worthington, 4 7.5 µg of trypsin was added at 2-h intervals) and processed as described (33) . Phosphopeptide mapping was performed as described previously (23, 34) . PhosphorImager analysis of radioactive spots was used for quantification of phosphopeptides.


RESULTS

Site Specificity and Stoichiometry of Op18 Phosphorylation during Mitosis

A previous study has shown that mitosis is associated with multisite phosphorylation of Op18 on up to four distinct Ser residues (29) . Op18 phosphoisomers can be resolved on a native PAGE system that separates proteins according to the charge differences introduced by each phosphate group (23, 29) . This method reveals that about 90% of all Op18 molecules are nonphosphorylated in interphase cells and that the remaining 10% are phosphorylated on one site (Fig. 1A). In contrast, cells blocked in mitosis by nocodazole treatment contain Op18 that is phosphorylated on at least two sites, with most phosphoisomers phosphorylated on three or four sites. We have previously reported a similar pattern of phosphoisomers in mitotic cells recovered after ``mitotic shake off,'' which suggests that multiphosphorylation of Op18 is indeed associated with mitosis, even without nocodazole treatment (29) .


Figure 1: Site-mapping analysis of Op18 phosphoisomers in mitotic HeLa cells. A, exponentially growing HeLa cells were either left untreated (Interphase) or treated with nocodazole for 16 h (M-Block). Op18 was thereafter purified from either adherent (Interphase) or non-adherent (M-Block) cells and phosphoisomers of Op18 resolved by a native PAGE system. After transfer to nitrocellulose filter, Op18 was revealed as described under ``Materials and Methods.'' The arrows indicate nonphosphorylated Op18 (non-P) as well as migration of Op18 with various numbers of phosphate groups (1-P, 2-P, 3-P, and 4-P). B-F, interphase or M-blocked HeLa cells were labeled with [P]orthophosphate for 16 h, Op18-purified, resolved by a native PAGE, and transferred to a nitrocellulose filter. The indicated Op18 phosphoisomers were digested with trypsin, and phosphopeptides were resolved by two-dimensional separation on thin layer cellulose plates. The electrophoretic dimension (pH 8.9, cathode right), the chromatography dimension, and the location of sample application (+) are shown. Each plate was loaded with approximately 200 cpm and exposed for 14 days. The previously identified Ser (S16), Ser (S25), Ser/Ser (S16/25), Ser (S38), and Ser (S63) phosphopeptides are shown in panel C. S16`, S38`, and S16/S25` represent partially cleaved phosphopeptides. Results shown are representative for two independent experiments.



Op18 is phosphorylated during mitosis by specific CDKs, most likely by p34-cdc2 and p33-cdk2 on Ser and Ser, and on Ser and Ser by an as yet unidentified protein kinase (29) . The stoichiometry of Op18 phosphorylation by these two kinase systems during mitosis was determined by site-mapping of distinct phosphoisomers. Accordingly, P-labeled Op18 phosphoisomers from interphase and M-blocked HeLa cells (Fig. 1A) were separated by native PAGE and subjected to tryptic phosphopeptide mapping. Previous studies have identified all potential tryptic phosphopeptides of Op18, as well as a diphosphorylated peptide containing both Ser and Ser (Fig. 1C) (23, 24, 27) . Since Ser phosphorylation interferes with trypsin cleavage between amino acids 14 and 15, the Ser and Ser peptides migrate differently in the electrophoretic dimension, which allows independent identification of Ser and Ser phosphorylation (Fig. 1C) (24) . Fig. 1B shows that monophosphorylated Op18 derived from interphase cells contains phosphopeptides corresponding to all four previously mapped Ser residues. PhosphorImager analysis of the plate reveals that Ser contains 21%, Ser contains 6%, Ser contains 65%, and Ser contains about 8% of the total radioactivity. M-blocked cells do not contain monophosphorylated Op18, but interestingly, the diphosphorylated Op18 phosphoisomers contain only the Ser and Ser peptides (Fig. 1D). As expected, the Ser and Ser peptides derived from diphosphorylated Op18 contain a similar amount of radioactivity on their two CDK target sites. Analysis of the tri- and tetraphosphorylated Op18 species reveals phosphorylation of all four previously identified Ser residues. As shown in Fig. 1E, the Ser peptide is absent in triphosphorylated Op18, demonstrating that Ser is phosphorylated to completion in triphosphorylated Op18, since the Ser peptide is replaced by the Ser/Ser peptide. Finally, as can be predicted for tetraphosphorylated Op18, the S25 peptide is absent, since all Op18 is phosphorylated on both Ser and Ser, which results in the Ser/Ser peptide. Hence, site mapping of all phosphoisomers of Op18 shows that the two CDK target sites, Ser and Ser, are phosphorylated to completion during mitosis. Moreover, quantification of Ser and Ser phosphopeptides shows that both are phosphorylated to a high stoichiometry, but analysis of triphosphorylated Op18 reveals that the Ser contains more radioactivity than Ser (taking into account that half of the radioactivity of the Ser/Ser peptide is on Ser; Ser contains 14% and Ser contains 23% of the total radioactivity).

Analysis of the Phenotype of Kinase Target Site-deficient Mutants of Op18

As shown above and in a previous study, phosphorylations of four distinct sites of Op18 are subject to dramatic cell cycle-dependent fluctuations (29) . To address the potential function of these phosphorylation events, we constructed and expressed Op18 mutants containing Ala substitutions of each of the phosphorylated Ser residues. Previous experiments have shown that mutations of Ser and Ser block cell growth, and this prompted us to optimize a system allowing regulated expression of cDNA inserts cloned into the episomal EBV-based vector pMEP4 (30) . The hMTIIa promotor of this vector can be suppressed by nontoxic levels of EDTA, while high level expression can be induced by Cd (see ``Materials and Methods'' and Fig. 2). Increased expression of Op18 in Cd-treated cells transfected with either pMEP-Op18-wt or Ala-substituted derivatives thereof are shown by Western blot analysis in Fig. 2, leftpanel. PhosphorImager analysis of the results reveals that EDTA-suppressed transfected cells express about the same amount of recombinant Op18 as the endogenous gene product, which is 1100 ng/mg total cell proteins in the K562 erythroleukemia cell line. Moreover, Cd induction results in expression levels that are more than 10-fold higher than the endogenous gene product. These results also show that the introduced mutations do not alter the expression levels of the proteins.


Figure 2: Regulated ectopic expression of wild type and mutated Op18. K562 cells were transfected with the indicated pMEP4-Op18 constructs and ``stable'' transfectants were selected by cultivation with hygromycin and EDTA for 6 days as described under ``Materials and Methods.'' Cells were thereafter cultured for 24 h with EDTA (50 µM) (rightpanels) or Cd (0.1 µM) (leftpanels). Ectopic expression of Op18 was analyzed by Western blot analysis using anti-Op18:34-149 and I-Protein A. The positions of Op18 migrating at 19 kDa and a triphosphorylated (Refs. 25 and 38) form of Op18 migrating at 23 kDa is indicated. An autoradiograph exposed for 24 h is shown. As a control for equal loading the same filter was also probed with the PC10 monoclonal antibody (upperpanels). S25,38A, S25A,S38A; S16,63A, S16A,S63A; S16,25,38,63A, S16A,S25A,S38A,S63A.



Substitution of the CDK target sites Ser and Ser with Ala results in a dominant Op18 mutant that blocks cell division (30) . The phenotype of these Op18 mutants, i.e. a G2/M block followed by endoreduplication, was observed in both the K562 cell line and the BL-42 Burkitt's lymphoma cell line. To delineate the potential importance of Ser and Ser-specific phosphorylation, we analyzed DNA synthesis and the cell cycle distribution of K562 cells transfected with various Op18 derivatives. The results of this analysis are shown in Fig. 3. Only minor effects of the pMEP-Op18 constructs were observed in the presence of EDTA, and as expected, the cell cycle profile of the vector Co-transfected cells is not altered after addition of Cd. In agreement with our previous study, high level expression of Op18-wt results in only minor alterations of the cell cycle profile. However, addition of Cd to cells expressing either the S16A,S63A, S25A,S38A, or S16A,S25A,S38A,S63A Op18 mutants resulted in a drastic decrease in DNA synthesis and accumulation of cells in the G2/M phase of the cell cycle within 24 h. Most importantly, it appears that expression of the S16A,S63A mutant is as efficient at causing a G2/M block as the previously characterized CDK target site-deficient S25A,S38A mutant. Moreover, the similarities between the S16A,S63A, S25A,S38A, and S16A,S25A,S38A,S63A Op18 mutants are also evident at the 72-h time point, since a pronounced endoreduplication response is observed with all these mutants (Fig. 3, rightpanels).


Figure 3: Mutation of the ``non-CDK'' phosphorylation sites Ser and Ser results in a transient G2 block followed by endoreduplication. K562 cells were transfected with the indicated pMEP4-constructs, and hygromycin-resistant cell lines were selected as in Fig. 2. Cells were thereafter cultured in the presence of EDTA (50 µM), in medium alone (None), or in Cd (0.1 µM), as indicated. [H]Thymidine incorporation of cells cultured for 24 h (2 10 cells/well), using the indicated culture conditions, are presented as percentage of incorporation in the presence of EDTA. It should be noted that cells transfected with mutated Op18 routinely incorporated 20-30% less [H]thymidine per cell in the presence of EDTA, as compared with cells transfected with vector Co or Op18-wt. DNA profiles of cells cultivated for the indicated time with EDTA or Cd are also shown. The results are representative for six inde-pendent experiments. Op18-S25,38A, Op18-S25A,S38A; Op18-S16,63A, Op18-S16A,S63A; Op18-S16,25,38,63A, S16A,S25A,S38A,S63A.



To delineate the importance of Serversus Ser phosphorylation, the effect of single Ala substitutions at these sites was investigated. The results in Fig. 4show that expression of both wild type Op18 and the S16A mutant have only minor effects on the cell cycle profile, while expression of the S63A mutant caused a more pronounced inhibition of DNA synthesis and a substantial G2/M block. However, although the S63A mutation alone exhibits a pronounced phenotype, the result reveals that the phenotype of the S16A,S63A mutant is even more dramatic at both the 24- and 72-h time points. The differences between the S16A and S63A mutants outlined in Fig. 4 were reproducible, and Western blot analysis revealed that both mutants were expressed at equal levels (data not shown).


Figure 4: The phenotype of Op18 mutants with single site substitutions of Ser and Ser. [H]Thymidine incorporation and DNA profile of K562 cells expressing the indicated pMEP4 construct were assessed as in Fig. 3. Op18-S16,63A, Op18-S16A,S63A.



Analysis of Cell Cycle Dynamics Suggests That both the S16A,S63A and S25A,S38A Op18 Mutants Cause an Immediate G2/M Block

The experiments outlined above show that expression of the S16A,S63A mutant results in rapid accumulation of cells with G2/M content of DNA, followed by endoreduplication, and this phenotype appears similar to the one caused by the CDK target site-deficient S25A,S38A mutant. To further analyze cell cycle dynamics and to search for potential phenotypic differences between Op18 mutants we analyzed incorporation of BrdU during the entire 20-h period of induced expression of Ala-substituted Op18 derivatives. Subsequent dual parameter flow-cytometric analysis of BrdU incorporation and DNA content allowed identification of cycling and noncycling cells in various stages of the cell cycle. The result in Fig. 5A shows that most cells expressing wild type Op18 incorporates BrdU during the 20-h period and that the minor population of BrdU-negative cells have a G1 content of DNA. In contrast, cells expressing all the indicated Ser to Ala-substituted Op18 derivatives contain BrdU-negative cell populations with both G1 and G2/M content of DNA. Most importantly, the results clearly show that all cycling cells are blocked in G2/M by expression of these Op18 mutants. It seems reasonable to assume that the BrdU-negative cell populations with G2 content of DNA represent cells that were in the G2 phase at the time of Cd addition and that these cells were immediately blocked by the mutant Op18 protein. This interpretation agrees with the rapid kinetics of Cd-induced expression of the hMTIIa promotor, which result in elevated Op18 levels within a few hours of induction (data not shown). In conclusion, the result in Fig. 5 shows that expression of all kinase target site-deficient mutants tested results in a more or less immediate block in cell division, and this block seems independent of expression of the mutants during the preceding S-phase. Moreover, it appears that the phenotypes of the S16A,S63A and the CDK target site-deficient S25A,S38A mutants are identical. The observed phenotype of the S16A,S25A,S38A,S63A mutant is in line with this interpretation, since mutation of all four available phosphorylation sites results in a phenotype indistinguishable from the other mutants.


Figure 5: Analysis of BrdU incorporation in cells expressing Op18 ``kinase site-deficient'' mutants reveals an immediate block in cell division. K562 cells were transfected with the indicated pMEP4 constructs, and hygromycin-resistant cell lines were selected as in Fig. 2. Op18 expression was thereafter induced with Cd in the presence of BrdU for 20 h. Cell fixation, staining with anti-BrdU, and flow cytometric dual parameter analyses of BrdU incorporation versus DNA content are described under ``Materials and Methods.'' The results are representative for two independent experiments. S25,38A, S25A,S38A; S16,63A, S16A,S63A; S16,25,38,63A, S16A,S25A,S38A,S63A.



Previous morphological studies of cells transfected with the Op18-S25A,S38A mutant and induced with Cd for 24 h revealed that about two-thirds of the cells appeared as normal healthy G2 cells (30) . The remaining cells appeared as mitotic cells, lacking the nuclear envelope but without signs of spindle formation. Transmission electron micrographs revealed various degrees of aberrant chromosome aggregations, which appeared as compacted nuclei in light microscopy (30) . To compare the morphology of cells expressing the S25A,S38A, S16A,S63A, and S16A,S25A,S38A,S63A Op18 mutants, respectively, we analyzed May-Grunwald-Giemsa-stained cytocentrifuge preparations of transfected cells. The results in show that expression of all these Op18 mutants resulted in essentially the same frequency of cells blocked in M-phase and of mitotic cells with a compacted nucleus. Thus, morphological examination of G2/M-blocked cells also suggest that the phenotypes of the S25A,S38A, S16A,S63A, and S16A,S25A,S38A,S63A Op18 mutants are the same.

Deletion of an N-terminal Region of Op18 Abolishes the Phenotype of the S63A Mutant

The phenotype of kinase target sites mutants is most likely due to the absence of a phosphate group(s) but may also, in some cases, be due to the creation of a ``pseudo-substrate site'' or some other alteration that interferes with specific kinase systems. To address these questions we expressed both full-length and N-terminal deleted Op18 derivatives with or without the S63A mutation. Fig. 6A shows high level expression of all these derivatives in Cd-induced cells (lanesd and e), as compared with the endogenous gene product and that the N-terminal deleted Op18 proteins are expressed at almost the same levels as the full-length Op18 protein (lanesb and c). Ala substitution of Ser, but not Ser, is sufficient to induce a substantial G2/M block (see above). To determine if this phenotype is resistant to an N-terminal deletion, the cell cycle profile of cells expressing Op18-4-55 was compared with that of cells expressing Op18-4-55/S63A. The result in Fig. 6, B and C, shows that expression of Op18-4-55/S63A does not alter the DNA distribution compared with vector Co or Op18-4-55. For comparison, we also included Op18-wt- and Op18-S63A-transfected cells in this experiment. As expected, the S63A mutation caused a profound G2/M block as compared with the wild type construct. Thus, the G2/M block caused by the S63A mutation requires the N-terminal region of Op18, which suggests that a region distal to the mutation is critical for the observed phenotype.


Figure 6: The phenotype of the S63A mutant requires the N-terminal part of Op18. K562 cells were transfected with the indicated pMEP4-Op18 constructs, and hygromycin-resistant cell lines were selected as in Fig. 2. Op18 expression was thereafter induced with Cd for 24 h. A, ectopic expression of Op18 was analyzed by Western blot analysis as in Fig. 2 (the minor band migrating below full-length Op18 (lanesb and c) is due to proteolytic cleavage). The positions of Op18 migrating at 19, 23, and 15 kDa (4-55) are indicated. As a control for equal loading, the same filter was also probed with the PC10 monoclonal antibody (upperpanel). Phosphorylation of the deleted Op18 proteins was analyzed by P labeling of cells followed by immunoprecipitation. As anticipated, while the Op18-4-55 protein incorporated the expected amount of [P] label, the Op18-4-55/S63A protein was completely nonphosphorylated (data not shown). DNA profiles of transfected cells cultivated for 24 (B) and 72 h (C) with Cd are shown.



The S25A,S38A Mutation Interferes with Phosphorylation of Ser and Ser

Since the G2/M transition is associated with multisite phosphorylation of Op18 by two distinct kinase systems, it is possible that mutations of some sites interfere with phosphorylation of others. To test this idea, we resolved Op18 phosphoisomers by native PAGE from Cd-induced cells transfected with Op18-wt, Op18-S25A,S38A, Op18-S16A,S63A, or Op18-S16A,S25A,S38A,S63A (Fig. 7). The resulting blot of Op18 phosphoisomers reveal that overexpression of the wild type protein results in a substantial increase of the fraction of monophosphorylated Op18 as compared with phosphorylation of the endogenous gene product (see Fig. 1A and Ref. 29). This finding is striking, but the low levels of multisite-phosphorylated Op18 suggest that the phenomenon is not a result of the minor alteration of the cell cycle profile caused by overexpression of the wild type protein.


Figure 7: Analysis of Op18 phosphoisomers in Op18-transfected cells. K562 cells were transfected with the indicated pMEP-Op18 constructs and hygromycin-resistant cell lines were selected as in Fig. 2. Cells were thereafter cultured in the presence of Cd (0.1 µM) for 24 h, and phosphoisomers of Op18 were analyzed by separation on native PAGE as in Fig. 1A. Only the recombinant Op18 gene product is visualized on the presented autoradiograph, since the transfected Op18 derivatives are expressed at 10-20-fold higher levels than the endogenous Op18 protein. S25,38A, S25A,S38A; S16,63A, S16A,S63A; S16,25,38,63A, S16A,S25A,S38A,S63A; non-P, nonphosphorylated.



Cells expressing the S25A,S38A, S16A,S63A, or S16A,S25A,S38A,S63A Op18 mutants for 24 h all have G2/M content of DNA. Analysis of the Op18 phosphoisomers derived from these mutants reveals profound differences in the stoichiometry and distribution of phosphoisomers. First, the S16A,S25A,S38A,S63A protein is not phosphorylated at all, as could be anticipated. Second and most importantly, while the S25A,S38A protein is mainly nonphosphorylated, a major fraction of S16A,S63A protein is phosphorylated on both of its remaining Ser and Ser sites, as revealed by the predominant species of diphosphorylated protein. Induced expression of these Op18 mutants blocks between 65 and 80% of all cells in G2, and the remaining cells are blocked in mitosis ( and Ref. 30). Hence, the presence of mitotic cells readily explains diphosphorylation on the two non-mutated sites of the S16A,S63A mutant. However, it was unexpected that the diphosphorylated isomer is absent in Op-18-S25A,S38A-expressing cells, since diphosphorylation of Ser and Ser is prominent in nontransfected mitotic cells (Fig. 1). These results suggest that mutation of the CDK sites interferes with phosphorylation of Ser and Ser. It follows that the observed phenotype of the S25A,S38A Op18 mutant may be caused by interference with Ser and Ser phosphorylation, which would explain the similarity of the phenotypes of the S16A,S63A and S25A,S38A Op18 mutants.


DISCUSSION

Op18 has been studied by several groups due to its rapid phosphorylation in response to a stimulation of a variety of receptor systems (7, 8, 9, 14) and dramatic up-regulation in various neoplasms (12, 13, 18, 19, 20, 21) . The potential function of Op18 during receptor signaling is still unknown, but our present and previous study present genetic evidence for an essential function of this protein during cell division (30). Hence, our data have revealed that mutation of the phosphorylated Ser residues, which are all subject to cell cycle-regulated fluctuations, results in dominant Op18 mutants that block cell division. As argued in a previous study, the CDK site-deficient Op18 mutant is likely to mediate its phenotype by interfering with the function of the endogenous gene product, i.e. Op18-S25A,S38A is a dominant negative mutant (30) . This conclusion was based on the observation that antisense mRNA-mediated suppression of Op18 expression results in a G2/M block similar to Op18-S25A,S38A-expressing cells and that the phenotype of the S25A,S38A mutant was resistant to an extensive COOH-terminal deletion. Moreover, it was also shown that the Op18 mutant did not act by sequestering of the endogenous gene product, which suggests that the mutant acts by sequestering a putative interacting protein. The minor, but still significant, phenotype observed by overexpression of wild type Op18 is also in line with a sequestering mechanism as previously discussed (30) , since overexpression of the wild type may result in a fraction of Op18 that is nonphosphorylated on critical sites.

We have observed cell cycle-regulated phosphorylation on all four residues of Op18 that are phosphorylated in intact cells and identified CDKs as one of the kinase systems involved (23, 29) . The evidence that Ser and Ser are physiological substrates for at least some members of the CDK family includes the following: (i) S-phase progression is associated with increased phosphorylation of these residues, which reaches its peak during mitosis ( Fig. 1and Ref. 29); and (ii) both Ser and Ser contain CDK consensus phosphorylation sites and are efficiently phosphorylated in vitro by at least two of the members of the CDK family, namely p34-cdc2 and p33-cdk2 (23, 29) . Hence, Ser and Ser are likely physiological targets for CDKs. However, two lines of evidence exclude CDKs as the kinases phosphorylating Ser and Ser; first, these sites are not phosphorylated by the CDKs tested in vitro (e.g. p34-cdc2-, p33-cdk2-, and p13-suc1-precipitated kinases) (23, 29) . Second, the sequences surrounding Ser (KRASGQA) and Ser (RRKSHEA) lack the Pro residues and the other features that are essential for phosphorylation by CDKs (6, 35) . Thus, the kinase system(s) involved in cell cycle-regulated Ser and Ser phosphorylation is still unknown, but there are many possible candidates, e.g. MPM-2 epitope kinases, calcium/calmodulin kinase II, or NimA (4, 5, 36, 37, 38) .

The present report extends our previous studies of cell cycle-regulated phosphorylation of Op18 (29, 30) . First, site-mapping analysis of all phosphoisomers present in mitotic cells demonstrates that both of the CDK target sites, Ser and Ser, are phosphorylated to completion. Second, a major fraction of Op18 is tri- or tetraphosphorylated with Ser phosphorylated to a somewhat higher stoichiometry than Ser during mitosis. Third, phosphorylation of Ser and Ser as well as the CDK sites is shown to be functionally important for cell division. Our previous study of CDK site-deficient mutants of Op18 revealed a clear cut dominant phenotype of single site mutations of Ser and Ser and an even more pronounced phenotype of a double mutant. This study shows that the S16A mutant has only minor effects on the cell cycle profile as compared with wild type Op18, while expression of the S63A mutant caused a pronounced inhibition of DNA synthesis and a substantial G2/M block (Fig. 4). Interestingly, the combination of the S16A and S63A mutations resulted in a more dramatic phenotype that was indistinguishable from that of the S25A,S38A mutant. Thus, these genetic data suggest that all four kinase target sites of Op18 are functionally important during cell division, although the S16A mutation alone has an almost undetectable phenotype.

A possible concern in interpreting the results from overexpression of kinase target site mutants of Op18 is that such mutants may interfere with specific kinase systems, e.g. by acting as a pseudo-substrate site. In the case of the S25A,S38A mutant, in vitro phosphorylation assays have shown that Ala substitutions of Ser or Ser do not interfere with CDK-mediated phosphorylation of the alternative Op18 sites (29) , nor have we detected inhibition of CDK phosphorylation of histone H1 in vitro in the presence of the Op18-S25A,S38A mutant protein (data not shown). In the present study we analyzed the phenotype of N-terminal deleted Op18 derivatives, with or without the S63A mutation (Fig. 6A). The result showed that the phenotype of the S63A mutation requires the N-terminal region of Op18. Since a region distal to the mutation is critical for the phenotype, we assume that the phenotype is not caused by generation of a putative inhibitory pseudo-substrate site. This interpretation is in line with our observation that single site mutations of at least three distal cell cycle-regulated phosphorylation sites (Ser, Ser, and Ser) are sufficient to cause a profound phenotype on the level of cell cycle regulation, i.e. G2/M block followed by endoreduplication.

As outlined above, multisite phosphorylation of Op18 during mitosis involves CDKs and a distinct but as yet unidentified protein kinase system. Initially it was surprising that mutations of the target sites for these protein kinases result in an identical phenotype on all levels tested. However, analysis of Op18 phosphoisomers by native PAGE revealed that Ser and Ser were only weakly phosphorylated in cells expressing the S25A,S38A CDK sites mutant, while cells expressing the S16A,S63A mutant contained a large fraction of Op18 diphosphorylated on the Ser and Ser CDK sites (Fig. 7). Cells blocked at G2/M by either of these mutants contain about 25% of M-blocked cells (). Therefore, multisite phosphorylation would be predicted on the intact sites of the mutated protein. While this was the case for cells expressing the S16A,S63A mutant, it was not for the S25A,S38A mutant. Hence, mutation of the CDK sites Ser and Ser interferes with phosphorylation of Ser and Ser in transfected cells with the consequence that the S25A,S38A as well as the S16A,S63A mutated protein lacks diphosphorylation on Ser and Ser. It follows that the expressed S25A,S38A protein is identical to the S16A,S25A,S38A,S63A protein with respect to the complete absence of multiphosphorylation, which may explain the identical phenotype. The S16A,S63A protein is heavily phosphorylated on its intact CDK target sites, but this phosphorylation does not seem to influence the observed phenotype, which appears identical to that of the S25A,S38A and S16A,S25A,S38A,S63A mutants. The simplest interpretation of these observations is that phosphorylation of Ser and Ser is essential during a point at G2/M transition and that the observed phenotype of the S25A,S38A CDK site-deficient mutant is caused by interference with multiphosphorylation of Ser and Ser.

Deficiency of the two CDK target sites of Op18 may interfere with phosphorylation of Ser and Ser by at least two mechanisms. First, CDK dependent prephosphorylation on Ser and Ser may be a prerequisite for Ser and Ser phosphorylation during G2/M transition. Second, phosphorylation on the CDK sites may by involved in the activation of the unidentified Op18 Ser and Ser kinase. Discrimination between these two alternatives has to await the future identification of the protein kinase(s) responsible. However, the finding that cell division requires phosphorylation of Op18 by at least two distinct protein kinase systems suggests that Op18 is directly involved in some aspect of G2/M transition. Such an involvement is consistent with the rapid block in cell division caused by the kinase target site-deficient mutants and the finding that this block does not require expression of the mutants during the preceding S-phase (Fig. 5). Since both CDK target sites are phosphorylated to completion in mitotic cells, it is tempting to speculate that CDKs have an on/off function that allows phosphorylation of Ser and Ser, while the Ser/Ser kinase(s) may mediate the phosphorylation event that is essential for G2/M transition. If this is the case, the observation that the S25A,S38A, S16A,S63A, or S16A,S25A,S38A,S63A Op18 mutants all result in an identical phenotype would be predicted since all are defective in multisite phosphorylation on Ser and Ser.

Most previous studies on the Op18 protein have been concerned with phosphorylation in response to extracellular signals. As outlined in the introduction section, both Ser and Ser are clearly substrates for receptor-regulated kinase systems, but the role(s) of these phosphorylation events is still obscure. The genetic evidence in this and our previous study (30) demonstrates the importance of a complex multisite phosphorylation of Op18 during G2/M transition. A distinct type of multisite phosphorylation is observed after triggering of the T-cell antigen receptor, involving increased phosphorylation of Ser and Ser but not of Ser and Ser(24, 27) . Thus, it seems that a plethora of protein kinases converge on Op18. An important extension of the studies on Op18 will be to determine if phosphorylation of this protein is of functional importance on other levels of cell regulation besides G2/M transition.

  
Table: M-phase frequency and nuclear morphology in cells expressing various kinase site-deficient mutants of Op18



FOOTNOTES

*
This work was supported by the Swedish Natural Science Research Council, Lion's Cancer Research Foundation, University of Umeå (LP 797/91), the Swedish Society for Medical Research, and the Foundation for Medical Research at the University of Umeå. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: CDK, cyclin-dependent kinase; BrdU, 5-bromo-2`-deoxyuridine; EBV, Epstein-Barr virus; hMTIIa promotor, human metallothionein IIa promotor; Op18, oncoprotein 18; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Victoria Shingler for critical reading of the manuscript.


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