Mitotic Phosphorylation of the Lamin B Receptor by a Serine/Arginine Kinase and p34cdc2*

(Received for publication, September 15, 1996, and in revised form, November 5, 1996)

Eleni Nikolakaki Dagger , Juergen Meier §, George Simos §par , Spyros D. Georgatos §** and Thomas Giannakouros Dagger Dagger Dagger

From the Dagger  Laboratory of Biochemistry, School of Chemistry, The Aristotelian University of Thessaloniki, Thessaloniki 54 006, Greece, § Programme of Cell Biology, European Molecular Biology Laboratory, 69 017 Heidelberg, Germany, and ** Department of Basic Sciences, Faculty of Medicine, The University of Crete, Heraclion 71 110, Crete, Greece

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that is modified at interphase by a nuclear envelope-bound protein kinase. This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH2-terminal domain of LBR and regulates its interactions with other nuclear envelope proteins. To compare the phosphorylation state of LBR during interphase and mitosis, we performed phosphopeptide mapping of in vitro and in vivo 32P-labeled LBR and analyzed a series of recombinant proteins and synthetic peptides. Our results show that LBR undergoes two types of mitotic phosphorylation mediated by the RS and the p34cdc2 protein kinases, respectively. The RS kinase modifies similar sites at interphase and mitosis (i.e. Ser76, Ser78, Ser80, Ser82, Ser84), whereas p34cdc2 mainly phosphorylates Ser71. These findings clarify the phosphorylation state of LBR during the cell cycle and provide new information for understanding the mechanisms responsible for nuclear envelope assembly and disassembly.


INTRODUCTION

The nuclear lamina is a filamentous meshwork underlying the inner nuclear membrane (1, 2). In most cells this structure is a heteropolymer of type A and B lamins (3) linked to the inner nuclear membrane through integral membrane proteins. These lamin-binding proteins include the lamin B receptor (LBR1 or p58; Ref. 4) and the lamina-associated polypeptides (5).

LBR possesses a long, hydrophilic NH2-terminal domain protruding into the nucleoplasm, eight hydrophobic segments that are predicted to span the membrane, and a hydrophilic COOH-terminal domain (6, 7). The NH2-terminal domain of LBR contains distinct sites for protein kinase A and p34cdc2 kinase phosphorylation (8, 9) as well as a stretch rich in arginine-serine (RS) motifs (10). The RS motifs are specifically modified by a protein kinase that co-isolates with LBR and is part of a multimeric complex (8, 10). This LBR complex also includes the nuclear lamins and three polypeptides with molecular masses of 18 (p18), 150 (p150), and 34 (p34/p32) kDa, respectively (for pertinent information see Refs. 8, 10, and 12). The latter protein has been shown to interact with the splicing factor 2 (SF2) as well as with the HIV-1 proteins Rev and Tat (13-15). Phosphorylation of LBR by the RS kinase completely abolishes binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex (11).

At the onset of mitosis, the structure of the nuclear envelope is dramatically altered. The nuclear lamina depolymerizes as a result of hyperphosphorylation of the nuclear lamins at specific sites involved in lamin-lamin (16), lamin-chromatin (17), and lamin-membrane (5) interactions. Following depolymerization, the bulk of type A lamins disperse in the cytoplasm, whereas type B lamins remain bound to remnants of the nuclear envelope. At the same time, the nuclear envelope membranes break down into vesicular structures (1). Apart from lamin hyperphosphorylation, Courvalin et al. (9) also reported that LBR is phosphorylated on serine and threonine residues during mitosis.

As the events responsible for nuclear membrane breakdown are not completely understood and in light of the fact that LBR is phosphorylated by the RS kinase during interphase, we found it important to examine the specific modifications of LBR during mitosis. Results presented below reveal that during mitosis LBR is phosphorylated by both RS and p34cdc2 protein kinases.


EXPERIMENTAL PROCEDURES

Materials

Phosphocellulose and Affi-Gel 10 were purchased from Whatman Biosystems Ltd., United Kingdom, and Bio-Rad, respectively. Peptides R0 (70SSPSRRSRSRSRSRSPGRPAKG91), R1 (61KQRKSQSSSSSPSRRSRSRS80), R2 (78SRSRSRSPGRPAKG91), and R4 (182KIFEAIKTPEKPSSKT197) were made at the Protein Sequencing and Peptide Synthesis Facility of the European Molecular Biology Laboratory, Heidelberg, Germany. R0 peptide was coupled to Affi-Gel 10 as described previously (11). Recombinant p34cdc2/cyclin B was purchased from New England Biolabs Ltd., United Kingdom. Histone H1 was obtained from Boehringer Mannheim GMbH, Germany. [gamma -32P]ATP (6000 Ci/mmol) as well as [32P]phosphate (10 mCi/ml) were purchased from ICN Pharmaceuticals Ltd., United Kingdom. The anti-LBR antibody aR1, raised against the peptide R1, was prepared and affinity-purified as described previously (8). An anti-cyclin B antibody was kindly provided by Ingrid Hoffmann (Germany Cancer Research Center, Heidelberg, Germany). All other chemicals were purchased from Sigma.

Construction of Plasmids and Expression of Fusion Proteins

The pGEX-2T bacterial expression vector (Pharmacia Biotech Inc.) was used to construct plasmids that encode the wild type NH2 terminus (wtNt) and three mutated forms (wtNtA71, wtNtA84, and wtNtA188) of the NH2-terminal domain of chicken LBR (6) fused with glutathione S-transferase (GST). To generate the cDNA coding for wtNt (amino acids 1-205), 30 cycles of the polymerase chain reaction were performed as described (11). Full-length LBR cloned to the EcoRI site of Bluescript SK- was used as a template. The LBR-SK- clone was a generous gift of G. Blobel (Rockefeller University, New York) and H. J. Worman (Columbia University, New York). The sense primer contained nucleotides +1 to +21 of LBR preceded by a BamHI site. CAGTA was added 5' to the BamHI site. The antisense primer was complementary to nucleotides +598 to +615 of LBR. A complementary stop codon was added 5' to this sequence, preceded by an EcoRI site. GC was added 5' to the EcoRI site. The polymerase chain reaction product was purified using the QIAEX gel extraction kit (QIAGEN Inc., Chatsworth, CA). Purified DNA was digested with EcoRI and BamHI, repurified, and ligated into the BamHI/EcoRI site of pGEX-2T. Escherichia coli strains XL-1 Blue were transformed by standard methods.

An oligonucleotide-directed in vitro mutagenesis system (Altered Sites®II In vitro Mutagenesis system, Promega, Corp., Madison, WI) was used to mutate the sites that are potentially phosphorylated by p34cdc2/cyclin B protein kinase. Using the oligonucleotides 5'-TCTGGAAGGAGCACTTGAGGA-3', 5'-GACCAGGAGCTCTGGATCG-3', and 5'-TTTCTCCGGAGCTTTTATTGC-3', Ser71, Ser84, and Thr188 were mutated to Ala. The mutated cDNAs (wtNtA71, wtNtA84, and wtNtA188) were sequenced and subcloned into the pGEX-2T expression vector as described previously for wtNt. GST fusion proteins were produced in bacteria and purified as described (11). A fusion protein missing the RS motifs (deletion of residues 75-84; construct termed GST-Delta RSNt) as well as a protein containing the five arginine-serine repeats of LBR fused to GST (residues 75-84; construct termed GST-RS) was generated as described previously (11).

Purification of LBR Kinase

LBR kinase was isolated from turkey erythrocyte nuclear envelopes as described previously (11). Briefly, the 1 M NaCl extract of nuclear envelopes (following dilution to 0.3 M and clarification by centrifugation) was applied to a phosphocellulose column previously equilibrated with 20 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The bound proteins were eluted by a linear (0.3-1 M) NaCl gradient. Kinase-containing fractions were pooled, the salt concentration was adjusted to 0.35 M, and the material was further chromatographed through an Affi-Gel 10 column containing the R0 peptide. The column was subsequently washed with 0.9 M NaCl, and elution of the kinase activity was accomplished by a linear (0.9-2.2 M) NaCl gradient. The active fractions were pooled, concentrated with an Amicon device, and used in subsequent experiments.

Cell Culture and Synchronization; in Vivo and in Vitro Phosphorylation

Chicken hepatoma cells, DU249, were grown according to Meier and Georgatos (18). To obtain mitotic cells, the cultures were synchronized by a double block of 2 mM thymidine (16 h) and 20 ng/ml nocodazole (4 h) as described elsewhere (19). Mitotically arrested cells were detached by mechanical agitation. For in vivo 32P labeling, the cells were incubated in suspension for 3 h with 1 mCi/ml 32Pi in phosphate-free medium containing nocodazole, washed with cold phosphate-buffered saline (155 mM NaCl, 20 mM sodium phosphate, pH 7.4), and collected by centrifugation. To obtain interphase cells, cultures were grown under similar conditions as above except that thymidine and nocodazole were not included in the growth medium. After the end of incubation with 32Pi, the dishes were vigorously washed two times with cold phosphate-buffered saline to remove mitotic cells. Adherent interphase cells were collected with a rubber policeman. Both mitotic and interphase cells were lysed in 1 ml of 50 mM Tris-HCl (pH 7.4), 0.2% SDS, 1% Triton X-100, 100 mM NaCl, 50 mM NaF, 0.1 mM sodium orthovanadate, 2 mM EDTA, and a mixture of protease inhibitors (8). The lysates were clarified by centrifugation at 12,000 × g for 10 min, and then immunoprecipitation of LBR was carried out as described previously (8).

In order to obtain mitotic cell extracts, cells were harvested by centrifugation at mitosis, washed once in ice-cold KHM buffer (78 mM KCl, 50 mM Hepes-KOH (pH 7.0), 4 mM MgCl2, 8.37 mM CaCl2, 10 mM EGTA, 1 mM dithiothreitol, 20 µM cytochalasin B, and 1 mM phenylmethylsulfonyl fluoride), resuspended at 0 °C in KHM buffer, and Dounce-homogenized. Membrane-free cytosol was prepared by ultracentrifuging the samples at 400,000 × g for 1 h at 4 °C. Immunodepletion of mitotic extracts with an anti-cyclin B antibody was performed essentially as described by Hoffmann et al. (20). Briefly, 25 µl of protein A-Sepharose were incubated with antiserum for 2 h at 4 °C and washed three times with phosphate-buffered saline containing 1% Triton X-100. Extracts were incubated twice with the Immunobeads for 2 h at 4 °C on a rotator and recovered after centrifugation for 30 s in a microcentrifuge. The immunoprecipitates were washed three times with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% Triton X-100 and subsequently used as a source of p34cdc2/cyclin B protein kinase.

For p34cdc2/cyclin B phosphorylation, 1 µl of the enzyme preparation (activity, 2000 units/ml; 1 unit is the amount of p34cdc2/cyclin B required to catalyze the transfer of 1 pmol of phosphate to histone H1 in 1 min at 30 °C) was incubated with 6 µg of GST-wtNt or with 1.5 µg of electroeluted LBR in a buffer composed of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, and 50 µM [gamma -32P]ATP (6000 Ci/mmol) in a reaction volume of 25 µl.

For RS kinase phosphorylation, 6 µl of the enzyme preparation (activity, 10 units/ml; 1 unit is the amount of enzyme required to catalyze the transfer of 0.1 nmol of phosphate to 1.5 µg of electroeluted LBR in 30 min at 30 °C) was incubated with 6 µg of GST-wtNt in a buffer composed of 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 200 mM NaCl, 1 mM dithiothreitol, and 50 µM [gamma -32P]ATP (6000 Ci/mmol) in a reaction volume of 25 µl.

Samples were incubated for 30 min at 30 °C, and the reaction was stopped by adding the appropriate volume of 5 × Laemmli buffer (21) and heating at 95 °C for 3 min. Electroeluted LBR was obtained from urea-insoluble nuclear envelopes as described previously (11).

Phosphopeptide Mapping and Phosphoamino Acid Analysis

Proteolytic peptide mapping was performed essentially as described by Luo et al. (22) and Simos and Georgatos (8). Briefly, immunoprecipitates of in vivo phosphorylated LBR or in vitro phosphorylated GST-wtNt were run on SDS-PAGE and then transferred to a nitrocellulose sheet. The radioactive LBR/GST-wild type NH2 terminal bands were excised, soaked in 0.5% polyvinylpyrrolidone 360 in 100 mM acetic acid for 1 h at 37 °C and washed extensively with water. The protein was digested by trypsin in 50 mM NH4HCO3 at 37 °C overnight. The released peptides were dried, resuspended in water, and loaded on a cellulose TLC plate (Eastman Kodak Co.). Electrophoresis (in the first dimension) was run at pH 8.9 (1% ammonium carbonate) for 1 h at 500 V; ascending chromatography (in the second dimension) was performed using as a solvent a mixture of 1-butanol/pyridine/glacial acetic acid/water at a ratio of 75:50:15:60.

For phosphoamino acid analysis, the tryptic digest was treated with 5.7 M HCl at 110 °C for 90 min, dried, and subjected to electrophoresis on cellulose TLC plates at pH 3.5 (pyridine/glacial acetic acid/water, 1:7:992) for 45 min at 1500 V.

Other Methods

SDS-PAGE was performed according to Laemmli (21) using 12% gels. Dried gels were exposed to Kodak x-ray film with intensifying screens. Protein concentration was determined by the method of Bradford (23).


RESULTS

To determine whether LBR is a substrate for p34cdc2 protein kinase, electroeluted LBR as well as salt-washed nuclear envelopes preheated at 60 °C for 10 min (to inactivate the endogenous RS kinase) were used as substrates for in vitro phosphorylation assays. Fig. 1 shows that p34cdc2 modifies both the envelope-associated and the purified LBR protein. Under these conditions p34cdc2 also phosphorylated lamin A, lamin B, and residual histones H1 and H5 left behind after salt extraction of the nuclear envelopes.


Fig. 1. In vitro phosphorylation of nuclear envelope proteins and purified LBR by p34cdc2 protein kinase. 1.5 µg of electroeluted LBR or 15 µg of salt-washed nuclear envelopes (NE) preheated at 60 °C for 10 min were incubated with [gamma -32P]ATP and p34cdc2 protein kinase. Samples were analyzed by SDS-PAGE and stained with Coomassie Blue (left panel) or autoradiographed (right panel). NE* shows the phosphorylation of nuclear envelope proteins preheated at 60 °C for 10 min in the absence of p34cdc2 protein kinase. Arrowheads indicate the positions of lamins A and B. Brackets indicate the positions of histone H1 isoforms, histone H5, and core histones H3, H2B, H2A, and H4 (from top to bottom). Bars on the left indicate molecular masses (in kDa). Note the phosphorylation of lamins A and B and of histones H1 and H5 by p34cdc2 protein kinase.
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Inspection of the amino acid sequence of chicken LBR revealed the presence of three potential p34cdc2 phosphorylation sites (Ser71, Ser84, and Thr188; see Fig. 2A) conforming to the consensus Ser/Thr-Pro-X-X (24). To assess the ability of p34cdc2 to modify these sites, we synthesized three peptides (R1, R2, and R4) modeled after the published sequence, each one containing one potential phosphorylation site (Fig. 2A). As shown in Fig. 2B, the phosphorylation of purified LBR was inhibited by peptides R1 and R2, which acted as substrates for the kinase. In contrast, peptide R4 was poorly phosphorylated by p34cdc2 and did not significantly inhibit the phosphorylation of purified LBR. To express these results quantitatively, the same type of in vitro phosphorylation assays was performed using a range of peptide concentrations. Data presented in Table I document that R1 was the strongest inhibitor of p34cdc2-mediated phosphorylation of LBR and the best substrate for p34cdc2, whereas R4 was the weakest inhibitor and the poorest substrate for the kinase.


Fig. 2. Phosphorylation of purified LBR by p34cdc2 protein kinase in the presence of various synthetic peptides. A, amino acid sequences of the peptides used. Consensus motifs are shown in bold, and the asterisk denotes predicted phosphorylation sites. The relative position of the peptides in the LBR molecule is schematically indicated. Black boxes along the LBR sequence, numbered with Roman numerals, represent potential transmembrane domains. B, 1.5 µg of electroeluted LBR were incubated with p34cdc2 protein kinase in the presence of 0.5 mM of each peptide as described under "Experimental Procedures." Samples were subsequently analyzed by SDS-PAGE on 10-20% gradient gels and autoradiographed. The upper panel shows the phosphorylation of LBR and the lower panel, the phosphorylation of the added peptides. Only the relevant parts of the autoradiogram are shown.
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Table I.

Concentration of synthetic peptides required for 50% inhibition of the LBR phosphorylation (IC50) by exogenously added p34cdc2 protein kinase and relative phosphorylation rates (RPR) of the respective peptides by p34cdc2


Peptide IC50a RPRb

mM
 61KQRKSQSSSS<UNL>S</UNL>PSRRSRSRS80 (R1) 0.14 100
 78SRSRSR<UNL>S</UNL>PGRPAKG91 (R2) 0.40 60
182KIFEAIK<UNL>T</UNL>PEKPSSKT197 (R4) 0.60 11

a 1.5 µg of electroeluted LBR were incubated with p34cdc2 protein kinase and [gamma -32P]ATP as described under "Experimental Procedures," in the presence of different concentrations of R1, R2, and R4. Phosphorylation of LBR was monitored with autoradiography.
b Peptides R1, R2, and R4 were incubated at a concentration of 100 µM with p34cdc2 protein kinase and [gamma -32P]ATP. Phosphorylation of the peptides was monitored with autoradiography.

In agreement with these observations, only phosphoserine could be detected when in vitro phosphorylated LBR was analyzed by phosphoamino acid analysis (Fig. 3A). Pursuing this point further, we expressed in E. coli a fusion protein consisting of GST and the NH2-terminal domain of LBR (residues 1-205; construct termed GST-wtNt) as well as various derivatives of this fusion protein in which Ser71, Ser84, and Thr188 were changed to Ala (constructs termed GST-wtNtA71, GSTwtNtA84, and GST-wtNtA188, respectively). The wild type and the three mutated proteins were used as substrates in in vitro phosphorylation assays. p34cdc2 could efficiently phosphorylate GST-wtNt, GST-wtNTA84, and GST-wtNtA188, whereas the phosphorylation of GST-wtNtA71 was significantly impaired (Fig. 3D). This was not due to a global misfolding of the polypeptide chain induced by the replacement of Ser71 because both GST-wtNt and GST-wtNTA71 were efficiently modified by the RS kinase (Fig. 5A).


Fig. 3. Identification of the site on LBR that is phosphorylated in vitro by p34cdc2 protein kinase. A, phosphoamino acid analysis following in vitro phosphorylation of electroeluted LBR by p34cdc2 protein kinase. 32P-Labeled LBR was analyzed by SDS-PAGE, blotted onto nitrocellulose filters, and subsequently excised and subjected to hydrolysis and phosphoamino acid analysis as described under "Experimental Procedures." Migration of phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) standards is shown at left. B, SDS-PAGE analysis and Coomassie Blue staining of GST, GST fusion protein containing the NH2-terminal domain of LBR (GST-wtNt, amino acids 1-205), and similar fusion proteins except that Ser71, Ser84, and Thr188 were mutated to Ala (constructs termed GST-wtNtA71, GST-wtNtA84, and GST-wtNtA188, respectively). The full-length fusion protein migrates with an apparent molecular mass of approximately 51 kDa. The lower bands represent degradation products (see also Ye and Worman (7) and Nikolakaki et al. (11)). C, immunoblotting of bacterially expressed proteins using an affinity-purified anti-LBR antibody raised against peptide R1 (aR1). The blots were stained using an alkaline phosphatase-conjugated goat anti-rabbit antibody. Note that in addition to full-length fusion protein, aR1 also reacts with degradation products. D, in vitro phosphorylation of bacterially expressed proteins by p34cdc2 protein kinase. The samples were analyzed by SDS-PAGE and autoradiographed. Molecular mass standards are shown at left (in kDa).
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Fig. 5. Mitotic cell extracts contain both p34cdc2 and RS protein kinase activities. Autoradiograms of in vitro-phosphorylated histone H1, GST-wtNt, GST-wtNtA71, GST-RS, and GST-Delta RSNt (for nomenclature, see text and legend to Fig. 3) by partially purified RS kinase (A), p34cdc2 protein kinase (B), cell extracts made from DU249 cells arrested in mitosis (C), mitotic cell extracts made from HeLa cells (D), mitotic cell extracts from HeLa cells pretreated with an anti-cyclin B antibody (E), and immunoprecipitates of mitotic HeLa cell extracts with the anti-cyclin B antibody (F). Additional bands represent phosphorylated proteins associated with protein A-Sepharose/anti-cyclin B beads. Molecular mass standards are shown at left (in kDa).
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To confirm these results we performed two-dimensional proteolytic peptide mapping. Fig. 4 shows that p34cdc2-phosphorylated GST-wtNt yielded one major phosphopeptide (peptide designated b), one phosphopeptide of moderate intensity (peptide designated c), and two minor phosphopeptides (peptides designated a and d). Phosphopeptide mapping of in vitro phosphorylated GST-wtNtA71, GST-wtNtA84, and GST-wtNtA188 by p34cdc2 revealed that the major phosphopeptide (b) corresponds to phosphorylation of Ser71, peptides a and c correspond to phosphorylation of Ser84, and peptide d corresponds to phosphorylation of Thr188. From the sum of all these observations it can be concluded that Ser71 of avian LBR is the major site phosphorylated by p34cdc2 protein kinase under in vitro conditions, whereas Ser84 is weakly modified by the enzyme. It is noteworthy that in some of our experiments we have been unable to detect the spot corresponding to Thr188. The very low extent of Thr phosphorylation could explain our inability to detect it when we performed phosphoamino acid analysis of in vitro phosphorylated LBR by p34cdc2 (see Fig. 3A).


Fig. 4. Tryptic phosphopeptide analysis of GST-wtNt and alanine mutants of GST-wtNt (for nomenclature, see text and legend to Fig. 3). In vitro phosphorylated fusion proteins, by p34cdc2 protein kinase, were transferred to nitrocellulose and digested with trypsin. The eluted phosphopeptides were separated by electrophoresis at pH 8.9 (horizontal direction; cathode to the right) and by ascending chromatography. Origins of sample application are marked by odot .
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That LBR can be directly phosphorylated by p34cdc2 protein kinase implies that the lamin B receptor protein has the potential of being an in vivo substrate for mitotic kinases. To explore this idea, we performed experiments using mitotic cell extracts. Fig. 5C shows that membrane-free cytosol prepared from nocodazole-arrested chicken hepatoma (DU249) cells contained both p34cdc2 and RS protein kinase activities. This can be deduced from the fact that mitotic extracts phosphorylated GST-Delta RSNt (which lacks the RS region of LBR and is not a substrate for the RS kinase), GST-wtNtA71 (which is not phosphorylated by p34cdc2), and GST-RS (a fusion protein consisting of GST and five RS repeats but missing the putative p34cdc2 site of LBR). The same results were obtained with mitotic extracts from HeLa cells (Fig. 5D). To confirm that p34cdc2/cylin B is truly the kinase responsible for mitotic LBR phosphorylation, we immunodepleted HeLa cell extracts with an anti-cyclin B polyclonal antibody. Fig. 5E shows that extracts pretreated with the anti-cyclin B antibody had lost their ability to phosphorylate histone H1 and GST-Delta RSNt and contained only the RS kinase activity, whereas a typical p34cdc2 pattern was obtained with the immunoprecipitated activity (Fig. 5F).

Pursuing this point further, interphase DU249 cells and cells arrested at prometaphase were labeled metabolically with [32P]orthophosphate, and the in vivo phosphorylated LBR was immunoprecipitated by aR1 antibodies (see "Experimental Procedures"). The level of phosphorylation was similar in interphase and mitotic cells (data not shown; see also Ref. 9). The 58-kDa bands corresponding to immunoprecipitated LBR were excised and processed for phosphoamino acid analysis and two-dimensional tryptic phosphopeptide mapping. Only phosphoserine could be detected, irrespective of whether phosphorylation occurred during the interphase or the prometaphase (data not shown; for a typical TLC profile see Fig. 3A). The maps of mitotically phosphorylated LBR and LBR modified at interphase were qualitatively similar (some residues phosphorylated at interphase were phosphorylated to a lower extent at mitosis, i.e. phosphopeptides 1, 3, 8, and 9) except for one spot that was present in the former but absent in the latter (Fig. 6, compare panels C and D). This spot represented the major phosphopeptide (phosphopeptide b) corresponding to Ser71 as shown by mixing equal counts/min of the tryptic digests, in vitro phosphorylated GST-wtNt by p34cdc2, and LBR modified in vivo at interphase (Fig. 6, compare panels B and C with panel E). This mix reproduced the phosphopeptide pattern of LBR that had been modified by mitotic kinases in vivo. The same mitotic pattern was also obtained by mixing equal counts of in vitro phosphorylated GST-wtNt by p34cdc2 and LBR modified in vivo at mitosis (data not shown). To confirm that Ser71 is the additional site phosphorylated at mitosis by p34cdc2, we performed the following experiment. Mitotic extracts (prepared from DU249 cells as described under "Experimental Procedures") were used to phosphorylate either GST-wtNt or GST-wtNTA71. The phosphorylated proteins were then analyzed by two-dimensional tryptic phosphopeptide mapping. The phosphopeptide map of in vitro phosphorylated GST-wtNt was identical to the map derived from mitotic LBR in vivo (data not shown), whereas the map of in vitro phosphorylated GST-wtNtA71 was similar to the map derived from interphase LBR phosphorylated in vivo; that is, phosphopeptide b was conspicuously missing (compare panels F and C). From the sum of these observations two major conclusions can be drawn. First, the RS and the p34cdc2 protein kinases are both responsible for the mitotic phosphorylation of LBR and second, Ser71 is the major site phosphorylated in vivo by p34cdc2.


Fig. 6. Tryptic phosphopeptide analysis of LBR following phosphorylation during interphase and mitosis in vivo or by RS and p34cdc2 protein kinases in vitro. A, GST-wtNt was phosphorylated in vitro by partially purified RS kinase. B, GST-wtNt was phosphorylated in vitro by p34cdc2 protein kinase. B is the same as the top right panel in Fig. 4. C, LBR was immunoprecipitated from 32P-labeled DU249 cells arrested at interphase. D, LBR was immunoprecipitated from 32P-labeled DU249 cells arrested at mitosis. E, equal Cerenkov counts of digests of in vitro phosphorylated GST-wtNt by p34cdc2 protein kinase (B) and in vivo phosphorylated LBR during interphase (C). F, GST-wtNtA71 was phosphorylated in vitro by mitotic extracts prepared from DU249 cells (as described under "Experimental Procedures"). Analysis of phosphopeptides was carried out as described in Fig. 4. Anode is on the left and cathode on the right. Origins of sample application are marked by odot .
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DISCUSSION

In this study we demonstrated that LBR undergoes mitotic phosphorylation and that the RS kinase is the main protein kinase responsible for this modification. Comparison of tryptic phosphopeptide maps of in vivo 32P-labeled LBR immunoprecipitated from chicken cells indicates that the enzyme modifies similar sites at interphase and mitosis. Some serine residues of the RS motif phosphorylated at interphase are phosphorylated to a lower extent at mitosis. Furthermore, we demonstrated that LBR is also a substrate for p34cdc2 protein kinase during mitosis. Using recombinant proteins produced in bacteria, phosphoamino acid analysis and two-dimensional phosphopeptide mapping of in vitro and in vivo 32P-labeled LBR, we have been able to demonstrate that Ser71 is the major site phosphorylated by p34cdc2 at mitosis. Courvalin et al. (9) reported that Thr188 is likely to be phosphorylated by this enzyme during mitosis. According to our results the extent of Thr phosphorylation is very low and most probably Thr188 represents a minor site modified by p34cdc2. In line with our observations is the fact that the phosphoamino acid analysis presented by Courvalin et al. (9) demonstrated that mitotic LBR contained mainly phosphoserine, whereas phosphothreonine was hardly detectable.

Previous reports have shown that the RS protein kinase is strongly associated with LBR, participating in a subassembly of nuclear envelope proteins termed "the LBR complex" (8, 11). The enzyme phosphorylates LBR in a constitutive fashion during interphase (8, 11) and belongs to a novel class of protein kinases that specifically modify arginine-serine (RS) dipeptide motifs. Other members of this novel class of enzymes include a kinase associated with small nuclear ribonucleoprotein particles, which phosphorylates the U1 small nuclear ribonucleoprotein 70-kDa protein and ASF/SF2 (25), and a cell cycle-regulated serine kinase (SRPK1, <UNL>SR</UNL> <UNL>P</UNL>rotein <UNL>K</UNL>inase 1) that can phosphorylate splicing factors of the SR family (26, 27). The SRPK1 activity was initially identified in nuclear extracts (26), and later the same group (27) also purified and characterized SRPK1 from cytosolic extracts. Thus, the cellular distribution of SRPK1 remains to be clarified. Interestingly, its fission yeast homologue, Dsk1, was found to be cytoplasmic in interphase cells, migrating to the nucleus before mitosis (28). Recently, Colwill et al. (29) reported that mammalian Clk/Sty, which is the prototype for a family of dual specificity kinases (termed LAMMER kinases), also interacts with members of the SR family of splicing factors and phosphorylates ASF/SF2.

The function of these enzymes appears to be the regulation of protein-protein interactions through phosphorylation of RS domains. In fact, LBR kinase regulates interactions among the components of the LBR complex (11), whereas SRPK1 and Clk/Sty regulate the intranuclear distribution of SR splicing factors (26, 29, 30). SRPK1 may also be responsible for the redistribution of splicing factors as cells enter mitosis (26).

LBR is also a substrate for p34cdc2 protein kinase, a key mitotic kinase. Several substrates for this enzyme have been identified to date (reviewed in Ref. 31). However, the detailed mechanisms by which p34cdc2 induces the profound structural changes characteristic of mitotic cells remain quite obscure. Although the physiological significance of LBR phosphorylation by p34cdc2 remains to be examined by other approaches, it is interesting to note here that, at least in vitro, the p34cdc2-modified LBR and the nonmodified protein do not significantly differ in their lamin B binding properties.2 This is in line with the fact that lamin B, although disassembled by p34cdc2 phosphorylation (32-34), remains associated with membrane vesicles during mitosis (1, 18).

On the other hand, LBR, together with the integral membrane protein Lap2, is the most obvious candidate to mediate the association of interphase nuclear membranes to chromatin (5, 19, 35, 36). The idea that the LBR complex mediates chromatin anchorage during interphase is further reinforced by the fact that lamin A has been found to interact with components of interphase chromatin (17, 37-40).

During mitosis, the nuclear envelope breaks down and the nuclear lamina is depolymerized (1, 32-34). Already, at prophase, binding of the membranous structures to chromosomes is weakened. At the end of mitosis, the first step in nuclear envelope reformation appears to be the binding of mitotic vesicles to the surfaces of chromosomes, followed by fusion of these vesicles and assembly of an envelope structure around chromatin (reviewed in Ref. 41). Taking into account that LBR is phosphorylated by the RS kinase and by p34cdc2 protein kinase and that the major phosphorylation target of p34cdc2 is Ser71 (which is located near the RS repeats), one might consider that there is some cross-talk between these two phosphorylation events. An intriguing possibility would be that phosphorylation of LBR, mediated by p34cdc2 protein kinase, together with RS phosphorylation function as a switch preventing premature membrane assembly around chromosomes. This idea is consistent with the previously reported observation that phosphorylation of Lap2 by mitotic cytosol inhibits its binding to chromosomes (5). Along these lines, it is also noteworthy that at least part of p34cdc2 and RS protein kinase activities is associated with chromosomes.3

Finally, we need to note the existence of a p34cdc2 phosphorylation site among other proteins containing an RS motif and, even more interestingly, that this site is located a few amino acids upstream of the RS repeats, as in the case of LBR (Table II). Some of these proteins have been shown to undergo RS-specific phosphorylation such as the splicing factors SC35 and SF2 (11, 26), whereas the phosphorylation status of the others remains to be examined in future studies.

Table II.

Existence of a p34cdc2 phosphorylation motif in RS-containing proteins

Predicted p34cdc2 phosphorylation sites are shown in bold and RS motifs are underlined. Numbers in parentheses correspond to the appropriate references.
·SSPSR<UNL>RSRSRSRSRS</UNL>PG Chicken LBR (6)
·RSPRRRR<UNL>RSRSRSRSRSRSRSRS</UNL>RY Human splicing factor SC35 (42)
·RSPSYG<UNL>RSRSRSRSRSRSRSRS</UNL>NS Human splicing factor SF2 (13)
·PSPSR<UNL>RSRSRSRSRS</UNL>KS Mussel sperm-specific protein PHI-2B (43)
·PSPTRRSSKS<UNL>RSKSRSRSRS</UNL>AS Mussel sperm-specific protein PHI-1 (43)
·KSPS<UNL>RSRSRSRSKS</UNL>NA Yeast cytoskeleton assembly control protein SLA1 (44)
·RSPNRGRGGSSGGPTTRSQSRSLS<UNL>RSRSRSRSRS</UNL>RG Human papillomavirus E2 protein (45)
·RTPCSFADQLLSTFIANNYLCYFYRRR<UNL>RSRSRSRSRSRSRS</UNL>PH Nuclear polyhedrosis virus polyhedral envelope protein (46)


FOOTNOTES

*   This work was supported in part by a grant from the German-Greek Cooperation in Science and Technology.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.

This article is dedicated to the memory of Prof. Nikolaos Alexandrou.


   Present address: Dept. of Neurosciences, Montreal General Hospital, Montreal H3G 1A4, Canada.
par    Recipient of a research bursary granted by the Commission of the European Communities in the framework of the BIOMED 1 program. Present address: Institut fur Biochemie I, University of Heidelberg, D-69120 Heidelberg, Germany.
Dagger Dagger    To whom correspondence should be addressed: Laboratory of Biochemistry, School of Chemistry, Aristotelian University of Thessaloniki, 54 006 Thessaloniki, Greece. Tel.: 30 31 997726; Fax: 30 31 997689; E mail: giannako{at}ccf.auth.gr.
1   The abbreviations used are: LBR, lamin B receptor; wtNt, wild type NH2 terminus; GST, glutathione S-transferase; SF, splicing factor; PAGE, polyacrylamide gel electrophoresis; SRPK1, SR protein kinase 1.
2   G. Simos and S. D. Georgatos, unpublished observations.
3   E. Nikolakaki, S. D. Georgatos, and T. Giannakouros, unpublished observations.

Acknowledgments

We thank G. Blobel and H. J. Worman for providing us with the LBR cDNA clone, Ingrid Hoffmann for the anti-cyclin B antibody, and Athina Pyrpasopoulou for the mitotic extracts from HeLa cells. We also thank J. G. Georgatsos and J. R. Woodgett for useful discussions and comments on the manuscript.


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