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Originally published In Press as doi:10.1074/jbc.M402546200 on June 18, 2004

J. Biol. Chem., Vol. 279, Issue 34, 35813-35821, August 20, 2004
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A Phosphorylation Cluster in the Chromatin-binding Region Regulates Chromosome Association of LAP2{alpha}*

Andreas Gajewski{ddagger}§, Edina Csaszar¶, and Roland Foisner{ddagger}||

From the {ddagger}Department of Medical Biochemistry, Medical University of Vienna and the Department of Biochemistry and Molecular Cell Biology, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, University of Vienna, A-1030 Vienna, Austria

Received for publication, March 5, 2004 , and in revised form, June 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
LAP2{alpha} is a LEM family protein associated with nucleoplasmic A-type lamins and chromatin in interphase. Like lamins and other lamina proteins LAP2{alpha} is cytoplasmic in metaphase, but it associates with chromosomes prior to nuclear envelope formation in late anaphase to telophase. In vitro phosphorylation analysis and mass spectrometry identified a cluster of at least three mitotic cyclin-dependent kinase 1 phosphorylation sites in the C-terminal chromatin-binding region of LAP2{alpha} as well as four additional potential sites in the cluster, some of which were targeted alternatively in LAP2{alpha} mutated at the major sites. LAP2{alpha} mutants containing serine -> alanine mutations at all seven sites revealed a clear phenotype. Mutated LAP2{alpha} remained associated with chromosomes throughout mitosis, but the dissociation of lamins into the cytoplasm and nuclear envelope disassembly were not affected. These data demonstrate the in vivo significance of mitotic phosphorylation for the dynamic behavior of LAP2{alpha} in the cell cycle and show that, unlike the interaction with lamins, the chromatin association of LAP2{alpha} is regulated by multiple mitosis-specific phosphorylation at sites clustered within a defined region in the C terminus of the protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In higher eukaryotes, lamins (1) and lamin-binding proteins form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane (2, 3). Lamins are also found in stable complexes in the nuclear interior (4–6), where they are involved in numerous functions (7, 8), including DNA replication (9), RNA polymerase II-dependent transcription (10), chromatin organization, and spacing of nuclear pores (11) and possibly RNA splicing (12). A growing number of lamin-associated proteins at the nuclear periphery and in the nucleoplasm regulate lamin complex assembly and function (7, 13, 14). Lamin-binding proteins in the inner nuclear membrane include the lamin B receptor (LBR, Ref. 15, emerin, Ref. 16, and various isoforms of lamina-associated polypeptide (LAP1,1 Refs. 17 and 18 and LAP2, Refs. 19–21). In the nuclear interior, lamins have been found to interact with histones (22, 23), retinoblastoma protein pRb (24, 25), and a nucleoplasmic LAP2 isoform, LAP2{alpha} (26).

The mouse and human LAP2 gene encodes six alternatively spliced isoforms (21), most of which are inner nuclear membrane proteins and closely related in structure to LAP2{beta} that binds B-type lamins (18). LAP2{alpha} shares only the N-terminal constant region with the other isoforms and contains a unique C terminus. LAP2{alpha} forms nucleoskeletal complexes with A-type lamins throughout the nuclear interior (26, 27). All LAP2 isoforms belong to the LEM (LAP-emerin-MAN1) protein family (28), characterized by the presence of a 40-residue long structural motif (LEM domain) (28–30). The LEM domain interacts with the DNA cross-bridging protein barrier-to-autointegration factor (BAF; Ref. 31) and an N-terminal LEM-like motif binds DNA (29). Thus, LAP2 isoforms interact with chromatin at the nuclear envelope and in the nucleoplasm and may regulate higher order chromatin structure.

Multicellular eukaryotes reversibly disassemble the nuclear lamina, nuclear pore complexes, and nucleoskeletal structures during mitosis (8, 32). Mitosis-specific phosphorylation of lamins (33, 34) was found to be essential for lamina disassembly by expressing lamin mutants with mutations in their cyclin-dependent kinase 1 (cdk1) target sites (35). Lamin-binding proteins, such as LAP2{beta} and LAP2{alpha}, are also phosphorylated in mitosis (18, 27). However, unlike for lamins, the role of mitosis-specific phosphorylation of lamin-binding proteins for their cell cycle-dependent dynamics has never been demonstrated in vivo.

LAP2{alpha} showed a particularly intriguing dynamic behavior in the cell cycle. Like lamins and LAP2{beta}, LAP2{alpha} was dispersed throughout the cytoplasm in metaphase cells, but associated with chromosomes early during nuclear reassembly, prior to accumulation of LAP2{beta} and assembly of membranes (18, 26, 27, 36). While the LEM and LEM-like motifs in the LAP2{alpha} N terminus were dispensable for the LAP2{alpha}-chromosome interaction, a chromatin-binding region in the unique C-terminal domain was found to be essential and sufficient for interaction (36, 37). Chromatin-binding LAP2{alpha} fragments dominantly inhibited nuclear assembly (37), suggesting that LAP2{alpha} relocalization to chromosomes is essential for proper nuclear assembly. In this study we focused on identifying molecular regulatory mechanisms, controlling the association of LAP2{alpha} with chromosomes. We identify three major mitosis-specific phosphorylation sites clustered in the core of the chromatin-binding region as well as four potential sites in the cluster. We further show that eliminating LAP2{alpha} phosphorylation by mutating serines to alanines at major and potential target sites renders the protein incapable of dissociating from chromosomes in mitosis. These studies demonstrate for the first time the in vivo significance of phosphorylation of a lamin-binding protein for its redistribution during mitosis and show that chromosome association, unlike its interaction with lamins, is regulated by phosphorylation of LAP2{alpha}.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of LAP2{alpha} Expression Plasmids—Mutations replacing serine to alanine residues in the LAP2{alpha} polypeptide were introduced into LAP2{alpha} cDNA using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) and pTD15, a tetracycline-dependent Tet-on expression plasmid encoding full-length LAP2{alpha} with a C-terminal Myc tag (36) as template. The following pairs of oligonucleotide primers were used: 5'-GGTCTCTACTGCAGCTGCTCCTTCACTGATTAAAG-3'and 5'-CTTTAATCAGTGAAGGAGCAGCTGCAGTAGAGACC-3' to replace serine 309; 5'-GTCCTGAGAGGGCCCATATTTCAGATCAATCGCC-3'and 5'-GGCGATTGATCTGAAATATGGGCCCTCCTCAGGA-3' to replace serine 344; 5'-CCATATTTCAGATCAAGCGCTTCTCTCCAGTAAAAGG-3' and 5'-CCTTTTACTGGAGAGAGGCGCTTGATCTGAAATATGG-3' to replace serine 350; 5'-GGAAAGCACTAGAAGAGGCTGAGAGCTCACAAC-3' and 5'-GTTGTGAGCTCTCAGCCTCTTCTAGTGCTTTCC-3' to replace serine 362; 5'-GAGCTCACAACTAATTGCTCCGCCACTTGCCCAG-3'and 5'-CTGGGCAAGTGGCGGAGCAATTAGTTGTGAGCT-3' to replace serine 369; 5'-GTTTCAAGAAACTGAATTCCTGGCTCCTCCAAGAAAAGTCCC-3' and 5'-GGGACTTTTCTTGGAGGAGCCAGGAATTCAGTTTCTTGAAAC-3' to replace serine 423; 5'-GGAAAGGGATTCAGGTGCCTTTGTGGCATTTCAG-3' and 5'-CTGAAATGCCACAAAGGCACCTGAATCCCTTTCC-3' to replace serine 443.

All mutations were confirmed by sequencing. For bacterial expression of mutated proteins, the respective cDNAs were subcloned from pTD15 derivatives into the bacterial expression vector pET-23a(+) (Novagen, Madison, WI) via NheI-XhoI.

Cell Culture and Transfections—HeLa, HeLa Tet-on (Clontech Laboratories, Palo Alto, CA), and CHO cells were routinely maintained in Dulbecco's modified Eagle's medium containing high glucose, 10% fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin (all from Invitrogen) at 37 °C in a humidified atmosphere with 8.5% CO2. Culture media for HeLa Tet-on cells were supplemented with 100 µg/ml G418 (Invitrogen), for transfected stable HeLa Tet-on clones with additional 200 µg/ml hygromycin B (Roche Applied Science). For induction of protein expression, 2 µg/ml doxycyclin (Sigma-Aldrich Chemie GmbH) were added to the medium for at least 24 h. Transfections were performed according to the manufacturer's instructions using LipofectAMINE 2000 reagent, Opti-MEM (Invitrogen), and DNA prepared with a Jet Star plasmid kit (Genomed, Bad Oeynhausen, Germany). Transient transfections were done in Falcon 4 chamber tissue culture glass slides (BD Biosciences, Franklin Lakes, NJ) for 4 h in transfection reagent containing 0.6 µg of DNA per chamber. For stable transfections, 5 x 105 HeLa Tet-on cells in 6-cm culture dishes were co-transfected with 0.4 µg of pTK-Hyg selection vector (Clontech Laboratories) and 6 µg of response plasmid. After 48 h of culture in complete medium, cells were split into ten 10-cm culture dishes and cultured in medium containing 200 µg/ml hygromycin B for 14 days. Single clones were transferred into 24-well plates and grown in the presence or absence of doxycyclin.

Chromosome Spreads—Mitotic cells harvested from an unsynchronized cell culture were incubated in 0.075 mM KCl for 20 min at room temperature and lysed by addition of 0.1% Tween 20. Samples were spun onto coverslips at 2,000 rpm for 3 min in a Cytospin 2 (Thermo Shandon, Pittsburgh PA), fixed in 2% formaldehyde for 10 min, and processed for immunofluorescence microscopy.

Immunofluorescence Microscopy—Cells were fixed in 2 or 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature, incubated with 50 mM NH4Cl in PBS, and permeabilized with 0.5% Triton X-100 in PBS for 5 min each. After incubation in PBS/0.2% gelatin for 30 min, primary and secondary antibodies were applied in PBS for 1 h each. Primary antibodies used were monoclonal antibodies to LAP2{alpha}, and LAP2{beta} (27), antiserum to LAP2{alpha} (37), monoclonal Myc 1–9E10.2 antibody (American Type Culture Collection CRL-1729), monoclonal lamin A antibody (133A2, abcam, Cambridge, UK). Secondary antibodies used were affinity-purified goat anti-rabbit or anti-mouse IgG conjugated to Alexa 488 or Texas Red (Jackson Immuno-Research Laboratories, West Grove, PA). DNA was stained with 1 µg/ml Hoechst dye 33258 applied together with the secondary antibody. Samples were mounted in Mowiol and viewed in a Zeiss Axiovert 100M equipped with either a LSM 510 confocal microscope or an Axiocam.

Protein Expression in Bacteria and in Vitro Phosphorylation—For bacterial protein expression Escherichia coli strain BL21 was transformed with pET-23a(+) (Novagen)-derived plasmids. Bacteria were grown in 100 ml of LB medium plus 100 µg/ml ampicillin until an OD600 of 0.6–0.8, and protein expression was induced with 0.5 mM isopropyl-{beta}-D-thiogalactopyranoside for 4 h. Bacteria were lysed in 5 ml of lysis buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1 mg/ml lysozyme, 0.1% Triton X-100, and 1 mM MgCl2) for 30 min at 37 °C, and centrifuged at 3,000 x g for 30 min. Pellets were resuspended in urea buffer (7 M urea, 100 mM NaCl, 20 mM HEPES pH 7.4, and 1 mM DTT) and stored frozen at -20 °C. For in vitro phosphorylation, samples were dialyzed into buffer A (50 mM HEPES pH 7.4, 5 mM MgCl2, 1 mM EGTA, 0.1% Triton X-100, 100 mM NaCl2, and 0.1 mM DTT). 80-µl samples were mixed with a 10-µl interphase or mitotic CHO cell extract or immunoprecipitated cdk1 (38), and 10 µl of an ATP/[{gamma}-32P]ATP mixture (1 µCi for 50 µl of a 1 mM ATP solution), incubated for 30 min at 37 °C, mixed with 3x SDS sample buffer, and analyzed by SDS-PAGE and autoradiography. The following inhibitors were used: olomoucine (84 µM, Calbiochem, La Jolla, CA); staurosporine (0.2 µM, Sigma); H7 (0.3 mM, Calbiochem); EDTA (10 mM, Sigma). Two-dimensional phosphopeptide mapping was done according to Boyle et al. (39) with slight modifications (38).

Immunoprecipitation of LAP2{alpha} and Mass Spectrometry—Mitotic or interphase cells (38) were lysed in 500 µl of buffer A containing 1% Triton X-100 and 0.4% SDS and pressed through a 27-gauge needle to shear DNA. Buffer A was added to reduce SDS concentration to 0.1%, and lysates were precleared by incubation with 20 µl of 50% protein G-Sepharose beads (Sigma) and brief centrifugation. 20 µl of protein G-Sepharose beads, preincubated in 100 µl of hybridoma supernatant containing antibody to LAP2{alpha} or to the Myc tag, were added and harvested by centrifugation following a 2-h incubation. The beads were washed five times in buffer A and proteins separated by SDS-PAGE. Coomassie-stained LAP2{alpha} bands were cut out from gels and digested as described elsewhere (40). High quality water for the in-gel digestion and the mass spectrometric experiments was prepared using an ELGA Maxima water purification system (Vivendy Water Systems, High Wycombe, Bucks, UK). Ammonium hydrogen carbonate was obtained from FLUKA (Sigma-Aldrich), DTT for the reduction of proteins before in-gel digestion was purchased from Roche Applied Science, iodoacetamide was supplied by Sigma, and trypsin was obtained from Roche Applied Science. Tryptic peptides were extracted by 5% formic acid, purified, and concentrated. The chromatographic media used was Poros 20 R2 (PerSeptive Biosystems, Foster City, CA) filled into the non-coated nanospray capillaries of Protana (Proxeon Biosystems A/S; Odense, Denmark). Hydrophilic peptides were captured by Poros Oligo material (PerSeptive Biosystems, Foster City, CA). Concentrated peptides were eluted with a mixture of 50% methanol, 5% formic acid directly into a metal-plated silica capillary (Proxeon Biosystems A/S; Odense, Denmark). The measurements were carried out on a QSTAR (Applied Biosystems; Foster City, CA) hybrid mass spectrometer. The instrument was externally calibrated with the MS/MS fragments of a synthetic peptide (ALILTLVS). First a peptide spectrum was obtained, and the charge state of the peptides was determined. Doubly or triply charged peptides were chosen for MS/MS experiments. Fragment ions were labeled according to the nomenclature proposed by Roepstorff and Fohlman (41). MS/MS spectra were interpreted with the aid of the Mascot (Matrix Science Ltd, London, UK) search engine.

Polyacrylamide Gel Electrophoresis, Transfer, and Detection—SDS-PAGE was performed according to Laemmli (42). For autoradiography gels were dried and exposed to an x-ray film. For immunoblotting, proteins were electrophoretically transferred onto nitrocellulose (0.2 µm; Schleicher and Schuell, Dassel, Germany) in 48 mM Tris-HCl, pH 9.4, 39 mM glycine using the Mini Transblot system (Bio-Rad). Primary antibodies used were: hybridoma supernatants of anti-LAP2 antibodies (27), monoclonal Myc 9E10 antibody (ATCC), polyclonal LAP2{alpha} antibody 245-2 (diluted 1:10,000; Ref. 37); secondary antibodies, alkaline phosphatase- or peroxidase-conjugated goat antibodies against the primary antibodies (diluted 1:7,500). For detection of proteins, the Proto-blot immunoscreening system (Promega) or the Super Signal ECL (Pierce) were used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
LAP2{alpha} Is Differentially Phosphorylated by Interphase and Mitotic Kinases in Vitro—We have previously shown that LAP2{alpha} isolated from metaphase cells had a slightly reduced mobility on SDS-PAGE as compared with LAP2{alpha} from interphase cells. Furthermore, isoelectric focusing revealed a more acidic pI of mitotic versus interphase LAP2{alpha} (27). As these properties were consistent with a mitosis-specific phosphorylation of LAP2{alpha}, we set out to test this possibility by various means. First, we performed in vitro phosphorylation assays using bacterially expressed, recombinant LAP2{alpha} as substrate, or histone H1 as a control, and mitotic or interphase cell extracts as a source for kinases. Samples were analyzed by SDS-PAGE and autoradiography (Fig. 1A) and specific protein phosphorylation (cpm per microgram of protein, Fig. 1B) was determined. Extracts of nocodazole-arrested metaphase cells phosphorylated both LAP2{alpha} and histone H1 to a 3–4-fold higher extent than interphase extracts. Endogenous LAP2{alpha} present in cell extracts was highly diluted and not detectable in these assays (data not shown). Three potent kinase inhibitors, olomoucine, staurosporine, and H7, which affect predominantly cdk-type kinases, protein kinase C, and a broad range of different kinases, respectively, reduced in vitro phosphorylation significantly, while the calcium chelator EDTA blocked kinase activity completely. Interestingly, unlike EDTA, none of the inhibitors significantly affected interphase kinase activity toward LAP2{alpha}. Thus, we concluded that LAP2{alpha} is primarily phosphorylated by mitotic kinases. cdk1 Phosphorylates LAP2{alpha} at Mitosis-specific Site(s) in Vitro—The sensitivity of mitotic LAP2{alpha} kinase activity to olomoucine suggested an involvement of mitosis-specific kinase cdk1. To test this hypothesis, we immunoprecipitated cdk1 from nocodazole-arrested metaphase cells using an antiserum generated against a synthetic peptide representing the C terminus of human cdk1 (43) and performed in vitro phosphorylation assays. Recombinant LAP2{alpha} (Fig. 2, A and B) and histone H1 (not shown) were significantly phosphorylated by cdk1 in vitro.



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  		FIG. 1.
LAP2{alpha} is heavily phosphorylated by mitotic cell extracts in vitro. A, recombinant LAP2{alpha} or histone H1 were incubated with mitotic or interphase cell extracts and [{gamma}-32P]ATP in the absence (Control) or presence of indicated kinase inhibitors and analyzed by SDS-PAGE (Coom.) and autoradiography (Autorad.). B, cpm on blots in A were detected by Cerenkov counting, the amount of protein was determined by densitometric scanning, and cpm per microgram of protein was calculated.

 



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  		FIG. 2.
LAP2{alpha} C terminus is phosphorylated by cdk1. Recombinant LAP2{alpha} fragments shown in A were incubated with immunoprecipitated cdk1 as shown in B. C, cdk1, mitotic, or interphase extract as indicated. D, with mitotic extract. Samples were analyzed by SDS-PAGE/autoradiography (B) or by two-dimensional phosphopeptide mapping (C and D). Arrows in peptide maps show mitotic, cdk1-dependent spot; arrowheads, a spot present in interphase and mitotic samples.

 
To demonstrate that cdk1 also represented a major kinase activity for LAP2{alpha} in whole mitotic cell lysates, we performed two-dimensional tryptic phosphopeptide mapping of in vitro phosphorylated LAP2{alpha}. The phosphopeptide analysis of LAP2{alpha} phosphorylated by whole mitotic cell lysate yielded several strongly labeled spots (Fig. 2C), indicating targeting of various phosphorylation sites during mitosis. Most of the phosphopeptides generated from LAP2{alpha} phosphorylated by interphase extracts were only weakly labeled, and interphase and mitotic peptide patterns were clearly different. While only one spot was detected in both mitotic and interphase samples (Fig. 2C, arrowhead), two of three major mitotic peptides were exclusively detected in mitotic samples, indicating mitosis-specific phosphorylation. Interestingly, one of these mitosis-specific peptides was also detected in phosphopeptide maps of LAP2{alpha} phosphorylated by immunoprecipitated cdk1 (arrow). Thus, the major phosphorylation sites in LAP2{alpha} were predominantly or exclusively targeted in mitosis and involve cdk1 as well as other kinases.

To narrow down the cdk1-dependent, mitosis-specific target site in LAP2{alpha}, we expressed LAP2{alpha} fragments covering different domains of the protein in bacteria and tested their in vitro phosphorylation by immunoprecipitated cdk1 (Fig. 2B) or mitotic extracts (Fig. 2D) using SDS-PAGE/autoradiography and two-dimensional phosphopeptide mapping, respectively. While the LAP2{alpha}-specific C-terminal region (amino acids 188–693) was phosphorylated as efficiently as full-length protein (amino acids 1–693) and produced the mitotic, cdk1-dependent spot in phosphopeptide maps (Fig. 2D, arrow), the constant N terminus (amino acids 1–187) was not phosphorylated to a considerable extent. Similarly, LAP2{alpha}-(1–264) was not phosphorylated by cdk1 and LAP2-(1–414) only weakly, while LAP2{alpha}-(410–693) was strongly labeled. Correspondingly, the cdk1-dependent spot was not detected in peptide maps of LAP2{alpha}-(1–414), but was clearly present in C-terminal truncation mutants (1–615). Altogether, these data narrowed down the cdk1 target site to a region between amino acids 410 and 615. Strikingly this region overlapped with a domain in LAP2{alpha} that has previously been found to mediate the association with chromatin during nuclear reassembly (36) (Fig. 3).



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  		FIG. 3.
In vivo phosphorylation of LAP2{alpha} analyzed by mass spectrometry. LAP2{alpha} immunoprecipitated from mitotic or interphase cells was subjected to mass spectrometric analysis. Drawing shows domain organization of LAP2{alpha} and cdk-1 consensus phosphorylation sites. Phosphorylated residues identified by mass spectrometry are indicated. Circles denote unambiguously identified sites, rectangles are potential sites. Ser309 was found phosphorylated only in the mutated protein. Bars indicate regions of LAP2{alpha} covered in the analysis of samples from metaphase (M) and interphase (I) cells. Numbers represent amino acid position in the human LAP2{alpha} sequence; letters are amino acid sequence in single letter code. Serine to alanine mutations in the mutated proteins are indicated.

 
Serine 423 Is the Main cdk1-dependent Phosphorylation Site in Vitro—Searching for minimal cdk1-consensus phosphorylation sites in the LAP2{alpha} primary sequence, serine/threonine followed by proline (44), revealed two potential targets between LAP2{alpha} amino acids 410 and 615, serine 423 and threonine 547, the former being an optimal consensus site with a basic residue at position +2 ((S/T)PX(K/R)) (Fig. 3). Thus, we hypothesized that serine 423 may be the predominant cdk1-dependent in vitro phosphorylation site in LAP2{alpha}. To confirm this hypothesis, we exchanged serine 423 with an alanine by site-directed mutagenesis and tested the in vitro phosphorylation of LAP2{alpha} S423A mutant by mitotic extract. The specific phosphorylation of LAP2{alpha} S423A was reduced ~2-fold compared with wild-type protein (Fig. 4A), and the mutated protein phosphorylated by mitotic cell extracts (Fig. 4B) or cdk1 (not shown) did not generate the cdk1-dependent spot in two-dimensional peptide maps (arrow). Endogenous LAP2{alpha} from mitotic cell lysates was not detectable at significant levels because of its low concentration in the assay, and it may incorporate radioactive phosphate less efficiently than recombinant protein, as it was already phosphorylated at mitotic sites. In contrast to serine 423, mutation of serine 309 or serine 369 did not significantly reduce the mitotic in vitro phosphorylation of LAP2{alpha}, although the extent of phosphorylation of double and triple mutants containing the S423A mutation plus S309A and/or S369A mutations was further reduced as compared with that of the single S423A LAP2{alpha} mutant. Thus, serines 309 and 369 may be targeted by mitotic kinases with different efficiencies in wild type and S423A mutants.



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  		FIG. 4.
Mutation of serine 423 to alanine reduced in vitro phosphorylation of LAP2{alpha} significantly. A, recombinant LAP2{alpha} containing serine -> alanine mutations at indicated residues were phosphorylated in vitro using mitotic extracts, and cpm/nmol protein was determined as in Fig. 1. B, two-dimensional phosphopeptide mapping of the LAP2{alpha} S423A mutant. Arrow denotes the cdk1-dependent phosphopeptide.

 
Mass Spectrometry Reveals a Cluster of at Least Three Mitotic Phosphorylation Sites in Vivo—Our in vitro assays have shown that cdk1 phosphorylated LAP2{alpha} predominantly at serine 423. To test if this site is also phosphorylated in vivo and to identify additional potential targets for mitosis-specific kinases, we immunoprecipitated LAP2{alpha} from nocodazole-arrested metaphase and from interphase cells in the presence of kinase and phosphatase inhibitors and analyzed the proteins by mass spectrometric sequencing. To minimize contamination of interphase cell fractions with mitotic cells we removed round mitotic cells from adherent interphase cells by vigorous shaking prior to cell harvest. Mass spectrometry confirmed serine 423 as a mitosis-specific in vivo phosphorylation site in LAP2{alpha}, as fragments containing phosphorylated serine 423 were consistently detected only in the mitotic, but not in several independent interphase protein samples (Fig. 5A). The relative molecular mass of the tryptic peptide containing the phosphorylated serine 423 differed by only 0.05 Da from a non-phosphorylated form with a completely different sequence. The resolution of the QSTAR mass spectrometer was not high enough to separate the triply charged ions, but Fig. 5A clearly shows that the fragment spectra of the ions at m/z 715 Da are different in the samples prepared from the mitotic and interphase cells. The peptide VIEEEWQQVDR was present in both interphase and mitotic samples. Additional fragment ions were only detected in the sample prepared from the mitotic cells. These ions can be assigned to the peptide FQETEFLS*PPR containing the phosphorylated serine 423. The ion generated by the loss of a phosphoric acid residue from the parent molecule was also detected (labeled with an asterisk in Fig. 5A). In addition we observed phosphorylation at the minimal cdk1 consensus site at serines 369 (Fig. 5B) and 350 (Fig. 5C), as well as at serine 443 (data not shown), which does not represent a known kinase consensus motif. With the exception of serine 443, for which we never got sequence coverage in interphase samples, we showed that these phosphorylation sites were targeted exclusively in mitosis. Thus, LAP2{alpha} was phosphorylated during metaphase at least at four sites, including three mitosis-specific potential cdk1-dependent sites. Interestingly, all sites clustered within a 100-amino acid long region in the center of the previously identified chromatin-binding domain of LAP2{alpha} (36). In addition the mass spectrometric analyses provided evidence that serines 344 and 362 (Fig. 3), located within the identified phosphorylation cluster, may also be targeted in mitosis. However, the quality of the fragment spectra was not good enough to unambiguously localize the target sites within these peptides. Aside from the phosphorylation sites in the chromatin-binding region, mass spectrometric analyses uncovered permanent cell cycle-independent phosphorylation of serines 65 and 66 within a protein kinase CKII consensus motif (Fig. 3).



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  		FIG. 5.
Positive ionization product ion spectra of tryptic peptides. Peptides shown are at m/z 715 Da (A), acquired from the samples prepared from mitotic (containing phosphorylated serine 423) and interphase cells: at m/z 783 Da (B), containing the phosphorylated serine 369 from mitotic cells; at m/z 761 Da (C), containing the phosphorylated serine 350 in mitotic cells; and at m/z 503 Da (D), containing the phosphorylated serine 309 in mutated LAP2{alpha} in mitotic cells. Ions generated by the loss of a phosphoric acid residue from the phosphorylated parent ion were labeled with an asterisk (*).

 
Alternative Phosphorylation Sites Can Be Used in Mutated LAP2{alpha}—The clustering of mitosis-specific phosphorylation sites in the chromatin-binding region of LAP2{alpha} suggested that the dissociation of the protein from chromosomes in metaphase, as described previously (27), may be regulated by phosphorylation. To test the physiological relevance of LAP2{alpha} phosphorylation, we generated various LAP2{alpha} mutants containing different combinations of mutated phosphorylation sites (serine to alanine exchanges) and expressed C-terminally Myc-tagged mutants in HeLa cells using the doxycyclin-inducible expression system. None of the cell clones expressing LAP2{alpha} mutant protein with different single or multiple serine to alanine mutations showed a phenotype like mislocalization or unusual reorganization of mutant versus wild-type endogenous proteins in the cell cycle (data not shown).

To analyze this phenomenon in more detail, we focused on generating stable clones expressing LAP2{alpha} mutants to analyze the phosphorylation of mutated LAP2{alpha} in vivo. We obtained a stable cell clone expressing the LAP2{alpha} phosphorylation mutant S344A/S362A/S369A/S423A/S443A, in which two of the three mitotic in vivo targets as well as the potential phosphorylation sites at serine 344, 362, and 443 (see above) were mutated (Fig. 3); yet we did not observe any clear cellular phenotype. We precipitated mutated LAP2{alpha} from lysates of mitotic cells using antibodies to the Myc tag. As expected mass spectrometry revealed phosphorylation of serine 350, the cdk1 target site identified also in wild-type LAP2{alpha}, but surprisingly, the mutated protein contained an additional phosphate group at the cdk1 target serine 309 (Figs. 3 and 5D), which was never observed to be phosphorylated in the wild-type protein. Thus, we concluded that upon mutation of cdk1 target sites within the phosphorylation cluster, the cells used a different alternative phosphorylation site in mutated LAP2{alpha}, thus probably retaining a partial phosphorylation-dependent regulation.

Expression of a 7-fold LAP2{alpha} Mutant Caused a Stable Cellular Phenotype.—In view of the observed alternative phosphorylation in LAP2{alpha} phosphorylation mutants, we decided to mutate all potential cdk1 target sites within the phosphorylation cluster (serines 309, 350, 369, and 423) as well as potentially phosphorylated sites revealed by mass spectrometry of wild-type proteins (seines 344, 362, and 443) (Fig. 3) in order to avoid any alternative phosphorylation of LAP2{alpha} phosphorylation mutants in the cluster region in vivo. The 7-fold LAP2{alpha} phosphorylation mutant with a C-terminal Myc tag was stably expressed in HeLa cells using the doxycyclin-dependent expression system. Immunoblot analyses of cell lysates of stable clones, using antibodies to Myc or to LAP2{alpha}, revealed that the expression of the mutated protein was tightly controlled by doxycyclin (Fig. 6A). The level of ectopic protein was 2–3-fold higher than that of endogenous protein as checked by densitometric scanning of bands on the blots. As a control, expression of LAP2{beta} did not change upon addition of doxycyclin.



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  		FIG. 6.
Expressed 7-fold mutated LAP2{alpha} permanently associates with chromosomes. Stable HeLa Tet-on cells expressing LAP2{alpha} S309A/S344A/S350A/S362A/S369A/S423A/S443A mutant were cultured in the absence (-) or presence (+) of doxycyclin and cell lysates analyzed by immunoblotting (A) using antibodies to Myc, to the LAP2 N terminus (LAP2), or to LAP2{alpha}, or processed for immunofluorescence microscopy (B, C, E, F) or for preparation of chromosome spreads (D) using antibodies as indicated. Epifluorescence (C) or confocal (B, D–F) images are shown. Bars in B (for B, C, E, F) and in D represent 10 µm.

 
In order to specifically detect the mutated LAP2{alpha} in mitotic cells we performed immunofluorescence microscopy using an antiserum to the Myc tag. Mutated LAP2{alpha} was predominantly localized on chromosomes in metaphase and anaphase, while endogenous LAP2{alpha} in untransfected cells was cytoplasmic at these stages of the cell cycle (Fig. 6B). In order to detect both ectopic mutated and endogenous wild-type LAP2{alpha} in stable cell clones we stained the cells with monoclonal antibody 9E10 to the Myc tag and antiserum to LAP2{alpha}, which detects both ectopic and mutated protein. These studies revealed that endogenous LAP2{alpha} was still released from chromosomes in cell clones expressing mutated protein (Fig. 6C). Interestingly, the LAP2{alpha} antibody seemed to react only weakly with mutated protein, which can be explained by tight association of mutated LAP2{alpha} with chromosomes causing epitope masking. Together these data indicated that mutated LAP2{alpha} bound tightly to chromosomes, but did not interfere with the dissociation of wild-type endogenous LAP2{alpha}. To confirm the tight association of mutated LAP2{alpha} with metaphase chromosomes, we generated chromosome spreads of stable clones and performed immunofluorescence microscopic analysis. The Myc-tagged protein was clearly detected all over the chromosomes (Fig. 6D, +Doxycyclin), whereas endogenous protein in cells not expressing the mutated LAP2{alpha} (Fig. 6D, -Doxycyclin) localized exclusively to the tips of chromosomes.

The LAP2{alpha}-binding partner in interphase cells, lamin A (Fig. 6E), and other lamina proteins (data not shown) were unaffected in mutant cells and were exclusively cytoplasmic during metaphase/anaphase. Thus, lamin A also efficiently dissociated from chromosomes and mutated LAP2{alpha}, indicating that the LAP2{alpha}-lamin interaction was not regulated by phosphorylation of LAP2{alpha}, but may involve lamin phosphorylation. Mutated, unphosphorylated LAP2{alpha} remained firmly bound to chromosomes throughout completion of mitosis, while wild-type LAP2{alpha} accumulated efficiently at chromosomes in telophase (Fig. 6C), similar to untransfected control cells (compare Refs. 27 and 36). In interphase, both mutated and wild-type protein localized throughout the nucleus, although the merged image indicated a more peripheral localization of endogenous versus mutated protein. Since mutated LAP2{alpha} in the center of the nucleus was weakly stained by the antiserum to LAP2{alpha},we concluded that also in interphase the epitopes in the mutated protein may be masked, possibly reflecting the strong association of mutated protein with chromosomes.

Altogether, we observed a mislocalization of mutated LAP2{alpha} at chromosomes in mitosis, demonstrating the essential role of LAP2{alpha} phosphorylation in its dynamic behavior during the cell cycle. Nuclear envelope reassembly following sister chromatid separation was, however, not affected, as indicated by the translocation of nuclear membrane protein LAP2{beta} to chromosomes in telophase and formation of a continuous nuclear envelope around chromatin in interphase (Fig. 6F).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this article we have shown that LAP2{alpha} is predominantly phosphorylated by mitotic kinases in its chromatin-binding domain, and demonstrated the in vivo significance of LAP2{alpha} phosphorylation in its dynamic behavior during mitosis.

In Vitro versus in Vivo Phosphorylation—Several observations indicated that serine 423 is the major cdk1 phosphorylation site in LAP2{alpha}. (i) Mutation of this site reduced in vitro phosphorylation by 50%. (ii) Serine 423 represents a classical cdk1 consensus motif followed by a basic residue in position +2 (44) and is conserved between mouse and humans. (iii) Mass spectrometry of LAP2{alpha} immunoprecipitated from mitotic and interphase cells confirmed serine 423 as a mitotic phosphorylation target in vivo.

Besides the major site at serine 423, we unambiguously identified two additional mitosis-specific minimal cdk1 consensus sites and one site of unknown kinase specificity and interphase modification. In addition we also obtained evidence for two additional phosphorylation sites of unknown kinase specificity in the vicinity of these sites. Although mass spectrometry did not allow distinguishing major from minor phosphorylation sites, serine 423 may also represent the major target site in vivo, assuming that all kinases phosphorylating LAP2{alpha} in vivo are also present in the mitotic extract used for the in vitro phosphorylation assays. Nevertheless, if serine 423 were the major target site in vivo, its phosphorylation was not sufficient for regulating LAP2{alpha} association with chromosomes, because expression of LAP2{alpha} mutants containing a single serine to alanine mutation at residue 423, behaved like the wild-type protein.

Previous studies by Dreger et al. (45) have identified five phosphorylated sites in the membrane-anchored LAP2 isoform, LAP2{beta}, during interphase in neuroblastoma Neuro2a cells. Although all sites were located in the N-terminal constant region of LAP2{beta}, which is identical to the N terminus of LAP2{alpha}, we did not find any of these sites phosphorylated in LAP2{alpha} in HeLa cells. Instead, we identified a permanent phosphorylation in the N terminus within a casein kinase II phosphorylation consensus motif. Thus, the isoforms LAP2{alpha} and LAP2{beta} may be differentially phosphorylated in the common N terminus, consistent with their different cellular localization, or alternatively, LAP2 proteins are differentially phosphorylated in a cell type-specific manner in interphase.

Significance of LAP2{alpha} Phosphorylation—The importance of lamina protein phosphorylation for nuclear disassembly has been postulated many times based on the observation that lamina proteins were phosphorylated in a mitosis-specific manner, that phosphorylation correlated with lamin-complex disassembly, and that phosphorylated proteins failed to interact with their binding partners (for review see Ref. 46). However, except for lamins (35), phosphorylation sites have not been identified in most lamina proteins, and the significance of phosphorylation for nuclear disassembly has not been demonstrated in vivo. Here, we show for the first time for a lamin-binding protein that phosphorylation is essential for its dissociation from chromosomes during mitosis in cells. LAP2{alpha} has previously been shown to localize to the cytoplasm in metaphase, but assembled at chromosomes prior to nuclear membrane formation (36). Furthermore, chromatin-binding LAP2{alpha} fragments dominantly inhibited nuclear envelope assembly in an in vitro assay (37). As the early targeting of LAP2{alpha} to chromosomes may be an essential step in nuclear assembly, knowledge of the molecular mechanisms regulating LAP2{alpha} redistribution is crucial for understanding functions of the protein.

Strikingly, our studies revealed mitotic phosphorylation of LAP2{alpha} at at least four sites clustered in a C-terminal domain that has previously been found to be essential and sufficient for chromatin binding of LAP2{alpha} (36). All four target sites as well as alternatively used sites in the mutant had to be replaced in order to detect a stable cellular phenotype. This may be caused by the fact that even minor phosphorylation in this region may restore partial regulation. Alternatively, mutated protein may form complexes with wild-type endogenous LAP2{alpha} and allow sufficient phosphorylation-dependent regulation of the LAP2{alpha} oligomer. Expression of a mutant LAP2{alpha} with seven mutated serines in the phosphorylation cluster revealed a stable cellular phenotype, in which mutated LAP2{alpha} was constitutively associated with chromosomes throughout cell division. Interestingly, the localization of the LAP2{alpha}-binding partner lamin A was not affected, indicating that the interaction of LAP2{alpha} with lamin A is not regulated by LAP2{alpha} phosphorylation. This observation is consistent with the localization of the lamin A-binding domain in the extreme C terminus of LAP2{alpha} (26), which was not found to be phosphorylated.

Although the 7-fold mutated LAP2{alpha} bound tightly to chromosomes throughout mitosis, nuclear assembly was not affected. This phenomenon can be explained by the proposed function of LAP2{alpha} in assembly. Based on our previous in vitro nuclear assembly studies, the binding of the LAP2{alpha} C terminus to chromosomes in early stages of nuclear assembly may target the DNA cross-bridging protein BAF to the chromosomal surface, which binds to the LAP2{alpha} N-terminal domain (for review see Ref. 46). In line with this model, addition of chromatin-binding LAP2{alpha} fragments missing the BAF interaction domain to the nuclear assembly reactions inhibited assembly, while bacterial full-length LAP2{alpha}, which bound both chromatin and BAF had no effect (37). Assuming that the LAP2{alpha} phosphorylation mutant can bind to BAF, one would not expect a phenotype in nuclear assembly. However, if this model is correct, it remains unclear, why LAP2{alpha} has to dissociate from chromosomes at the onset of mitosis.

Another important question concerns the individual contributions of the four LAP2{alpha} phosphorylation sites to the dynamics of the protein. First, multiple phosphorylation could simply ensure efficient dissociation. Second, sequential phosphorylation or dephosphorylation of the different sites during disassembly and assembly, respectively, may regulate different interactions of the protein and thus, regulate and fine-tune the assembly process.


   FOOTNOTES
 
* This study was supported by grants from the Austrian Science Research Fund (FWF, P15312 [GenBank] ), from the Jubiläumsfonds of the Austrian National Bank, and from the Österreichische Muskelforschung (to R. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a fellowship from the Deutsche Forschungsgemeinschaft (DFG). Back

|| To whom correspondence should be addressed: Max. F. Perutz Laboratories, University Depts. of the Vienna Biocenter, Dept. of Medical Biochemistry, Medical University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria. Tel.: 43-1-4277-61680; Fax: 43-1-4277-9616; E-mail: Roland.Foisner{at}univie.ac.at.

1 The abbreviations used are: LAP, lamina-associated polypeptide; LEM, LAP-emerin-MAN1 protein family; PBS, phosphate-buffered saline; DTT, dithiothreitol; CHO, Chinese hamster ovary cells; cdk, cyclin-dependent kinase; MS, mass spectrometry. Back


   ACKNOWLEDGMENTS
 
We thank Larry Gerace, The Scripps Research Institute, La Jolla, CA, for providing the antiserum to cdk1, and Fritz Propst, University of Vienna, for the antiserum to Myc.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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