(Received for publication, September 15, 1996, and in revised form, November 5, 1996)
From the 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
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.
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.
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.
[-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.
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-
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).
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 PhosphorylationChicken 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 [-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
[-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 AnalysisProteolytic 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 MethodsSDS-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).
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.
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.
|
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).
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).
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-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-
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.
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,
rotein
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.
|
This article is dedicated to the memory of Prof. Nikolaos Alexandrou.
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.