Phosphorylation of the Nuclear Lamins during Interphase and Mitosis

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THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1985 by The American Society of Biological Chemists, Inc. Vol. 260, No. 1, Issue of January 10, _{Printed in U.S.A.}pp. 624432,1985

Phosphorylation of the Nuclear Lamins during Interphase and Mitosis”

(Received for publication, July 12, 1984)

Yvonne Ottaviano and Larry GeraceS

From the Department of Cell Biologv and Anatomv. The Johns Hopkins University School of Medicine,

Baltimore, Maryland 21205 -” ” .

The nuclear lamina is a polymeric protein assembly that is proposed to function as an architectural frame- work for the nuclear envelope. Previous work sug- gested that phosphorylation of the major polypeptides of the lamina (the “lamins”) may induce disassembly of this structure during mitosis. To further investigate the possible involvement of phosphorylation in regu- lation of lamina structure, we characterized lamin phosphorylation occurring in mammalian tissue cul- ture cells during interphase and mitosis.

Phosphorylation occurs continuously throughout all interphase periods (coordinately with nuclear envelope growth), and takes place mainly on the assembled lam- ina. When the lamina is disassembled during cell divi- sion, the lamins are modified with approximately 1-2 molecules of associated phosphate. This level of mitotic phosphorylation is 4-7-fold higher than the average interphase level. Lamin phosphate occurs predomi- nantly as phosphoserine, and is distributed over nu- merous tryptic peptides, many of which are modified during both interphase and mitotic periods. Signifi- cantly, phosphorylation is the only detectable charge- altering postsynthetic modification of the lamins that occurs specifically during mitosis. The results of this study support the notion that phosphorylation is im- portant for regulation of interphase and mitotic lamina structure.

The nuclear lamina is a protein meshwork closely associated with the nucleoplasmic surface of the nuclear envelope, that forms a shell-like structure at the nuclear periphery (see Refs. 18, 19, and 22 for reviews). The lamina is a widespread or ubiquitous nuclear envelope component (Ref. 16, other Refs. in 20) that is postulated to provide a framework for nuclear envelope organization (20,21, 23). This structure has also been suggested to serve as a major chromatin anchoring site during interphase (20, 25, 36) that is possibly involved in organizing higher order chromatin domains (e.g. Refs. 2, 10).

Biochemical fractionation studies have demonstrated that in a number of different cells, the lamina is stable in both low- and high-ionic strength buffers, and requires neither phospholipid nor chromatin for its structural integrity (Refs. in 22). These features have permitted the isolation of lamina- enriched fractions from several cell types (e.g., 1, 12, 30, 31,

45,46). The lamina of rat liver nuclear envelopes is comprised predominantly of three 60-70-kilodalton polypeptides (lamins

A, B, and C) that are suggested to form a polymeric assembly

(20, 22).

Three analogous lamins have been detected in numerous *This work was supported by a Searle Scholars Award and a National Institutes of General Medical Sciences grant to L. G . 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 t o indicate this fact.

$ To whom correspondence should be addressed.

other vertebrate somatic cells by immunochemical and biochemical procedures (e.g. Refs. 22, 32, 46). These three polypeptides appear to be evolutionarily related, since two of the polypeptides (lamins A and C) are very similar by peptide mapping (28, 46), and since all three lamins cross-react with certain monoclonal antibodies (7, 32). While many vertebrate somatic cells contain three distinct lamins, certain other cell types appear to contain only one or two major lamin-related polypeptides (31, 32,40).

Studies on vertebrate tissue culture cells have demonstrated that the lamina undergoes major architectural changes during cell division (14, 20, 21, 29, 49). When the nuclear envelope is disassembled during mitotic prophase (15, 19, 44), the lamins are depolymerized and become dispersed throughout the cytoplasm as monomers (21). Subsequently, the lamins reassemble at the surfaces of the daughter cell chromosomes during telophase (12, 20, 29), when the nuclear envelope is reconstructed. In view of the skeleton-like structural and biochemical properties suggested for the interphase lamina, the reversible lamina depolymerization that occurs during cell division may regulate the mitotic disassembly and reconstruc- tion of the nuclear envelope (20, 22).

We previously observed that the lamins are modified by phosphorylation in both interphase and mitotic populations of tissue culture cells (21). During mitosis when they are disassembled, these polypeptides have a substantially elevated level of associated phosphate compared to interphase, sug- gesting that phosphorylation/dephosphorylation may be im- portant for regulating the mitotic reorganization of the lamina

(21). To obtain further insight into the possible role of phosphorylation in control of lamina structure, we have performed a detailed biochemical characterization of the lamin phosphorylation that occurs in vivo in CHO’ cells during interphase and mitosis. These results support the hypothesis that lamin phosphorylation is important for modulation of nuclear lamina structure during cell division, and also suggest that phosphorylation may be involved in regulation of interphase lamina organization.

EXPERIMENTAL PROCEDURES~ RESULTS

Interphase Lamin Phosphorylation-We examined several of the major physiological characteristics of lamin phospho- ’The abbreviations used are: CHO, Chinese hamster ovary; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; TPCK, L-1-tosylamido-2-phenylethyl chloro- methyl ketone.

*

“Experimental Procedures” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-2134, cite the authors, and include a check or money order for $1.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

624

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Nuclear

Lamin

Phosphorylation

625

rylation that takes place in growing interphase CHO cells, to

obtain information on possible functions of this interphase modi~cation. For this analysis, we investigated the state of assembly of the interphase phosphorylated lamins, and the cell cycle timing of phosphorylation.

Most of the interphase lamins in growing tissue culture cells occur in an assembled lamina structure, but a small pool of unassembled lamins (including newly synthesized proteins) is also present? To examine whether the phosphorylated interphase lamins occur in the assembled or unassembled pool, we lysed 32P-labeled CHO cells in a Triton-containing buffer in which the assembled lamina i s insoluble (21). Fob lowing centrifugation, the lamins were immunoadsorbed from supernatant and pellet fractions with affinity-puri~ed antibodies to determine the subcellular distribution of these polypeptides (Fig. 1). Cultures labeled with ~35S~rnethionine for various time periods were also fractionated by this procedure, to examine the characteristics of lamin assembly associated with growing CHO cells.

When interphase cells labeled for 16 h with [35S]methionine

are fractionated after Triton lysis, the labeled lamins (grossly reflecting the total lamin mass) occur almost exclusively in the pellet fraction containing the assembled lamina (Fig. lA, 16 h, s and p lanes). In contrast, newly synthesized lamins from cells pulse-labeled for 5 min with [35S]methionine occur to a large extent in the supernatant, apparently representing unassembled proteins (Fig. lA, 5‘, s, and p lunes). When

pulse-labeled cells are chased in medium containing nonra- dioactive methionine for 60 min, almost all of the labeled lamins A and

B

and the majority of lamin C now occur in the pellet fraction (Fig. lA, 5’

+

60’ chase, s, and p lanes), presumably reflecting entry of soluble proteins into the assembled lamina.

Lamin A is initially synthesized in uiuo in CHO cells (Fig.

lA, arrotus) and other cell lines as a species (lamin

A,)

that

migrates approximately 2000 daltons more slowly than lamin

A, and that is converted to the latter only subsequent to integration into a Triton-insoluble ~tructure.~ After a 5-min pulse label approximately half of lamin A, occurs in the pellet fraction (Fig. lA, 5‘, s, and p lanes, arrows) but has not yet been converted to lamin A. Much of this conversion has occurred after an additional 60 min (Fig. lA, 5’

+

60’ chase,

arrow). A similar apparent biosynthetic precursor to lamin A

is evident from in vitro translation of messenger RNA from tissue culture cells (34).

When CHO cells labeled with [32P]phosphate for 45 min are analyzed by this fractionation procedure, it is apparent that all of the initially incorporated phosphate is recovered after the fractionation procedure, and that the label occurs almost exclusively in the assembled lamina of the pellet (Fig.

IS,

45‘, compare t, s, and p lanes).

A

similar distribution of lamin-associated phosphate is observed in cells labeled with [”Plphosphate for 10 min, when a substantial fraction of lamins synthesized during the period of the pulse are still in the supernatant (Fig. lB, 10’ lanes). Fu~herrnore, in both labeling periods, only the lamin A species (which arises after integration into the lamina) is phosphorylated, and not the lamin A, species (which is partially soluble). These results indicate that at least most of the interphase lamin phosphorylation that we detect actually takes place on the lamina, and does not derive from proteins that are phospho~lated when soluble and only subsequently integrated into the assembled structure. (If phosphorylation occurred while the lamins were soluble, a significant proportion of the phospho-

Gerace, L., Comeau, C., and Benson, M., J. Cell S c i , in press,

rylated lamins should occur in the supernatant in the 10-min

32P pulse label, and lamin A, should be phosphorylated.) These results do not exclude the possibility that unassembled lamins are phosphorylated, but if this occurs, it must be quantitatively minor.

To determine whether the interphase lamin phosphorylation that we detect is restricted to a specific segment of the cell cycle, such as the period of DNA synthesis or

_Gz

phase, or whether it occurs continuously throughout interphase as does lamin biosynthesis: we examined lamin phosphorylation in synchronized cell populations. Metaphase CHO cells were obtained by selective mechanical detachment and at progressive 2-h intervals over a period of one cell cycle, individuai cell aliquots were pulse labeled with [3*P]phosphate prior to immunoadsorption of the lamins and SDS-gel electrophoresis (Fig. 2 ) .

In our synchronized populations, the earliest time when a significant number of cells have entered S phase as determined by [3H]thymidine incorporation (Fig. 2, bottom) is 7 h after metaphase.

DNA

replication reaches a peak at 11 h, and cells begin entering mitosis in substantial numbers at 15 h (at which point 15% of the population is in the prophase-telophase period). Therefore, the 1-5-h samples contain GI phase

cells, and 15 h populations are enriched in Gz cells. As shown in Fig. 2, the total incorporation of 32P into the lamins is roughly similar at all progressive periods of interphase ex- amined from 1 to 15 h, with no pronounced peaks of phosphate inco~oration at specific cell cycle stages. Furthermore, the lamins at each synchronized interphase time point incorpo- rate a pro~rtionately similar level of phosphate label as the proteins in an asynchronous exponentially growing interphase sample (Fig. 2, EXP lane). Since this continuous pattern of lamin phosphorylation through interphase parallels the pat- tern of interphase nuclear envelope growth (19,39) and lamin biosynthesis~ much of the interphase phospho~lation may be related to lamina restructuring and/or assembly.

Biochemical Analysis of Lamin-associated Phosphate-We compared the salient phosphorylation characteristics of the lamins in interphase and metaphase cells, to identify chemical differences that occur between these two cell cycle populations, and to establish a basis for future studies of lamin protein kinases. For interphase phosphate labeling, we used asynchronous cultures of exponentially growing interphase cells, which reflect the “average” interphase phosphorylation characteristics of the lamins. In our metaphase cell popula- tions, phosphate labeling of the lamins occurs within 30 min prior to mitotic selection, and therefore represents lamin phospho~lation that takes place during or immediately prior to the period of prophase disassembly of the lamina (see “Experimental Procedures”). The apparent rate of incorporation of phosphate into the three lamins (collectively) of interphase cells is roughly 20% of the level of the prophase- metaphase incorporation with a 45-min label {data not shown).

As shown in Fig. 3, treatment of 32P-labeled immunoad- sorbed lamins with bacterial alkaline phosphatase before gel electrophoresis results in complete removal of the labeled phosphate from both interphase and mitotic proteins (Fig. 3, 32P,

I

and M, +APase lanes). The disappearance of radioactive label from alkaline phosphatase-treated samples is not a consequence of proteolytic degradation, since %-labeled lam- ins are intact following phosphatase treatment (Fig. 3, ”S, I

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626

Nuclear Lamin

Phosphorylation

La A-

La B-

La

C-

A)

35S

16 hr

SI

60'chase

S

P

S

P

s

P

(NI)

B) 32P

45/

lo1

t

S

P

t

S

P

Flc;. 1 . Subcellular distribution of the interphase phosphorylated lamins. Cultures o f interph;~se C'HO

cells were labeled with [""Slmethionine ( A ) for either I 6 h or 5 min, or with [~"'I']phosphate (HI for 45 min or 1 0 min. One sample o f cells that was laheled with [""Slmethionine for 5 min was suhsequently chased lor 60 min in nonradioartive medium containing 0.005 M methionine ( A , 5 '

+ 60'

rhnsr). Cells were then lysed in a huffer containing 1'; Triton S-100 and fractionated hv centrifugation to ohtain supernatants ( S I and pellets (PI. For "I'

samples. one-half of the total cells ( t ) was frozen immediatelv after harvesting, and proressed for imm~lnoadso~)ti~)n in parallel with the other samples to examine whether in vitro dephosphorvlation ocrurred during frartionntion o f lysates. Irnn~unnadsorl,ed lamins ( I d . LnH, and I d ' ) were then electrophoresed o n a n SDS pel a n d nppropriatr.ly visualized. The apparent prerursor to lamin A (lamin A,) is indicated hv downward pointing orrows l r . . ~ . A . 5' .\

and p Innvs). Nonimmune guinea pig Ig(; incuhated with a sample of ["'Slmethionine-laheled rells ( A . .VI Innvl. or with [:"l']lalwled cells (data not shown) does not result in selertion offi0-71-kilodalton laheled hands. tlemonstratinp the antihod?: specificity nf our adsorption procedure. For the IO-rnin "*P-Iaheling condition. the effective duration o f t h e pulse is artrtallv less than 5 rnin, since little radioactive phosphate is inrorporatetl into the lamins during the first :i min of the laheling period (data not shown). A minor amount of :"I'-lal)eletl lamins orrurs in the supernatant for the IO-min laheling condition, hut not for the 45-min sample. We attrihute this t n the presenre o f prophase cells (containing disassernhled lamins) in the 10-min sample, which are virtually ahsent in rultures Iaheled lor 45 min (presumably due to the longer period of phosphate starvation in the Intterl.

demonstrate that the lamin-associated phosphate does not scribed above, suggest that the lamin-associated phosphate

occur as ADP-ribose ( X ) , which would he insensitive to could occur as phosphoserine, phosphothreonine, or phospho- alkaline phosphatase hydrolysis. tyrosine (3, 27). To determine which of these phosphorylated

Most or all of the phosphate incorporated into the inter- amino acids is present in the lamins during interphase and phase and mitotic lamins remains protein-associated upon metaphase, individual '"P-labeled lamins purified hy immu- incuhation in acid, and is hydrolyzed by alkaline treatment noadsorption and SDS-gel electrophoresis were hydrolyzed in

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Nuclear Lamin Phosphorylation

627

1

3

5

7

9

11

13

15 EXP

6 - 4 -

2-

1 3 5 7 9 11 13 15

HOURS AFTER MITOSIS

FIG. 2. Phosphorylation of the lamins through the cell cycle. Metaphase CHO cells were selected from mbnolaver cultures by mechanical detachment, and aliquots of 1 X

lo6

cells were plated into Petri dishes and

returned to 37 "C culture. At the end of 1 h, and at 2-h intervals thereafter, individual dishes were labeled for 45 min with [R2P]phosphate and cells were harvested. A culture of 1.2 X lo6 asynchronous exponentially growing

interphase cells ( E X P lane) was labeled with phosphate under identical conditions. The lamins ( L a A , LaR. and

L a 0 were suhsequently immunoadsorhed from solubilized samples and were electrophoresed on an SDS gel prior to visualization by autoradiography. In parallel with the '*P labeling, separate cell aliquots ('2 X lo5 cells) were laheled with [I4C]thymidine, to monitor incorporation of trichloroacetic acid-insoluble counts (reflecting DNA renlication). The autoradioeram of 32P-laheled lamins is shown above the corresponding determination of ["C] thymidine incorporation for each time point.

sional separation on cellulose thin-layer plates to resolve phosphoserine, phosphothreonine, and phosphotyrosine. As shown in Fig. 4, phosphoserine is the predominant phospho- rylated amino acid in all three lamins from both interphase and mitotic cells. Phosphothreonine is a considerably more minor component, and labeled phosphotyrosine is absent. No pronounced difference is noted in the phosphoserine/phosphothreonine ratio for any of these samples.

We compared the specific sites on the lamins that are phosphorylated during interphase and mitosis by performing two-dimensional tryptic peptide mapping on individual '"P-

labeled lamins purified by immunoadsorption and gel electrophoresis (Fig. 5). Because of the short-term phosphate labeling conditions used in this experiment, the peptide maps describe the rate of phosphorylation (including phosphate turnover) of specific tryptic peptides, but not necessarily the absolute level of peptide-associated phosphate. Also, since a single tryptic peptide can contain several phosphorylation sites, each phosphorylated spot on the two-dimensional maps represents a unique phosphorylated configuration of a specific peptide, but not necessarily a unique phosphorylation site.

A large number of distinct phosphorylated tryptic peptides (as many as 12) are reproducibly obtained in maps of all three

lamins from interphase and mitotic cells (Fig. 5). However, in each case much of the phosphate label occurs on a small number (2 or 3) of peptides. The interphase and mitotic phosphorylated peptide maps for lamin A are similar to the corresponding maps for lamin C, but distinct from those for lamin B, as expected from previous peptide mapping studies on the lamins (28, 46).

When the interphase phosphorylated peptide map for each lamin is compared to the mitotic map for the same polypep-

tide, many common spots (both major and minor) are apparent (Fig. 5). The most extensive similarities occur for lamin B, where most of the interphase and mitotic phosphorylated spots co-migrate and are present in the same relative intensities in both samples. In contrast, while common phosphorylated spots also occur in the interphase and mitotic maps for lamins A and C, the relative intensities of these common spots differ between the interphase and mitotic samples. Furthermore, interphase lamins A and C each contain two major phosphorylated peptides (Fig. 5, I panels, arrows) that

do not appear in the corresponding mitotic cell peptide maps, and the mitotic lamins A and C samples contain a major phosphorylated spot (Fig. 5,

M

panel, arrows) that is not

evident in interphase samples. Therefore, some of the major phosphorylated sites on lamins A and C may be modified during either interphase or mitosis specifically (and not during both periods). This would provide cell cycle stage-specific markers for i n uiuo phosphorylation of these polypeptides.

We previously determined the relative levels of interphase and mitotic phosphorylation of the lamins in CHO cells, using steady-state labeling with ["Hlleucine and ["P]phosphate

(21). T o quantitate the absolute level of lamin phosphoryla-

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35s

Nuclear Lamin Phosphorylation

32P

I

M

I M I M

La

A

. .

_...

lyr(P)

- +

-

+

-

+

APase " . Thr(P1

(.)

Ser(P)

La

B

La

C

FIG. 3. Sensitivity of lamin-associated phosphate to alkaline phosphatase. The lamins ( I d . ImH. and I d ' ) were immu- noadsorbed from interphase and mitotic CHO cells that had been labeled with [""SJmethionine (for 16 h ) or with ["'I'jphosphate (for

45 min). Samples were then incuhated with (+) o r without (-)

I.:schc,richia coli alkaline Phosphatase (Al'asa) and suhsequently elec- trophoresed on an SDS gel and appropriately visualized.

interphase. This represents a 4-7-fold relative increase for mitotic phosphorylation of the lamins, in agreement with the values we previously obtained (21).

The analysis described above demonstrates that a high level of phosphorylation is a significant mitotic-specific biochemical characteristic of the lamins. T o investigate whether additional post-translational modification(s) besides phosphorylation are also associated with these proteins specifically during mitosis, we analyzed interphase and mitotic lamins labeled with ["'SSjmethionine by two-dimensional nonequilibrium pH gradient/SDS-gel electrophoresis (Fig. 6). Most common postsynthetic protein modifications that are readily reversible in

vivo (including phosphorylation) induce a change in protein

isoelectric point (52), and would be detectable by this two- dimensional analysis.

Examination of immunoadsorbed interphase lamins on a two-dimensional gel (Fig. 6, I, -AP panel) reveals two pre- dominant charge isoforms for interphase lamins A and C, and a single major form for lamin

€3.

CHO cell lamins A and C

are isoelectric a t approximately pH 7-7.5, while lamin I3 has an isoelectric point near pH 6.0 (21). Lamins A and C show minor satellite spots adjacent to the major charge species (a phenomenon that is especially evident in Fig. 6, + A P p a n e k ) . These minor satellite spots do not necessarily represent actual

in vivo charge isoforms of the lamins, but could result from

nonuniform binding of ampholytes to these polypeptides, for example (8).

When the two-dimensional electropherograms of the interphase and mitotic lamins are compared (Fig. 6 ,

I

and

M,

-AP

FIG. 4. Phosphorylated amino acids present in t h e laminn. The three lamins ( I d , IAR. and /A(') were immunoadsort)ed from R'P-labeled interphase ( I ) and metaphase ( M I cells (see "Experimen-

tal Procedures"). Individual lamin hands from SI)S gels of these immunoadsorbed samples were then eluted. partially hydrolyzed in 6

N HCI, and separated on cellulose thin-layer plates by electrnphoresis

( E ) followed hy chromatography ((7. The migration positions o f nonradioactive phosphoserine. phosphothreonine. and phosphotvro- sine ohtained on a parallel plate (stained with ninhydrin) are indi- cated. Approximately equal ?'I' counts were analyzed for each sample.

panels), it is apparent that the lamins from mitotic cells migrate as more acidic isoelectric species than the interphase polypeptides, as previously observed (21). This acidic charge shift is consistent with the increased level of mitotic lamin phosphorylation (Table I). In this two-dimensional analysis, mitotic lamin I3 occurs almost exclusively as a single species (Fig. 6,

M,

-AP panel), which actually appears as a closely spaced doublet upon short fluorographic exposure (data not shown). Mitotic lamins A and C are predominantly one or

two major charge isoforms (Fig. 6,

M.

-AI'pand).

We took advantage of the observation that the interphase and mitotic lamin-associated phosphate can be quantitatively removed with alkaline phosphatase (Fig. 3 ) to determine whether the mitotic charge shift of the lamins is entirely the result of phosphorylation. For this experiment, immunoadsorbed interphase and mitotic lamins were treated with al-

kaline phosphatase before two-dimensional gel electrophoresis (Fig. 6, +AP panels). The interphase lamins A and C

incubated with alkaline phosphatase each shift to a single predominant spot (Fig. 6,

I,

+APpanel) that co-migrates with

the more basic of the two charge isoforms of these respective polypeptides in the interphase untreated sample (Fig. 6 , I ,

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I

La

A

La

B

0 La C

C

m

P

.)

.

*

I + M

e - "

interphase alkaline phosphatase-treated sample.

Significant Iy, these results demonstrate that phosphoryla-

t i c ~ n is the only detectable charge-altering modification of the lamins that occurs specilically during mitosis. Since we find

no wiclrnce I'or other major mitot ic-specific postsvnt hetic modificat ions 01' the Itlmins besides phosphorylation, this result st renkfl hens o u r hypothesis that lamin phosphorylation is important I'or mediating disassemt)ly of the nr~clear lamina

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630 Nuclear

Lamin Phosphorylation

('HO cells were grown in medium rontaining ["'I'lphosphate lor

'$8 h. ant1 the lamins were immunoadsort)ed from exponentially grow- ing interphase populations (1) and synrhronized metaphase cells (M). _v) Al'ter electrophoresis on an S I X gel and staining with Coomassie

P

Hlue, the protein mass in individual lamin bands was determined v) colorimetrically using bovine serum albumin as a standard (17). Subsequent Iv, the moles o f phosphate in each lamin were determined I y scintillation count inn. Values represent the average oltwo separate

experiments (whirh in all rases dillered by no more than 1 0 % ) .

~~ ~ ~~~~ ~ ~- ~~ M I MfI ~ ~ ~~ ~ ~ ~ ~ _ ~~~~ _ _ ~ m r d /',fm111 lnrnirl 1,amin A 2.2 0.46 4.R 1,amin 13 1.9 0.27 7.0 l a m i n (' 1.4 0 . 3 3 4.2 _ _ _ _ _ _ _ ~ . ~

get her with the absolute levels of interphase and mitotic lamin phosphorylat.ion discussed above (Table I), suggest that less than 50% of lamins A and C are phosphorylated in interphase cells, and occur mainly in a monophosphorylated form, while almost all of these polypeptides are phosphorylated in mitotic cells, and are found as di- and triphosphorylated species.

Similarly, essentially all of the mitotic lamin B may be modified as a mono- or diphosphorylated species.

DISCUSSION

We have characterized interphase and mitotic phosphorylation of the major structural components of the nuclear lamina (the lamins) to further investigate the possible relationship of phosphorylation to modulation of lamina structure. In a

large number of other biological systems, protein phospho-

rvlation/dephosphorylation is known to be an important

mechanism for regulation of protein function (reviewed in Kef. 9).

The interphase lamin phosphorylation that we detect takes place predominantly on a Triton-insoluble lamina structure, and is not significantly present in the intracellular pool of unassemhled lamins. This presumably reflects the presence of specific lamin protein kinases associated with the lamina structure (and nuclear envelope) of growing interphase cells. Protein kinase activities have been described in preparations of isolated rat. liver nuclear envelopes (3.5, 48), but it has not

I

0 - La A

r

M

-

La

B

La C I+M

I

M

been determined whether the lamins are among the _invitro

phosphorylation products obtained with these predominantly

G C , stage nuclear envelopes. In addition to lamins of growing acidic

"

+ +

t t

"AP

+AP

$. basic

tissue culture cells, the apparent lamin of amphibian oocyte nuclear envelopes has been shown to be phosphorylated _in

I ~ i U O ( 3 1 1.

Using synchronized populations of interphase cells, we determined that lamin phosphorylation occurs at roughly simi-

lar rates at all stages of interphase, and is not restricted to

any specific cell cycle period such as S phase (where it could be related to DNA replication) or G, phase (where it could be related to preparation for mitosis). Since the lamins are

phosphorylated continuously throughout interphase in con- cert with the continuous patterns of lamin biosynthesis:' and nuclear envelope growth (19, 39), it is possible that a significant part of this interphase phosphorylation is related to

growth and/or restructuring of the lamina.

Our biochemical analyses indicate that the interphase (as-

sembled) lamins have approximately 0.27-0.46 mol of associated phosphate/mol of protein, while the mitotic (disassembled) proteins have 1.4-2.2 mol of phosphate/mol of lamin (4-7-fold higher than interphase for each protein). Most or

all ofthe interphase lamins A and C that are phosphorylated (approximately one-third of the population) occur as appar-

FIG. 6. Two-dimensional gel analysis of interphase and mi-

totic lamins. C H O rells were Ial~eled with [ '"S]mc*thioninv. ;Ind metaphase cells were ohtainetl by selective mc*rhanicaI tlc.t;lc.hmc.nt. The three lamins were then immunoadsorl~ed from solrhilizcd mc*t;v phase ( M ) cells and exponentially growing interphase ( ~ 1 1 s I / , . I n - munoadsorhed samples were separated on nonequllihium pH gr;v dient electrophoresis (NI.:/'H(;I.')/SI)S two-dimrnsional ~ c . 1 ~ . c.it hcsr without ( - A / ' ) or with (+AI', treatment with Irartc*rinl alkaline phosphatase prior to elertrophoresis. %laterial was visu;llizcd try fluorography. For hot h -AI' and +Al' rondititrns. we rlert rophorrsc-d mixturesofinte~~haseantlmitotirsamples~/+M~aswe11asseperntc~ interphase ( 1 ) and mitotic ( M I samples. Major rhnrge isoforms olthc. lamins that are apparent in mixed immunoadsor1)ed samples o f interphase and mitotic cells ( - A / ' and +A/'prrnds) are intlicatetl by

nrrorcx Memhers o l a pair o f adjacent rhargr isolorms protrclt)ly tlifler hv one phosphate molecule.

ently monophosphorylated species, while essentially all of the mitotic lamins A and C apparently have 2 or 3 molecules of

associated phosphate.

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Nuclear

Lamin Phosphorylation

631

protein-protein associations among the lamins to permit addition of new subunits. In the context of the phosphorylation data described above, a low level of phosphorylation (one molecule of phosphate/lamin) of a restricted subpopulation of the assembled proteins during interphase could locally diminish the affinity of interaction between specific lamin polypeptides, and permit the insertion of new subunits and/

or rearrangement of assembled subunits. In contrast, a higher level of lamin phosphorylation during mitosis (several molecules of phosphate/lamin) could reduce lamin-lamin affinity to a greater extent. Since the entire mitotic lamin population would be modified in this fashion, generalized depolymerization of the lamina could be favored.

Aside from the significantly different levels of phosphate associated with interphase and mitotic lamins, other biochemical characteristics of lamin phosphorylation are qualitatively similar (although not identical) in both cell populations. All of the detectable phosphate associated with the interphase and mitotic proteins occurs in a phosphomonoester linkage (sensitive to alkaline phosphatase hydrolysis), as phosphoserine, and to a considerably lesser extent, as phosphothreonine. In addition, two-dimensional tryptic peptide maps of ‘”P-labeled interphase and mitotic lamins suggest that many phosphorylated sites on each lamin are modified during both the interphase and mitotic periods. Lamins A and C, however, also show specific qualitative and quantitative differences between interphase and mitotic phosphorylation patterns.

Our peptide mapping analysis does not resolve whether lamin phosphorylation during interphase and mitosis is ac- complished by the same or distinct protein kinase(s). The differences between the phosphorylation patterns of interphase and mitotic lamins A and C that we observe could conceivably result from the same protein kinase acting on a substrate with different accessibility or conformation properties in the different cell cycle populations. Moreover, the activity of lamin phosphatases, in addition to lamin kinases, is likely to be important for modulating the levels and patterns of lamin phosphorylation occurring in uiuo at different cell cycle periods. At least the metaphase lamins are the targets of phosphatase activity in uiuo, since they are extensively dephosphorylated by early GI phase (21).

To investigate the possibility that other charge-altering postsynthetic modifications besides phosphorylation are specifically associated with the mitotic lamins, we analyzed interphase and mitotic proteins on two-dimensional gels. These experiments demonstrate that a high level of phosphorylation is the only detectable charge-altering modification that distin- guishes the interphase and mitotic lamins, providing support for our hypothesis that phosphorylation/dephosphorylation mediates the structural dynamics of the lamina during cell division. However, to conclusively prove this hypothesis, it will be necessary to investigate these processes with different approaches, such as utilizing in uitro systems to study nuclear envelope assembly and disassembly.

In addition to the nuclear lamins, phosphorylation of other proteins has been correlated with entry of cells into meiosis

or mitosis. For example, certain nuclear proteins such as histones (24) exist in a hyperphosphorylated state during metaphase, and a discrete class of phosphate-containing epi- topes present on a large number of cellular polypeptides appears specifically in mitotic cells, as defined by certain monoclonal antibodies (11).4

The two monoclonal antibodies described by Rao and co-workers with this specificity (11) do not react significantly with immunoadsorbed mitotic lamins (B. Burke and L. Gerace, unpublished), sug- gesting that the mitotic lamin kinase may be distinct from the kinase modifying this other class of polypeptides.

Phosphorylation may be important in triggering initiation of cell division at a primary hierarchical level. “Maturation promoting factor,” a cytoplasmic activity that induces entry into meiotic (38, 47) or mitotic (41) division after it is mi- croinjected into appropriate cells, is possibly activated by phosphorylation (53). Furthermore, maturation promoting factor may itself be a protein kinase, since it induces a burst of protein phosphorylation following injection into mature oocytes immediately prior to the entry of these cells into division (53). Hence, it is possible that a network of phosphorylation reactions is important for regulating initiation of mitosis and meiosis (discussed in Refs. 37, 41, and 53), and that some of these phosphorylation reactions directly mediate the structural reorganization of cellular organelles (such as the nuclear envelope) which takes place during prophase.

Acknowledgments-We thank Mary Jean Benson for assistance in determining the absolute levels of lamin phosphorylation; Vann Ben- nett for helpful suggestions on tryptic peptide mapping; Arlene Daniel for assistance with manuscript preparation; and Brian Burke for useful comments. We also are grateful to Gunter Blobel in whose laboratory the observations on in uiuo biosynthesis and assembly of the lamins were originally made.

REFERENCES

1. Aaronson, R. P., and Blobel, G. (1975) Proc. Natl. Acad. Sci. U. S. A. 7 2 ,

2. Benyajati, C., and Worcel, A. (1976) Cell 9,393-407

3. Bitte, L., and Kahat, D. (1974) Methods Enzymol. 30,563-590

4. Banner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46,83-88

5. Bostock, C. J., Prescott, D. M., and Kirkpatrick, J. B. (1971) Exp. Cell Res. 6. Boyd, A,, and Simon, M. (1982) Annu. Reu. Physiol. 44,501-517

7. Burke, B., Tooze, d . , and Warren, G. (1983) EMBO J. 2 , 361-367

8. Cann, J. R. (1979) in Electrokinetic Separation Methods (Righetti, P., Van

Oss, C. J., and Vanderhoff, J. W., eds) pp. 369-387, Elsevier/North- Holland, New York

1007-1011

6 8 , 163-168

10. Cook, P. R., and Brazell, I. A. (1976) J. Cell Sei. 22,287-302

11. Davis, F., Tsao, T., Fowler, S., and Rao, P. (1983) Proc. Natl. Acad. Sci. U.

13. Elder, J. H., Pickett, R. A. 11, Hampton, d . , and Lerner, R. A. (1977) J.

12. Dwyer, N., and Blobel, G. (1976) J. Cell Biol. 70, 581-591

15. Erlandson, R. A., and de Harven, E. (1971) J . Cell Sci. 8 , 353-397 14. Ely, S., D’Arcy, A., and Jost, E. (1578) Exp. Cell Res. 1 1 6 , 325-331

16. Fawcett, D. W. (1966) Am. J . Anat. 1 1 9 , 129-146

17. Fenner, C., Traut, R. R., Mason, D. T., and Wikman-Coffelt, J. (1975)

Anal. Biochem. 63,595-602

18. Franke, W. W., Scheer, U., Krohne, G., and Jarasch, E.-D. (1981) J . Cell

Biol. 91.39s-50s

19. Fry, D. J. (1976) in Mammalian Cell Membranes (Jamieson, G. A,, and Robinson, D. M., eds) Vol. 2, pp. 197-265 Butterworths Boston 20. Gerace, L., Blum, A., and Blobel, G. (1978)

i.

Cell Biol. 7 9 , 546-566 21. Gerace, L., and Blobel, G. (1980) Cell 19,277-287

22. Gerace, L., and Blobel, G. (1982) Cold Spring Harbor Symp. Qunnt. Bid.

46,967-978

23. Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95,

826-837

24. Gurley, L. R., D’Anna, J. A., Halleck, M. S., Barham, S. S., Walters, R. A,, Jett, J. J., and Tobey, R. A. (1981) in Protein Phosphorylation (Rosen,

0. M., and Krebs, E. G., ed) Book B, pp. 1073-1093, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

9. Cohen, P. (1982) Nature (Lond.) 296,613-620 S. A . 80,2926-2930

Biol. Chem. 252,6510-6515

25. Hancock, R., and Hughes, M. (1982) Biol. Cell. ,44, 201-212

26. Hayalshi, O., and Ueda, K. (1977) Anu. Reu. Brochem. 46.95-116

27. Hunter, T., and Sefton, B. M. (1980) Proc. Natl. Acad. Sei. U. S. A . 77, 28. Kaufmann, S. H., Gibson, W., and Shaper, J. H. (1983) J . Biol. Chem. 2 5 8 , 29. Krohne, ,G., Franke, W. W., Ely, S., D’Arcy, A., and Jost, E. (1978a) 30. Krohne, G., Franke, W., and Scheer, U. (157813) Exp. Cell Res. 1 1 6 , 85-

31. Krohne, G., Dabauvalle, M.-C., and Franke, W. W. (1981) J . Mol. Biol.

32. Krohne, G., Debus, E., Osborn, M., Weber, K., and Franke, W. W. (1984) 33. Laemmli, U. K. (1970) Nature (Lond.) 2 2 7 , 680-685

34. Laliberte, T., Dagenais, A., Filion, M., Bibor-Hardy, V., Simard, R., and

35. Lam, K., and Kasper, C . (1979) Royal, A. (1984) J. Cell Bid. 9 6 , 980-985 Biochemistry 1 8 , 307-311

36. Lehkowski, J., and Laemmli, U. (1982) J . Mol. Bid. 156,325-344

37. Maller, J., and Sadler, S. (1982) in Protein Phosphorylation (Rosen, 0. and

Krebs, E., eds) Book 2, pp. 1127-1141, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Borun, T. (1972)). Cell Biol. 5 5 , 433-44; 1311-1315

2710-2719

Cytobrologre 18, 22-38

102 151, 121-141

Exp. Cell Res., 1 5 0 , 47-55

38. Masui, Y., and Markert, C. (1971) J. Exp. Zool. 1 7 2 , 129-146

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