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p34cdc2 Kinase-mediated release of lamins from nuclear ghosts is inhibited by cAMP-dependent protein kinase
EXPERIMENTAL
CELL
RESEARCH
20 1, 494-499
(19%)
p34cdc2Kinase-Mediated Release of Lamins from Nuclear Ghosts Is Inhibited by CAMP-Dependent Protein Kinase STEPHEN MOLLOY AND MELWN Institute
for
Cell
and
Tumour
Biology,
German
Cancer
Research
Centre,
LITTLE’ ZNF
280,
D-6900
Heidelberg
1, Germany
.yeast (S. pombe) was shown to be phosphorylated by a mitotically active kinase that was ts in a cdc2 ts mutant strain [8]. Of the sites phosphorylated in M-phase, mutational analysis shows that of Ser-16 alone to be sufficient to depolymerize in uitro-assembled lamin polymers [9]. Subsequent dephosphorylation with phosphatase 1 resulted in the reformation of the polymer. Whether phosphatase 1 plays a role in lamin dephosphorylation in viva is unknown, but there is evidence of a phosphatase 1 requirement for exit from mitosis [lo, 111. PKA and PKC both phosphorylated lamin B, in vitro, producing phosphopeptides that also occur in viuo [7], suggesting a physiological function for these phosphorylations; neither, however, phosphorylated any of the major M-phase sites on B,. In addition, there are constitutively phosphorylated sites on chicken B, and B, that are dephosphorylated in M-phase. The microinjection of p34cdc2/cyclin B into an amphibian oocyte will cause it to rapidly enter mitosis. Somatic rat cells, however, proceeded to prophase but no further [12]. In mammalian oocytes, PKI, a specific inhibitor of PKA Press, Inc. [13], induces maturation whereas PKA activation is inhibitory [ 141. PKA inhibition and ~34”~“~ activation seemto be complementary inductive pathways since the INTRODUCTION co-injection of both ~34~~~~and PKI results in nuclear envelope breakdown, an effect that is not observed The lamin filaments of the nuclear lamina are important for the integrity of the nuclear envelope and may be when either is injected alone; there is also a marked reduction in the level of lamin phosphorylation when involved in the organization of chromatin and tranPKI is injected into REF-52 cells [ 151. So while it seems scription (for reviews see [l, 21). During mitosis the Atype lamins are completely solubilized and dispersed likely that multiple kinases act upon the lamins during through the cytoplasm, while the B-type remains asso- progression through the cell cycle [16], a phosphatase ciated with membrane elements [3]. The disassembled activity may also be required for the removal of inhibitory phosphate groups from PKA sites. In this paper we mitotic lamins are hyperphosphorylated [4], a state that is reversed upon exit from mitosis [3,5]. At least some of present evidence that supports a role for the dephosphorylation of PKA sites, either on the lamins or on an the ultrastructural changes associated with the lamina integral component of nuclear ghosts that interacts in mitosis seem to be the result of direct phosphorylawith the lamins during lamin depolymerization. tion by ~34’~” kinase (for review see [6] ). Bacterially expressed avian lam:n B, is phosphorylated by ~34”~“’ kinase on sites known to be phosphorylated in mitosis MATERIALS AND METHODS [7], and a chicken lamin protein expressed in fission
During mitosis the lamins are found in a hyperphosphorylated and soluble state. ~34~~~ kinase (MPF), a protein kinase complex with a pivotal role during mitosis, has been found to phosphorylate the lamins and, in some cases, though not all, to cause depolymerization of the lamina in vitro. Due to the variety of protein interactions in the lamina, there is a probable requirement for multiple enzyme activities to effect its breakdown in mitosis. Using nuclear ghosts as substrate, we have fractionated a Xenopus mitotic extract into a lamin-releasing fraction (~34 edcz kinase) and a fraction that inhibits ~34”~“~ kinase-mediated lamin release if the nuclear ghosts are first preincubated in it. The lamin-release-inhibiting activity in the ~34~“~ kinase-depleted mitotic extract is, in turn, inhibited if PKI, a protein kinase inhibitor specific for PKA, is included in the preincubation reaction mixture. Furthermore, a similar degree of inhibition can be achieved by using purified PKA to preincubate the nuclear ghosts. This suggests that dephosphorylation of PKA substrate sites is 0 1992 Academic necessary for lamin depolymerization.
1 To whom
reprint
requests
should
Preparation of rat liver nuclear ghosts (based on Krohne and Franke [1’7]). The livers were removed from medium-sized male rats, cut into small pieces in ice-cold medium A (440 mM sucrose, 10 mM
be addressed.
0014-4827/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Tris . HCl, pH 7.4, 70 mM KCl, 2 mM MgC&, 0.2 mM spermine, 0.5 mM spermidine, 2 mM DTT, 200 PM PMSF), passed through an embryo crusher, and homogenized in a tight-fitting potter. Passing through a muslin gauze removed the connective tissue. The nuclei were centrifuged through a 72% sucrose cushion at 16,000 rpm (Beckman SW.27) at 4”C, settling at the bottom of the tube. This sedimented material was pooled and washed in a wash buffer (medium A without sucrose), transferred to a DNase buffer (50 mM Tris * HCl, pH 7.8,2 mM MgCl,, 4 mM CaCl,, 1 mM DTT, 200 PM PMSF) at a concentration of lo6 nuclei per milliliter, to which benzone nuclease (Benzonase, Merck, Darmstadt) was added to a concentration of 1 U per milliliter, incubation was conducted at 37°C for 30 min. The nuclei were then extracted with 500 mM NaCl at 4°C and washed well with modified Ringer’s (MMR), 100 mM NaCl, 1.6 mM KCl, 1 mM MgSO,, 2 mM CaCl,, 6 mM Hepes, pH 7.2, 100 pM EDTA, giving nuclear ghosts essentially devoid of chromatin and chromatin-associated elements. Preparation of p34”&’ kinase from Xenopus oocytes. Each frog received 700 U HCG subcutaneously and about 12 h later mature eggs were collected in ice-cold 100 mM NaCl. The eggs were dejellied in a 2% cysteine solution (pH 7.8) [18] and washed four times in 10 vol cold 100 mM NaCl and three times in 2 vol 240 mM 2-glycerophosphate, 60 mM EGTA, 45 mM MgCl,, 1 mM DTT. They were then packed by centrifugation at 4°C for 15 s at lOOg, and all excess buffer was removed. Lysis was then carried out by centrifugation at 10,OOOg for 10 min at 4°C. The cytosolic fraction was then centrifuged for a further 60 min at 120,OOOg [lS]. The cytosolic fraction was again removed and diluted in 1 vol ~13 buffer (50 mM Tris*HCI, pH 7.4, 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 5 mM EGTA, 10 pg/ml leupeptin, 10 Mg/ml aprotinine, 10 rg/ml soy bean trypsin inhibitor (SBTI), 100 PM benzamidine); l/6 vol of washed p13”““’ Sepharose beads, prepared according to Meijer and Pondaven [20], was then added and the whole was mixed gently at 4°C for 30 min. The beads were then pelleted and washed extensively with p13 buffer, the last wash being performed with 100 mM Tris (pH 7.4), 20 mM MgCl,, 2 mM CaCl,, 160 mM 2-glycerophosphate, 12 mM EGTA, after which all excess buffer was removed. To elute the bound activity 1 bed vol of a p13s”c’ solution was added to the washed beads to a concentration of 2 mg/ml and this was agitated gently at room temperature for 15 min. After centrifugation at 10,OOOg for 5 min at 4°C the histone Hl kinase activity in the aqueous fraction was estimated to be 0.56 pmol units per milliliter using the method of Meijer and Pondaven [20]. Preparation of p34’dc2 kinase-depleted mitotic extract (dme). After the dejellying step (see above), the eggs were washed extensively with MMR1/4 (25 mM NaCl, 0.4 mM KCl, 0.25 mM MgSO,, 0.5 mM CaCl,, 1.25 mM Hepes, pH 7.2, 25 mM EDTA). They were then washed once with ice-cold acetate buffer (100 mM potassium acetate, 2.5 mM magnesium acetate, 60 mM EGTA, 5 rglml cytochalasin D, 1 mM DTT, 100 FM PMSF, pH 7.2), after which they were lysed, centrifuged, and stored as described above for the preparation of cdc2 kinase [21]. Preparation of cycling extracts. Dejellied eggs were washed with an ice-cold acetate buffer, as above, and activated in a Perspex activation chamber between two 10 X lo-cm electrodes 1 cm apart, by a 2-V pulse for 10 s, as described by Karsenti et al. [18]. They were then incubated at room temperature for 50-55 min and centrifuged at 4°C for 45 min at 100,OOOg. Aliquots of the supernatant were taken at activation times, t, of +55, f65, +85, and +95 min. The p34’“’ kinase activity was removed with Sepharose-pl3”“” beads as in the preparation of the cdc2 kinase described above. Lamin-releasing assay. Nuclear ghosts were washed in 1X MMR + 100 pM PMSF and were resuspended either in the ~34’~“‘kinase-depleted extract containing 100 PM ATP or in a PKA buffer (110 mM piperazine-N,N’-bis[2-ethanesulfonic acid] (Pipes), pH 6.9, 5 mM MgCl,, 50 PM CAMP, 200 PM ATP) at a concentration of 5 X lo7 per
RELEASE
BY
495
PKA
milliliter; PKI (rabbit sequence; Sigma, Munich) was added from a 100X stock in 1X MMR to a concentration of 5 mM [22]. Where included, the broad-spectrum phosphatase inhibitors were added from a 10X concentrated cocktail to give a working concentration of 1 mM sodium fluoride, 500 pM sodium vanadate, and 50 mM 2-glycerophosphate. PKA was then added to a concentration of 50 pglml to the appropriate samples followed by incubation at 37°C for 40 min. Samples with the depleted extracts were incubated at 30°C for 1 h. The nuclei were then washed in PBS-l% Triton X-100 at 0°C followed by PBS alone. After pelleting for 5 min at 450g the ghosts were resuspended in the eluted ~34’~~’ kinase fraction at a concentration of 3 X lo7 per milliliter. An ATP-regenerating system was added to a concentration of 500 PM ATP, 40 mM creatine phosphate, and 40 rglml creatine kinase. Incubations were carried out at 30°C. Aliquots were removed at 30 and 80 min. The aliquots were incubated on ice with 1 vol PBS-2% Triton X-100 for 10 min and then centrifuged at 15,000g for 10 min at 4°C. The supernatants were then aspirated, precipitated with methanol/chloroform according to Wessel and Fliigge [23], and assayed for lamin content by immunostaining Western blots of 8% polyacrylamide gels [24] as described by Burnette [25] using the antilamin monoclonal antibody PKB8 [26] as the primary antibody. Zmmunofluorescence. After incubation with ~34’~“’ kinase, nuclei or nuclear ghosts were put on ice for 5 min. One volume of PBS-l% Triton X-100 was then added before incubating for a further 5 min. The sample was fixed by the addition of 4 vol of 3% formaldehyde, 2% sucrose in PBS and incubated at room temperature for 5 min. The fixed samples were then absorbed onto polylysine-coated immunofluorescence slides, with care taken to avoid drying out. The slides were then washed in PBS for 5 min, and 50 ~1 of the primary antibody solution, a 1:50 dilution of PKB8 in PBS, was added followed by incubation at room temperature for 30 min. They were then washed twice with PBS followed by incubation at room temperature for 30 min with a secondary antibody solution, a 1:50 dilution of a fluoresceinelabeled goat anti-mouse antibody containing 5 pg/ml of the Hoechst Chromatin Dye 33258 in PBS. After washing in PBS and removing excess liquid, the samples were mounted in 40 ~1 Citifluor mountant (Amersham, Braunschweig, Germany).
RESULTS
To investigate enzymatic activities responsible for depolymerization of the nuclear lamina during mitosis, we prepared a high-speed cytoplasmic fraction from the oocytes of X. laevis. Whole rat liver nuclei, rather than purified lamin filament preparations, were used as substrate, in order to more closely resemble the in vivo situation. Upon incubation of nuclei with mitotic extract, a significant release of lamins occurred into the aqueous phase (data not shown). A similar level of lamin depolymerization was achieved when these whole nuclei were incubated with partially purified ~34”~“’ kinase that had been extracted from the Xenopus mitotic extract using pl3-coated Sepharose beads (see Materials and Methods) (Figs. la-lc). When the nuclei were incubated with PKA, however, no comparable level of depolymerization was achieved (Figs. Id-lf). In order to exclude the possibility that intranuclear enzyme activities may be participating in the observed release of lamins, nuclear ghosts were used, these are nuclei that have been digested by nucleases and thoroughly washed to remove most chromatin remnants and chromatin-associated elements (Fig. 1). In contrast to
496
FIG. 1. j-l) Incubation fluorescent with ~34’~“’ ghosts, per
MOLLOY
AND
LITTLE
Release of lamins from rat liver nuclei and nuclear ghosts after incubation with ~34’~” kinase: (a-f) nuclei; (g-l) ghosts. (a-c and with ~34’~” kinase; (d-f and g-i) incubation with CAMP-dependent protein kinase. (Top) Phase-contrast microscopy; (middle) microscopy with Hoechst Chromatin Dye 33258; (bottom) indirect immunofluorescence with anti-lamin mAb PKB8. Incubations kinase (1.12 pmol units/pi) or CAMP-dependent protein kinase (50 pg/ml) were carried out at a concentration of 5 X lO*nuclei, or milliliter for 60 min at 30°C.
the bright ring around the periphery of intact nuclei after immunofluorescent staining with an anti-lamin antibody (Fig. If), the nuclear ghosts displayed an intense, uniform immunofluorescence over their surfaces (Fig. li). A significant proportion of the fragile ghosts appeared to have collapsed during their preparation and subsequent incubations, particularly after treatment with the ~34”~“~ kinase. This is presumably due to greater fragility of the ghosts after removal of the lamins, and this would also explain the increased amount of debris seen in these preparations. This loss of mechanical integrity probably results in a greater leaching of chromatin-associated remnants out of the ghosts (Fig. lh), resulting in a virtual absence of chromatin-associated fluorescence (Fig. lk). As with the nuclei, the nuclear ghosts that had been incubated with CAMP-dependent protein kinase showed no noticeable decrease in lamin content (Fig. li), whereas those incubated with p34”dC2 kinase only showed an extremely faint lamin-associated fluorescence (Fig. ll), suggesting that there is
no substantial difference between nuclei and ghosts as regards the ability of ~34”~“~ kinase to break down the lamina. While ~34~~~~ kinase alone seems to effect lamin release from nuclear envelopes, the results from other groups using both nuclei and filaments as substrate for p34cdc2 kinase suggest that it is not sufficient in all cases, and some evidence for the regulatory role of PKA has been reported. We therefore examined the effects of preincubating nuclear ghosts with mitotic extracts that had been depleted of ~34”~“~ kinase, with and without the specific PKA inhibitor and broad spectrum phosphatase inhibitors. Figure 2a (0 min) shows the ~34”~” kinase activity of the extract before depletion. The four points between 60 and 95 min in Fig. 2a show the cycling competence of an extract made from a small fraction of the eggs used to make the depleted extract; this shows that the M-phase extract had all the enzymatic components needed to execute at least one cell cycle. Figure 2b shows the degree of ~34”~“~ kinase depletion achieved
INHIBITION
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TABLE
1
Summary of Lamin-Releasing
Substrate
mire. after activation.
whole extract
FIG. 2. Cycling ~34’~” kinase activity of a mitotic ployed for the preparation of a ~34’~‘~ kinase-depleted p34’d’Z activity of an oocyte extract after electroshock. amount of ~34’~” kinase activity before and after depletion arose-pl3”“” beads, of a cycling-competent extract.
x3-depleted extract
extract emfraction. (a) (b) Total with Seph-
with the Sepharose-p13sUC1 beads, a reduction to approximately 0.3% of its original level. We found that incubation of the ghosts with the depleted extract alone leads to virtually no lamin release after either 30 or 80 min (Fig. 3a, lanes 1 and 6), while incubation with ~34’~“’ kinase alone led to optimal amounts of lamin release from the ghosts (Fig. 3a, lanes 2 and 7). However, given that the levels of lamin release by equivalent amounts of whole M-phase extract and p34”d”2 kinase are very similar (data not shown), it was surprising to find that preincubation of the ghosts with the ~34~~“’ kinase-depleted extract greatly diminished the amount of lamins released when they were subsequently washed and incubated with ~34”~” kinase (Fig. 3a, lanes 3 and 8). However, if PKI, a specific inhibitor of PKA, is included in the preincubation mix, then optimal levels of lamin release are restored (Fig. 3a, lanes 4
J-”
i8, 1
23456
78910
12
3
FIG. 3. Effect of preincubating rat liver nuclear ghosts with a p34”“2 kinase-depleted mitotic extract or CAMP-dependent protein kinase on the release of lamins from rat liver nuclear ghosts with p34”d’2 kinase. (a) Effect of p34”“’ kinase-depleted mitotic extract (dme) and CAMP-dependent protein kinase inhibitor (PKI). Lane 1, ghosts incubated with dme; lane 2, ghosts incubated with purified p34cdc2 kinase; lane 3, preincubation of ghosts with dme followed by incubation with ~34”~“; lane 4, as for lane 3, except for the inclusion of 5 fiA4 PKI in the preincubation mix; lane 5, as for lane 4 except for the inclusion of 1 mM sodium fluoride, 500 &f sodium vanadate, and 50 mM 2-glycerophosphate in the preincubation mix. t = 30 min for lanes 1-5 and t = 80 min for lanes 6-10. (b) Effect of PKA. Lane 1, incubation with ~34”~“; lane 2, as for lane 1, except that ghosts were first preincubated with 50 pg/ml PKA (Sigma); lane 3, incubation with PKA alone. Markers, A, B, and C refer to lamins A, B, and C, respectively. Activity of ~34’~” kinase was 0.57 pmol units per microliter.
Nuclear Nuclear Nuclear Nuclear
ghosts ghosts ghosts ghosts
Preincubation None dme dme + 5 pM PKI dme + 5 pM PKI + 1 mM sodium fluoride + 500 pM sodium vanadate + 50 mM 2glycerophosphate
Experiments Primary incubation p34cdc2 p34cdc2 p34cdc2 p34cdc2
kinase kinase kinase kinase
Level of lamin release Optimal Basal Optimal Basal
and 9). The further inclusion of broad-spectrum phosphatase inhibitors (1 mM sodium fluoride, 500 pM sodium vanadate, and 50 mM 2-glycerophosphate) in the preincubation mix has the effect of greatly reducing the amount of lamin release. The simplest explanation for this is that the PKA is phosphorylating some component of the ghosts and inhibiting ~34”~“’ kinase-mediated lamin depolymerization. To test this hypothesis, ghosts were preincubated with PKA before incubation with ~34”~~’ kinase. This resulted in a substantial decrease in the amounts of lamins released from that in the positive control (Fig. 3b). A summary of these results is shown in Table 1. DISCUSSION
In an assay system similar to that described here, Newport and Spann [ 191 have shown that nuclear membrane vesicularization, chromosome condensation, and lamina depolymerization are independent and separable biochemical processes. However, there is a great disparity in the depolymerization ability of lamins from different sources in in vitro assays. Bacterially expressed avian lamins are released quite readily [ 91, while others are not depolymerized at all [27]. This may not always be explained by differences in the activities used, but by the ambient phosphate profile of lamins from different sources. ~34’~“’ kinase-mediated phosphorylation has been shown to be both necessary and sufficient for the depolymerization of bacterially expressed lamins [9]. However, as these carry no post-translational modifications of any kind, any inhibitory modifications that might be present on some lamins in uiuo (e.g., constitutive interphase phosphorylation) would go unnoticed. Furthermore, while ~34”~“’ kinase has been shown to phosphorylate avian lamin B, on all M-phase-specific sites [7], only a subset of these is phosphorylated by p34”dC2 kinase in uiuo [28]. Treatment of avian I-phase cells with okadaic acid, a potent inhibitor of phosphatase types 1 and 2A, induces
498
MOLLOY
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the phosphorylation of all but one of the M-phase-specific peptides found in lamin B,. Although this is not sufficient to disassemble lamin B, from the lamina, it points to a role for other activities. In the work described here, a mitotic extract that has been depleted of its ~34”~“’ kinase (Fig. 2) has been shown to contain an activity that modifies some component of the nuclear envelope such that subsequent incubation with purified ~34”~“~ kinase leads to greatly decreased levels of lamin depolymerization (Fig. 3a). This effect is inhibited by levels of PKI that would also inhibit PKA and is mimicked by using purified PKA instead of ~34’~“~ kinase-depleted extract (Fig. 3b). The question of why there should be two antagonistic activities present at the same time in these extracts may be explained by the fact that during extract preparation a fraction of the eggs routinely activate (i.e., are released from meiotic block) spontaneously and start to cycle, meaning that many of the activities downregulated during mitosis would begin to increase to a degree dependent on the proportion of eggs activated. Exit from mitosis has also been shown to have a requirement for phosphatase 1 [29, 10, 111; it was therefore interesting to note that the inclusion of phosphatase inhibitors in the preincubation mix led to a great reduction in lamin release. There is evidence that the interaction between lamin B and the membrane is regulated by the phosphorylation state of an integral membrane protein (~58) in avian erythrocytes [30]. It has also been indicated that there is a 54-kDa inner nuclear membrane protein in chicken whose interaction with the lamina is controlled by cell cycle-dependent, post-translational modification, most likely phosphorylation [31]. So there is growing evidence that enzymatic activities other than MPF may affect the lamina. Perhaps most intriguing of all is that PKC-mediated phosphorylation of the lamins is sufficient to restore an interphase state to the lamina, even in the presence of still-elevated levels of MPF [32]. PKC shares none of the important mitotic phosphorylation sites in lamins with MPF, but it does phosphorylate Ser-395 in human lamin C, which is almost adjacent to Ser-392, a residue phosphorylated by MPF. Steric interference between the two sites, when phosphorylated, is therefore a distinct possibility. Since PKC and PKA phosphorylate several of the same sites in avian lamin B, [7], it is quite possible that we are also observing the same kind of inhibition here, in vitro. The presence of a PKA site(s) on the lamins that must be dephosphorylated for optimal ~34~~~’ kinasemediated lamin release could explain the performance variability of lamin substrates from different sources and if our nuclear ghosts contain lamins which are not phosphorylated at PKA site(s), why preincubation with PKA has an inhibitory effect on lamin release in our
LITTLE
assay. The possibility that PKA is exerting its effect on lamin release indirectly cannot, of course, be excluded. We thank Dr. G. Krohne (Heidelberg) for his generous gift of the PKB8 anti-lamin mAb and Dr. L. Meijer and his colleagues for their kind help to M.L. at Roscoff with ~34”~~’ kinase techniques and for generously providing materials.
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CELL
RESEARCH
20 1, 494-499
(19%)
p34cdc2Kinase-Mediated Release of Lamins from Nuclear Ghosts Is Inhibited by CAMP-Dependent Protein Kinase STEPHEN MOLLOY AND MELWN Institute
for
Cell
and
Tumour
Biology,
German
Cancer
Research
Centre,
LITTLE’ ZNF
280,
D-6900
Heidelberg
1, Germany
.yeast (S. pombe) was shown to be phosphorylated by a mitotically active kinase that was ts in a cdc2 ts mutant strain [8]. Of the sites phosphorylated in M-phase, mutational analysis shows that of Ser-16 alone to be sufficient to depolymerize in uitro-assembled lamin polymers [9]. Subsequent dephosphorylation with phosphatase 1 resulted in the reformation of the polymer. Whether phosphatase 1 plays a role in lamin dephosphorylation in viva is unknown, but there is evidence of a phosphatase 1 requirement for exit from mitosis [lo, 111. PKA and PKC both phosphorylated lamin B, in vitro, producing phosphopeptides that also occur in viuo [7], suggesting a physiological function for these phosphorylations; neither, however, phosphorylated any of the major M-phase sites on B,. In addition, there are constitutively phosphorylated sites on chicken B, and B, that are dephosphorylated in M-phase. The microinjection of p34cdc2/cyclin B into an amphibian oocyte will cause it to rapidly enter mitosis. Somatic rat cells, however, proceeded to prophase but no further [12]. In mammalian oocytes, PKI, a specific inhibitor of PKA Press, Inc. [13], induces maturation whereas PKA activation is inhibitory [ 141. PKA inhibition and ~34”~“~ activation seemto be complementary inductive pathways since the INTRODUCTION co-injection of both ~34~~~~and PKI results in nuclear envelope breakdown, an effect that is not observed The lamin filaments of the nuclear lamina are important for the integrity of the nuclear envelope and may be when either is injected alone; there is also a marked reduction in the level of lamin phosphorylation when involved in the organization of chromatin and tranPKI is injected into REF-52 cells [ 151. So while it seems scription (for reviews see [l, 21). During mitosis the Atype lamins are completely solubilized and dispersed likely that multiple kinases act upon the lamins during through the cytoplasm, while the B-type remains asso- progression through the cell cycle [16], a phosphatase ciated with membrane elements [3]. The disassembled activity may also be required for the removal of inhibitory phosphate groups from PKA sites. In this paper we mitotic lamins are hyperphosphorylated [4], a state that is reversed upon exit from mitosis [3,5]. At least some of present evidence that supports a role for the dephosphorylation of PKA sites, either on the lamins or on an the ultrastructural changes associated with the lamina integral component of nuclear ghosts that interacts in mitosis seem to be the result of direct phosphorylawith the lamins during lamin depolymerization. tion by ~34’~” kinase (for review see [6] ). Bacterially expressed avian lam:n B, is phosphorylated by ~34”~“’ kinase on sites known to be phosphorylated in mitosis MATERIALS AND METHODS [7], and a chicken lamin protein expressed in fission
During mitosis the lamins are found in a hyperphosphorylated and soluble state. ~34~~~ kinase (MPF), a protein kinase complex with a pivotal role during mitosis, has been found to phosphorylate the lamins and, in some cases, though not all, to cause depolymerization of the lamina in vitro. Due to the variety of protein interactions in the lamina, there is a probable requirement for multiple enzyme activities to effect its breakdown in mitosis. Using nuclear ghosts as substrate, we have fractionated a Xenopus mitotic extract into a lamin-releasing fraction (~34 edcz kinase) and a fraction that inhibits ~34”~“~ kinase-mediated lamin release if the nuclear ghosts are first preincubated in it. The lamin-release-inhibiting activity in the ~34~“~ kinase-depleted mitotic extract is, in turn, inhibited if PKI, a protein kinase inhibitor specific for PKA, is included in the preincubation reaction mixture. Furthermore, a similar degree of inhibition can be achieved by using purified PKA to preincubate the nuclear ghosts. This suggests that dephosphorylation of PKA substrate sites is 0 1992 Academic necessary for lamin depolymerization.
1 To whom
reprint
requests
should
Preparation of rat liver nuclear ghosts (based on Krohne and Franke [1’7]). The livers were removed from medium-sized male rats, cut into small pieces in ice-cold medium A (440 mM sucrose, 10 mM
be addressed.
0014-4827/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
494
INHIBITION
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Tris . HCl, pH 7.4, 70 mM KCl, 2 mM MgC&, 0.2 mM spermine, 0.5 mM spermidine, 2 mM DTT, 200 PM PMSF), passed through an embryo crusher, and homogenized in a tight-fitting potter. Passing through a muslin gauze removed the connective tissue. The nuclei were centrifuged through a 72% sucrose cushion at 16,000 rpm (Beckman SW.27) at 4”C, settling at the bottom of the tube. This sedimented material was pooled and washed in a wash buffer (medium A without sucrose), transferred to a DNase buffer (50 mM Tris * HCl, pH 7.8,2 mM MgCl,, 4 mM CaCl,, 1 mM DTT, 200 PM PMSF) at a concentration of lo6 nuclei per milliliter, to which benzone nuclease (Benzonase, Merck, Darmstadt) was added to a concentration of 1 U per milliliter, incubation was conducted at 37°C for 30 min. The nuclei were then extracted with 500 mM NaCl at 4°C and washed well with modified Ringer’s (MMR), 100 mM NaCl, 1.6 mM KCl, 1 mM MgSO,, 2 mM CaCl,, 6 mM Hepes, pH 7.2, 100 pM EDTA, giving nuclear ghosts essentially devoid of chromatin and chromatin-associated elements. Preparation of p34”&’ kinase from Xenopus oocytes. Each frog received 700 U HCG subcutaneously and about 12 h later mature eggs were collected in ice-cold 100 mM NaCl. The eggs were dejellied in a 2% cysteine solution (pH 7.8) [18] and washed four times in 10 vol cold 100 mM NaCl and three times in 2 vol 240 mM 2-glycerophosphate, 60 mM EGTA, 45 mM MgCl,, 1 mM DTT. They were then packed by centrifugation at 4°C for 15 s at lOOg, and all excess buffer was removed. Lysis was then carried out by centrifugation at 10,OOOg for 10 min at 4°C. The cytosolic fraction was then centrifuged for a further 60 min at 120,OOOg [lS]. The cytosolic fraction was again removed and diluted in 1 vol ~13 buffer (50 mM Tris*HCI, pH 7.4, 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 5 mM EGTA, 10 pg/ml leupeptin, 10 Mg/ml aprotinine, 10 rg/ml soy bean trypsin inhibitor (SBTI), 100 PM benzamidine); l/6 vol of washed p13”““’ Sepharose beads, prepared according to Meijer and Pondaven [20], was then added and the whole was mixed gently at 4°C for 30 min. The beads were then pelleted and washed extensively with p13 buffer, the last wash being performed with 100 mM Tris (pH 7.4), 20 mM MgCl,, 2 mM CaCl,, 160 mM 2-glycerophosphate, 12 mM EGTA, after which all excess buffer was removed. To elute the bound activity 1 bed vol of a p13s”c’ solution was added to the washed beads to a concentration of 2 mg/ml and this was agitated gently at room temperature for 15 min. After centrifugation at 10,OOOg for 5 min at 4°C the histone Hl kinase activity in the aqueous fraction was estimated to be 0.56 pmol units per milliliter using the method of Meijer and Pondaven [20]. Preparation of p34’dc2 kinase-depleted mitotic extract (dme). After the dejellying step (see above), the eggs were washed extensively with MMR1/4 (25 mM NaCl, 0.4 mM KCl, 0.25 mM MgSO,, 0.5 mM CaCl,, 1.25 mM Hepes, pH 7.2, 25 mM EDTA). They were then washed once with ice-cold acetate buffer (100 mM potassium acetate, 2.5 mM magnesium acetate, 60 mM EGTA, 5 rglml cytochalasin D, 1 mM DTT, 100 FM PMSF, pH 7.2), after which they were lysed, centrifuged, and stored as described above for the preparation of cdc2 kinase [21]. Preparation of cycling extracts. Dejellied eggs were washed with an ice-cold acetate buffer, as above, and activated in a Perspex activation chamber between two 10 X lo-cm electrodes 1 cm apart, by a 2-V pulse for 10 s, as described by Karsenti et al. [18]. They were then incubated at room temperature for 50-55 min and centrifuged at 4°C for 45 min at 100,OOOg. Aliquots of the supernatant were taken at activation times, t, of +55, f65, +85, and +95 min. The p34’“’ kinase activity was removed with Sepharose-pl3”“” beads as in the preparation of the cdc2 kinase described above. Lamin-releasing assay. Nuclear ghosts were washed in 1X MMR + 100 pM PMSF and were resuspended either in the ~34’~“‘kinase-depleted extract containing 100 PM ATP or in a PKA buffer (110 mM piperazine-N,N’-bis[2-ethanesulfonic acid] (Pipes), pH 6.9, 5 mM MgCl,, 50 PM CAMP, 200 PM ATP) at a concentration of 5 X lo7 per
RELEASE
BY
495
PKA
milliliter; PKI (rabbit sequence; Sigma, Munich) was added from a 100X stock in 1X MMR to a concentration of 5 mM [22]. Where included, the broad-spectrum phosphatase inhibitors were added from a 10X concentrated cocktail to give a working concentration of 1 mM sodium fluoride, 500 pM sodium vanadate, and 50 mM 2-glycerophosphate. PKA was then added to a concentration of 50 pglml to the appropriate samples followed by incubation at 37°C for 40 min. Samples with the depleted extracts were incubated at 30°C for 1 h. The nuclei were then washed in PBS-l% Triton X-100 at 0°C followed by PBS alone. After pelleting for 5 min at 450g the ghosts were resuspended in the eluted ~34’~~’ kinase fraction at a concentration of 3 X lo7 per milliliter. An ATP-regenerating system was added to a concentration of 500 PM ATP, 40 mM creatine phosphate, and 40 rglml creatine kinase. Incubations were carried out at 30°C. Aliquots were removed at 30 and 80 min. The aliquots were incubated on ice with 1 vol PBS-2% Triton X-100 for 10 min and then centrifuged at 15,000g for 10 min at 4°C. The supernatants were then aspirated, precipitated with methanol/chloroform according to Wessel and Fliigge [23], and assayed for lamin content by immunostaining Western blots of 8% polyacrylamide gels [24] as described by Burnette [25] using the antilamin monoclonal antibody PKB8 [26] as the primary antibody. Zmmunofluorescence. After incubation with ~34’~“’ kinase, nuclei or nuclear ghosts were put on ice for 5 min. One volume of PBS-l% Triton X-100 was then added before incubating for a further 5 min. The sample was fixed by the addition of 4 vol of 3% formaldehyde, 2% sucrose in PBS and incubated at room temperature for 5 min. The fixed samples were then absorbed onto polylysine-coated immunofluorescence slides, with care taken to avoid drying out. The slides were then washed in PBS for 5 min, and 50 ~1 of the primary antibody solution, a 1:50 dilution of PKB8 in PBS, was added followed by incubation at room temperature for 30 min. They were then washed twice with PBS followed by incubation at room temperature for 30 min with a secondary antibody solution, a 1:50 dilution of a fluoresceinelabeled goat anti-mouse antibody containing 5 pg/ml of the Hoechst Chromatin Dye 33258 in PBS. After washing in PBS and removing excess liquid, the samples were mounted in 40 ~1 Citifluor mountant (Amersham, Braunschweig, Germany).
RESULTS
To investigate enzymatic activities responsible for depolymerization of the nuclear lamina during mitosis, we prepared a high-speed cytoplasmic fraction from the oocytes of X. laevis. Whole rat liver nuclei, rather than purified lamin filament preparations, were used as substrate, in order to more closely resemble the in vivo situation. Upon incubation of nuclei with mitotic extract, a significant release of lamins occurred into the aqueous phase (data not shown). A similar level of lamin depolymerization was achieved when these whole nuclei were incubated with partially purified ~34”~“’ kinase that had been extracted from the Xenopus mitotic extract using pl3-coated Sepharose beads (see Materials and Methods) (Figs. la-lc). When the nuclei were incubated with PKA, however, no comparable level of depolymerization was achieved (Figs. Id-lf). In order to exclude the possibility that intranuclear enzyme activities may be participating in the observed release of lamins, nuclear ghosts were used, these are nuclei that have been digested by nucleases and thoroughly washed to remove most chromatin remnants and chromatin-associated elements (Fig. 1). In contrast to
496
FIG. 1. j-l) Incubation fluorescent with ~34’~“’ ghosts, per
MOLLOY
AND
LITTLE
Release of lamins from rat liver nuclei and nuclear ghosts after incubation with ~34’~” kinase: (a-f) nuclei; (g-l) ghosts. (a-c and with ~34’~” kinase; (d-f and g-i) incubation with CAMP-dependent protein kinase. (Top) Phase-contrast microscopy; (middle) microscopy with Hoechst Chromatin Dye 33258; (bottom) indirect immunofluorescence with anti-lamin mAb PKB8. Incubations kinase (1.12 pmol units/pi) or CAMP-dependent protein kinase (50 pg/ml) were carried out at a concentration of 5 X lO*nuclei, or milliliter for 60 min at 30°C.
the bright ring around the periphery of intact nuclei after immunofluorescent staining with an anti-lamin antibody (Fig. If), the nuclear ghosts displayed an intense, uniform immunofluorescence over their surfaces (Fig. li). A significant proportion of the fragile ghosts appeared to have collapsed during their preparation and subsequent incubations, particularly after treatment with the ~34”~“~ kinase. This is presumably due to greater fragility of the ghosts after removal of the lamins, and this would also explain the increased amount of debris seen in these preparations. This loss of mechanical integrity probably results in a greater leaching of chromatin-associated remnants out of the ghosts (Fig. lh), resulting in a virtual absence of chromatin-associated fluorescence (Fig. lk). As with the nuclei, the nuclear ghosts that had been incubated with CAMP-dependent protein kinase showed no noticeable decrease in lamin content (Fig. li), whereas those incubated with p34”dC2 kinase only showed an extremely faint lamin-associated fluorescence (Fig. ll), suggesting that there is
no substantial difference between nuclei and ghosts as regards the ability of ~34”~“~ kinase to break down the lamina. While ~34~~~~ kinase alone seems to effect lamin release from nuclear envelopes, the results from other groups using both nuclei and filaments as substrate for p34cdc2 kinase suggest that it is not sufficient in all cases, and some evidence for the regulatory role of PKA has been reported. We therefore examined the effects of preincubating nuclear ghosts with mitotic extracts that had been depleted of ~34”~“~ kinase, with and without the specific PKA inhibitor and broad spectrum phosphatase inhibitors. Figure 2a (0 min) shows the ~34”~” kinase activity of the extract before depletion. The four points between 60 and 95 min in Fig. 2a show the cycling competence of an extract made from a small fraction of the eggs used to make the depleted extract; this shows that the M-phase extract had all the enzymatic components needed to execute at least one cell cycle. Figure 2b shows the degree of ~34”~“~ kinase depletion achieved
INHIBITION
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TABLE
1
Summary of Lamin-Releasing
Substrate
mire. after activation.
whole extract
FIG. 2. Cycling ~34’~” kinase activity of a mitotic ployed for the preparation of a ~34’~‘~ kinase-depleted p34’d’Z activity of an oocyte extract after electroshock. amount of ~34’~” kinase activity before and after depletion arose-pl3”“” beads, of a cycling-competent extract.
x3-depleted extract
extract emfraction. (a) (b) Total with Seph-
with the Sepharose-p13sUC1 beads, a reduction to approximately 0.3% of its original level. We found that incubation of the ghosts with the depleted extract alone leads to virtually no lamin release after either 30 or 80 min (Fig. 3a, lanes 1 and 6), while incubation with ~34’~“’ kinase alone led to optimal amounts of lamin release from the ghosts (Fig. 3a, lanes 2 and 7). However, given that the levels of lamin release by equivalent amounts of whole M-phase extract and p34”d”2 kinase are very similar (data not shown), it was surprising to find that preincubation of the ghosts with the ~34~~“’ kinase-depleted extract greatly diminished the amount of lamins released when they were subsequently washed and incubated with ~34”~” kinase (Fig. 3a, lanes 3 and 8). However, if PKI, a specific inhibitor of PKA, is included in the preincubation mix, then optimal levels of lamin release are restored (Fig. 3a, lanes 4
J-”
i8, 1
23456
78910
12
3
FIG. 3. Effect of preincubating rat liver nuclear ghosts with a p34”“2 kinase-depleted mitotic extract or CAMP-dependent protein kinase on the release of lamins from rat liver nuclear ghosts with p34”d’2 kinase. (a) Effect of p34”“’ kinase-depleted mitotic extract (dme) and CAMP-dependent protein kinase inhibitor (PKI). Lane 1, ghosts incubated with dme; lane 2, ghosts incubated with purified p34cdc2 kinase; lane 3, preincubation of ghosts with dme followed by incubation with ~34”~“; lane 4, as for lane 3, except for the inclusion of 5 fiA4 PKI in the preincubation mix; lane 5, as for lane 4 except for the inclusion of 1 mM sodium fluoride, 500 &f sodium vanadate, and 50 mM 2-glycerophosphate in the preincubation mix. t = 30 min for lanes 1-5 and t = 80 min for lanes 6-10. (b) Effect of PKA. Lane 1, incubation with ~34”~“; lane 2, as for lane 1, except that ghosts were first preincubated with 50 pg/ml PKA (Sigma); lane 3, incubation with PKA alone. Markers, A, B, and C refer to lamins A, B, and C, respectively. Activity of ~34’~” kinase was 0.57 pmol units per microliter.
Nuclear Nuclear Nuclear Nuclear
ghosts ghosts ghosts ghosts
Preincubation None dme dme + 5 pM PKI dme + 5 pM PKI + 1 mM sodium fluoride + 500 pM sodium vanadate + 50 mM 2glycerophosphate
Experiments Primary incubation p34cdc2 p34cdc2 p34cdc2 p34cdc2
kinase kinase kinase kinase
Level of lamin release Optimal Basal Optimal Basal
and 9). The further inclusion of broad-spectrum phosphatase inhibitors (1 mM sodium fluoride, 500 pM sodium vanadate, and 50 mM 2-glycerophosphate) in the preincubation mix has the effect of greatly reducing the amount of lamin release. The simplest explanation for this is that the PKA is phosphorylating some component of the ghosts and inhibiting ~34”~“’ kinase-mediated lamin depolymerization. To test this hypothesis, ghosts were preincubated with PKA before incubation with ~34”~~’ kinase. This resulted in a substantial decrease in the amounts of lamins released from that in the positive control (Fig. 3b). A summary of these results is shown in Table 1. DISCUSSION
In an assay system similar to that described here, Newport and Spann [ 191 have shown that nuclear membrane vesicularization, chromosome condensation, and lamina depolymerization are independent and separable biochemical processes. However, there is a great disparity in the depolymerization ability of lamins from different sources in in vitro assays. Bacterially expressed avian lamins are released quite readily [ 91, while others are not depolymerized at all [27]. This may not always be explained by differences in the activities used, but by the ambient phosphate profile of lamins from different sources. ~34’~“’ kinase-mediated phosphorylation has been shown to be both necessary and sufficient for the depolymerization of bacterially expressed lamins [9]. However, as these carry no post-translational modifications of any kind, any inhibitory modifications that might be present on some lamins in uiuo (e.g., constitutive interphase phosphorylation) would go unnoticed. Furthermore, while ~34”~“’ kinase has been shown to phosphorylate avian lamin B, on all M-phase-specific sites [7], only a subset of these is phosphorylated by p34”dC2 kinase in uiuo [28]. Treatment of avian I-phase cells with okadaic acid, a potent inhibitor of phosphatase types 1 and 2A, induces
498
MOLLOY
AND
the phosphorylation of all but one of the M-phase-specific peptides found in lamin B,. Although this is not sufficient to disassemble lamin B, from the lamina, it points to a role for other activities. In the work described here, a mitotic extract that has been depleted of its ~34”~“’ kinase (Fig. 2) has been shown to contain an activity that modifies some component of the nuclear envelope such that subsequent incubation with purified ~34”~“~ kinase leads to greatly decreased levels of lamin depolymerization (Fig. 3a). This effect is inhibited by levels of PKI that would also inhibit PKA and is mimicked by using purified PKA instead of ~34’~“~ kinase-depleted extract (Fig. 3b). The question of why there should be two antagonistic activities present at the same time in these extracts may be explained by the fact that during extract preparation a fraction of the eggs routinely activate (i.e., are released from meiotic block) spontaneously and start to cycle, meaning that many of the activities downregulated during mitosis would begin to increase to a degree dependent on the proportion of eggs activated. Exit from mitosis has also been shown to have a requirement for phosphatase 1 [29, 10, 111; it was therefore interesting to note that the inclusion of phosphatase inhibitors in the preincubation mix led to a great reduction in lamin release. There is evidence that the interaction between lamin B and the membrane is regulated by the phosphorylation state of an integral membrane protein (~58) in avian erythrocytes [30]. It has also been indicated that there is a 54-kDa inner nuclear membrane protein in chicken whose interaction with the lamina is controlled by cell cycle-dependent, post-translational modification, most likely phosphorylation [31]. So there is growing evidence that enzymatic activities other than MPF may affect the lamina. Perhaps most intriguing of all is that PKC-mediated phosphorylation of the lamins is sufficient to restore an interphase state to the lamina, even in the presence of still-elevated levels of MPF [32]. PKC shares none of the important mitotic phosphorylation sites in lamins with MPF, but it does phosphorylate Ser-395 in human lamin C, which is almost adjacent to Ser-392, a residue phosphorylated by MPF. Steric interference between the two sites, when phosphorylated, is therefore a distinct possibility. Since PKC and PKA phosphorylate several of the same sites in avian lamin B, [7], it is quite possible that we are also observing the same kind of inhibition here, in vitro. The presence of a PKA site(s) on the lamins that must be dephosphorylated for optimal ~34~~~’ kinasemediated lamin release could explain the performance variability of lamin substrates from different sources and if our nuclear ghosts contain lamins which are not phosphorylated at PKA site(s), why preincubation with PKA has an inhibitory effect on lamin release in our
LITTLE
assay. The possibility that PKA is exerting its effect on lamin release indirectly cannot, of course, be excluded. We thank Dr. G. Krohne (Heidelberg) for his generous gift of the PKB8 anti-lamin mAb and Dr. L. Meijer and his colleagues for their kind help to M.L. at Roscoff with ~34”~~’ kinase techniques and for generously providing materials.
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