Lamin B Phosphorylation by Protein Kinase Cα and Proteolysis during Apoptosis in Human Leukemia HL60 Cells*

Protein phosphorylation plays an important role in signal transduction, but its involvement in apoptosis still remains unclear. In this report, the p53-null human leukemia HL60 cells were used to investigate phosphorylation and degradation of lamin B during apoptosis. We found that lamin B was phosphorylated within 1 h after addition of the DNA topoisomerase I inhibitor, camptothecin, and that lamin B phosphorylation preceded lamin B degradation and DNA fragmentation. Using a cell-free system we also found that cytosol from camptothecin-treated cells induced lamin B phosphorylation and degradation in isolated nuclei from untreated HL60 cells. Lamin B phosphorylation was prevented by the protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) but not by the Cdc2 inhibitor, flavopiridol. Phosphorylation of lamin B was inhibited by immunodepletion of PKCα from activated cytosol and was restored by addition of purified PKCα. PKCα activity also increased rapidly as lamin B was phosphorylated after initiation of the apoptotic response in HL60 cells. These data suggest that lamin B is phosphorylated by PKCα and proteolyzed before DNA fragmentation in HL60 cells undergoing apoptosis.

Protein phosphorylation plays an important role in signal transduction, but its involvement in apoptosis still remains unclear. In this report, the p53-null human leukemia HL60 cells were used to investigate phosphorylation and degradation of lamin B during apoptosis. We found that lamin B was phosphorylated within 1 h after addition of the DNA topoisomerase I inhibitor, camptothecin, and that lamin B phosphorylation preceded lamin B degradation and DNA fragmentation. Using a cell-free system we also found that cytosol from camptothecin-treated cells induced lamin B phosphorylation and degradation in isolated nuclei from untreated HL60 cells. Lamin B phosphorylation was prevented by the protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) but not by the Cdc2 inhibitor, flavopiridol. Phosphorylation of lamin B was inhibited by immunodepletion of PKC␣ from activated cytosol and was restored by addition of purified PKC␣. PKC␣ activity also increased rapidly as lamin B was phosphorylated after initiation of the apoptotic response in HL60 cells. These data suggest that lamin B is phosphorylated by PKC␣ and proteolyzed before DNA fragmentation in HL60 cells undergoing apoptosis.
The nuclear lamins are karyophilic proteins located at the nucleoplasmic surface of the inner nuclear membrane where they assemble in a polymeric structure referred to as the nuclear lamina (for review, see Refs. [1][2][3]. Lamins belong to the family of intermediate filaments, which share a tripartite organization consisting of a central ␣-helical rod domain of conserved size, flanked by N-and C-terminal non-␣-helical end domains of variable size and sequence (see Fig. 9). The lamina has been suggested to serve as a major chromatin anchoring site of nuclear scaffold-associated regions during interphase and possibly to be involved in organizing higher order chromatin domains. The lamina is a dynamic structure regulated by phosphorylation. Phosphorylation by p34 cdc2 kinase is key to the dissolution of the nuclear lamina during mitosis. Other lamin kinases include mitogen-associated protein kinases, c-AMP-dependent protein kinase (PKA) 1 and protein kinase C (PKC) (1)(2)(3). Major PKC phosphorylation sites have been mapped to serine residues located in close proximity to the nuclear localization signal in the C-terminal non-␣-helical region, and phosphorylation of these residues interferes with the nuclear transport of lamin B (2). The p34 cdc2 phosphorylation sites are on both sides of the central ␣-helical rod domain. While many mammalian cells contains three distinct lamins (lamins A, B, and C), human leukemia HL60 cells express primarily lamin B (4).
Lamin proteolysis during apoptosis has been reported in various cell lines treated with different stimuli. In human leukemia HL60 cells treated with etoposide (VP-16) (5) or camptothecin (CPT) (6), apoptosis is accompanied by diminished levels of lamin B. Etoposide is a topoisomerase II inhibitor (7) and CPT a topoisomerase I inhibitor (8). Both drugs are effective anti-cancer agents. Lamin B1 degradation was also reported to precede DNA fragmentation in apoptotic thymocytes (9) and in HeLa cells treated with anti-CD95 antibody (10). Lamin A and B proteolysis into 45-kDa fragments is also observed in apoptosis induced by serum starvation of ras transformed primary rat embryo cells (11) and in reconstituted cell-free systems (6,12). The site of lamin A and B cleavage yielding the 45-kDa fragment has recently been mapped to a conserved aspartate residue at position 230 (13,14) corresponding to a consensus sequence for caspases. Furthermore, lamin A has been shown to be cleaved by caspase 6 (Mch-2␣) but not caspase 3 (CPP32/YAMA) (13,15).
The death-related cysteine proteases of the caspase family play a central role in the execution phase of apoptosis (16 -19). Each caspase cleaves selectively a subset of cellular proteins. For instance, poly(ADP-ribose)polymerase is preferentially cleaved by caspase 3 (CPP32/Yama) (20,21), and lamin A can be cleaved by caspase 6 (Mch-2) (13,15). Interestingly, recent observations demonstrated that overexpression of mutant lamins A or B resistant to caspase cleavage delayed DNA fragmentation, suggesting that lamin cleavage participates in the activation of DNA fragmentation and nuclear apoptosis (14). Thus, lamins are presently the only known caspase substrates known to be directly involved in the execution phase of apoptosis.
Protein phosphorylation is probably important to regulate apoptosis (22). For instance, unscheduled activation of p34 cdc2 kinase, one of the lamin kinases (2), is associated with cytotoxic T lymphocyte-mediated apoptosis (23) and precedes CPT-and DNA damage-induced apoptosis in HL60 cells (24). In the present study, we investigated lamin B phosphorylation and degradation during apoptosis in response to camptothecin in HL60 cells and in a previously described cell-free system (6,(25)(26)(27). The identity of the lamin B protease is not known. Both caspases and the nuclear scaffold-associated serine protease have been suggested as candidate proteases (28,29).

MATERIALS AND METHODS
Chemicals, Drugs, and Antibodies-CPT, 7-hydroxystaurosporine (UCN-01), and flavopiridol were obtained from the NCI Drug Chemistry and Synthesis Branch. Drugs were freshly dissolved in dimethyl * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Anti-lamin B monoclonal antibody from mouse (101-B7) was purchased from Oncogene Research Products (Cambridge, MA) and antiprotein kinase C␣ (anti-PKC␣) polyclonal antibodies from rabbit was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-PKC monoclonal antibody 1.9 and recombinant PKC␣ from baculovirus were purchased from Life Technologies, Inc. The horseradish peroxidase-conjugated anti-mouse immunogloblin secondary antibody was purchased from Amersham Pharmacia Biotech.
Cell Culture, Drug Treatment, DNA, and Protein Labeling-Human promyelocytic leukemia HL60 cells were grown in suspension culture in RPMI 1640 medium supplemented with 10% fetal calf serum (Life Technologies, Inc.), 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin at 37°C in an atmosphere of 95% air and 5% CO 2 . For filter elution assays, HL60 cells were incubated with [ 14 C]thymidine for 1-doubling time (about 24 h). Cell cultures were then washed with fresh medium twice and chased in isotope-free medium overnight before drug treatment. Unless otherwise indicated, camptothecin treatments were with 5 M. For in vivo phosphorylation, HL60 cells were washed twice in phosphate-free RPMI 1640 medium containing 10% dialyzed fetal calf serum, resuspended in the same medium and incubated with 250 Ci of [ 32 P]orthophosphate/10 7 cells. Following 1-h incubation, the 32 P-labeled cells were washed twice and resuspended in phosphatefree RPMI 1640 with dialyzed serum for 30 min prior to drug treatment.
Isolation of Nuclei and Cytosol for Reconstituted Cell-free System Studies-We followed our previously published procedure (25,26). Briefly, untreated and treated cells (5 M camptothecin for 3 h) were spun down, rinsed three times in cold PBS (phosphate-buffered saline), and resuspended at a density of approximately 10 7 cells/ml in nucleus buffer (1 mM KH 2 PO 4 , 150 mM NaCl, 5 mM MgCl 2, 1 mM EGTA, 0.1 mM AEBSF, 0.15 unit/ml aprotinin, 1.0 mM Na 3 VO 4 , 5 mM HEPES, pH 7.4, 10% glycerol), including 0.3% Triton X-100. After incubation at 4°C for 10 min and gentle agitation, cellular mixes were centrifuged at 2,000 ϫ g for 10 min, rinsed once by centrifugation/resuspension in nucleus buffer without Triton X-100, and used as nuclei suspensions at a density of 1-2 ϫ 10 7 nuclei/ml. Supernatants were centrifuged at 10,000 ϫ g for 10 min and used as cytosol. [ 14 C]Thymidine-labeled cells were used to prepare nuclei for filter elution assay.
Filter Elution Assays for Measurement of DNA Fragmentation-DNA fragmentation related to apoptosis was measured by filter elution as described previously (25,30). Briefly, reaction mixtures were deposited onto protein-adsorbing filters (Metricel, Gelman Science, Ann Harbor, MI) and washed with 3 ml of nucleus buffer. This fraction (W) was collected. Lysis was performed with 5 ml of LS10 (2 M NaCl, 0.04 M Na 2 EDTA, 0.2% Sarkosyl, pH 10) followed by washing with 5 ml of 0.02 mM Na 2 EDTA, pH 10. The lysis (L) and EDTA (E) fractions were collected. All fractions (W, L, and E) and filters (F) were counted by liquid scintillation. DNA fragmentation was calculated as the percent of DNA eluting from the filter as: percent DNA fragmentation ϭ 100 ϫ (W ϩ L ϩ E)/(W ϩ L ϩ E ϩ F). All experiments were repeated at least two or three times.
Immunoblotting for Lamin B-After drug treatment, cells were washed in PBS and resuspended in reducing loading buffer (62.5 mM Tris-HCl, pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.003% bromphenol blue, 5% 2-mercaptoethanol) and sonicated for 20 s. The lysates containing 2.5 ϫ 10 5 cells were heated at 65°C for 15 min and then loaded in 12% SDS-polyacrylamide gels (precast gel from NOVEX, San Diego, CA). After electrophoresis, proteins were electrophoretically transferred from the gels to polyvinylidene difluoride membranes (Immobilon-P from Millipore Co., Bedford, MA) according to the manufacturer's  protocol. Membranes were incubated at room temperature for 1 h in the primary antibody solutions after blocking in 5% non-fat dry milk solution for 1 h, followed by 1-h incubation with secondary antibody. Bands were visualized by enhanced chemiluminescence (SuperSignal, Pierce).
In Vivo Lamin B Phosphorylation-After drug treatment, 32 P-labeled HL60 cells (10 7 cells/sample) were washed in phosphate-free RPMI 1640 medium without serum once, and the cell pellets were lysed in buffer A (PBS containing 1% Nonidet P-40, 1 g/ml leupeptin, 5 mM NaF, 1 mM Na 3 VO 4 , 2 mM AEBSF, 4 units/ml aprotinin, and 1% bovine serum albumin) with 0.4% SDS before sonication. The cell lysates were centrifuged at 14,500 rpm for 15 min, and the supernatants were mixed with 1.5 g of anti-human lamin B antibody and 20% protein G-Sepha- rose suspensions in lysis buffer followed by overnight mixing at 4°C. At the end of incubation, the immune complex was washed in buffer A and buffer A without bovine serum albumin. The immune complex was boiled for 10 min after adding 3 ϫ SDS loading buffer. Samples were analyzed in 12% SDS-PAGE. Protein gels were dried up and subjected to autoradiography after being dried up.
In Vitro Lamin B Phosphorylation in Cell-free System-The nuclei from untreated HL60 cells (1.5 ϫ 10 6 nuclei) were incubated with cytosol in the presence of [␥-32 P]ATP. After incubation, buffer A supplemented with 0.4% SDS was added to the samples before brief sonication. Afterward the procedures were the same as for in vivo lamin B phosphorylation.
Immunodepletion of PKC␣-Anti-PKC␣ antibody was incubated with protein G-Sepharose beads at 4°C for 3 h. The beads were collected by centrifugation. After removal of the supernatant, the beads were washed once with nucleus buffer and incubated with cytosol from CPT-treated cells (CPT-cytosol:antibody ϭ 5:1 (v/v)) overnight in a rotator at 4°C. The beads were subsequently pelleted by centrifugation at 10,000 ϫ g. The supernatant was subjected to immunoblotting for PKC␣ and was used as CPT-cytosol immunodepleted of PKC␣. Mock-depleted CPT-cytosol was made just using nuclei buffer to replace antibody.

Lamin B Degradation and Phosphorylation in Apoptotic
HL60 Cells-HL60 cells are remarkably sensitive to various apoptotic stimuli, including chemotherapeutic DNA-damaging agents (5, 31) such as the topoisomerase I inhibitor camptothecin, protein kinase inhibitors (25,32), and the Golgi poison, brefeldin A (33). Consistent with previous studies (31), Fig. 1A shows that camptothecin induces apoptotic DNA fragmentation in HL60 cells with rapid kinetics. Lamin B protein was cleaved with similar kinetics as the DNA fragmentation, yielding two cleavage bands (Fig. 1B) corresponding to 45-and 32-kDa polypeptides that were detected 3 h after the beginning of drug treatment. Since phosphorylation is critical for modulating lamin stability and the nuclear and chromatin structure both in mitosis and interphase (2), we studied lamin B phosphorylation during apoptosis in HL60 cells. As shown in Fig. 2, phosphorylation of the 69-kDa lamin B polypeptide increased rapidly during camptothecin treatment. Three hours after the beginning of treatment, the 32-kDa lamin B cleavage product was also phosphorylated ( Fig. 2A). These results indicate that lamin B phosphorylation occurs early during apoptosis and is associated with its degradation.
Cytosol from Apoptotic HL60 Cells Also Induced Lamin B Cleavage and Phosphorylation in Vitro-We next used a cellfree system that we previously established to demonstrate the role of serine proteases in triggering apoptotic DNA fragmentation (25,26,34). Consistent with our previous results, the cytosol from apoptotic HL60 cells induced DNA fragmentation in nuclei from untreated HL60 cells (Fig. 3A). Cytosol from apoptotic cells also cleaved lamin B from naive nuclei to a 45-kDa product (Fig. 3B).
Lamin B phosphorylation was then studied in the cell-free system after incubation of nuclei suspensions with cytosols from apoptotic or control cells in the presence of [␥-32 P]ATP. After immunoprecipitation with anti-lamin B antibody, samples were run on SDS-PAGE, and phosphorylated lamin B was analyzed by autoradiography and PhosphorImager (Molecular Dynamics). Fig. 4 shows that cytosol from apoptotic cells enhanced lamin B phosphorylation.
Lamin B Cleavage, but Not Phosphorylation, in the Cellfree System Can Be Inhibited by the Serine Protease Inhibitor DCI-We observed previously that DNA fragmentation induced by apoptotic cytosol could be inhibited by the serine protease inhibitor DCI (6). Fig. 5 shows while DCI blocked DNA fragmentation induced by CPT, lamin B cleavage also was abolished. The result suggested that serine protease activation was required for both DNA fragmentation and lamin B degradation. We next asked whether lamin B phosphorylation could be inhibited by DCI. Fig. 5C shows that lamin B phosphorylation was not affected by DCI. This finding suggests that protease activation does not affect lamin B phosphorylation.
Investigation of the Lamin B Kinase during Apoptosis in HL60 Cells-Cyclin B/Cdc2 (p34 cdc2 ) kinase is critical for lamin depolymerization during mitosis (2). We found that flavopiridol, a potent Cdk inhibitor (35,36) could not inhibit lamin B phosphorylation induced by cytosol from apoptotic cells even at high concentrations (100 M) (Fig. 6A). We also found that flavopiridol had not effect on either DNA fragmentation or lamin B cleavage in the cell-free system (Fig. 6, B and C). These observations suggested that p34 cdc2 kinase was not responsible for phosphorylation of lamin B by the apoptotic cytosol.
Protein kinase C has also been shown to phosphorylate lamin B in interphase cells (37). We first used the PKC inhibitor UCN-01 (38) to test whether lamin B phosphorylation during apoptosis is related to PKC. Fig. 7A shows that lamin B phosphorylation was inhibited by UCN-01 in a dose-dependent manner. An anti-PKC monoclonal antibody, which acts near the active site of PKC and inhibits PKC activity by more than 80% (39), was used next. This antibody strongly suppressed lamin B phosphorylation induced by apoptotic cytosol. Recombinant PKC␣ restored lamin B phosphorylation (Fig. 7B). A third type of experiment was performed to test whether lamin B phosphorylation in apoptotic cells extracts could be linked to PKC␣. Fig. 7C shows that after immunodepletion of PKC␣ from apoptotic cytosol, lamin B phosphorylation was reduced by about 90%. The efficiency of the immunodepletion was tested (Fig. 7C, lower panel) and showed that under these condition PKC␣ protein levels were almost undetectable. Together, the results shown in Fig. 7 suggested that PKC␣ was critical for lamin B phosphorylation by cytosol from apoptotic HL60 cells.
Protein Kinase C␣ Is Activated with a Similar Kinetics as Lamin B Phosphorylation during Camptothecin-induced Apoptosis in HL60 Cells-A recent study showed that PKC␣ is activated in cytosol from apoptotic HL60 cells (40). Now we tested whether camptothecin also induces PKC␣ activation in whole HL60 cells. As shown in Fig. 8, PKC␣ activity increased rapidly during the first hour after the beginning of camptothecin treatment. DISCUSSION The present study is the first report of lamin B phosphorylation during apoptosis. We found that lamin B is phosphorylated within 1 h after the addition of the apoptotic inducer (camptothecin) and that lamin B phosphorylation persists for several hours as lamin B is being cleaved, and DNA fragmentation and complete apoptosis take place (31). Various kinases are involved in lamin phosphorylation, including The C-terminal tetrapeptide referred to as the CaaX box (C ϭ Cys; a ϭ aliphatic amino acid; X ϭ any amino acid) is subject to three successive posttranslational modifications (farnesylation, proteolytic trimming, and carboxymethylation), which are required for association of nuclear lamins with the nuclear membrane (2). p34 cdc2 kinase, mitogen-associated kinase, PKC, PKA (2). In particular, in mitosis, Cdc2 kinase is believed to play a critical role in lamin phosphorylation and thus disassembly of lamin polymers (2,41). The present data suggest that PKC is critical for lamin B phosphorylation in HL60 cell during apoptosis for the following reasons. First, the PKC inhibitor, UCN-01 (38,42) effectively blocked the lamin kinase in vitro, while the cyclin kinase inhibitor flavopiridol (35) was inactive. Second, lamin B phosphorylation was inhibited by a monoclonal antibody directed against the active site of PKC. Third, immunodepletion of apoptotic cell extracts with anti-PKC antibody inhibited lamin B phosphorylation, while addition of excess PKC restored lamin B phosphorylation. Fourth and finally, total PKC␣ activity increased at the time of lamin B phosphorylation (40).
The lamin B protein kinase C phosphorylation sites have been mapped to serines 395 and 405 in HL60 cells following PKC activation by bryostatin (43) (Fig. 9). These sites are adjacent to the highly conserved central ␣-helical rod domain, which is thought to be responsible for the formation of a highly stable coiled-coil dimer between two lamin molecules. It is also next to the nuclear translocation signal sequence (NLS) (Fig.  9). In the case of chicken lamin B2, phosphorylation by PKC in this region has been shown to alter recognition of this sequence and block nuclear import of newly synthesized lamin polypeptides (2). Recently, PKC-mediated lamin B phosphorylation during interphase has been shown to promote lamin B solubilization and nuclear lamina disassembly (37). Thus, PKC-mediated lamin B phosphorylation during apoptosis is likely to affect nuclear and chromatin structure.
Proteolytic lamin degradation is a common and probably functionally important biochemical feature of apoptosis. It has been observed in all the apoptotic cell systems described to date, including HL60 cells treated with chemotherapeutic agents (5,6,34), activation-driven cell death of T cells (44), thymocyte apoptosis (9), and serum starvation in ras transformed embryo cells (11). Lamin degradation has also been reported in apoptosis induced by drICE in insect cells (45). Interestingly, a recent study of Rao et al. (14) demonstrated that overexpression of lamin A or B delayed nuclear apoptosis and DNA fragmentation in the case of p53-dependent apoptosis in rodent cells. These observations suggest that lamin cleavage plays an active role in the execution phase of apoptosis. Caspase 6 (Mch-2) has been shown to cleave lamin A at the conserved aspartic residue at position 230 (13,14), and the corresponding lamin B cleavage site has been mapped to Asp 231 (14). This site is located in the conserved ␣-helical rod domain (Fig. 9), and its cleavage would produce two polypeptides of 40.3 and 25.9 kDa, respectively. The observed 45-kDa fragment has been shown to correspond to the predicted 40.3 fragment (13,14). Another candidate lamin protease is the nuclear scaffold protease (46), which would be expected to cleave lamin B at tyrosine 377 and to yield two polypeptides of 43.2 and 23 kDa, respectively (Fig. 9). Therefore, it is possible that serine proteases (6) might also be involved in lamin B cleavage during apoptosis to produce the 32-kDa lamin B fragment that we observed in addition to the 45-kDa polypeptide in apoptotic HL60 cells. Since the observed sizes indicate that cleavage of lamin B during apoptosis occurs in the conserved ␣-helical rod domain, which is essential for lamin dimerization (Fig. 9), it is likely that lamin B cleavage should promote the dissolution of the nuclear lamina and affect nuclear condensation. This recent conclusion is consistent with the works of Rao et al. (14) and Lazebnik et al. (12), demonstrating an impairment of apoptotic chromatin condensation upon inhibition of lamin proteolysis. Chromatin condensation and DNA fragmentation might be related to the important function of the nuclear lamina as an anchorage structure for the chromatin scaffold-associated regions, which would organize the chromatin loop structures. Thus, it is possible that chromatin release from the nuclear lamina might facilitate the activity of nucleases and the cleavage and release of chromatin loops during apoptosis.