PKCd-dependent cleavage and nuclear translocation of annexin A1 by phorbol 12-myristate 13-acetate Yoon S. Kim 1, *, Jesang Ko 2, *, In S. Kim 1 , Sung-Wuk Jang 2 , Ho J. Sung 2 , Hye J. Lee 1 , Si Y. Lee 1 , Youngho Kim 3 and Doe S. Na 1 1 1 Department of Biochemistry and Molecular Biology; 2 Asan Institute for Life Sciences, and 3 Genome Research Center for Birth Defects and Genetic Diseases, University of Ulsan College of Medicine, Seoul, Korea Annexin A1 (ANX-1), a calcium-dependent, phospholipid binding protein, is known to be involved in diverse cellular processes, including regulation of cell growth and differen- tiation, apoptosis, and inflammation. The mitogen phorbol 12-myristate 13-acetate (PMA) induces expression and phosphorylation of ANX-1. However, the roles of ANX-1 in PMA-induced signal transduction is unknown. Here, we study the cellular localization of ANX-1 in the PMA-induced signal transduction process. We have found that PMA induces the cleavage of ANX-1 in human embryonic kidney (HEK) 293 cells, and that the cleaved form of ANX-1 translocates to the nucleus. The PMA- induced nuclear translocation of ANX-1 was inhibited by the protein kinase C (PKC)d-specific inhibitor rottlerin, indicating that PKCd plays a role in nuclear translocation of the cleaved ANX-1. We propose a novel mechanism of PMA-induced translocation of ANX-1 to the nucleus that may participate in the regulation of cell proliferation and differentiation. Keywords: annexin A1; PMA; cleavage; nuclear transloca- tion; PKCd. Annexins (ANXs) are a family of calcium-dependent, phospholipid-binding proteins. Several members of the ANX family are known to be involved in various physio- logical functions including anti-inflammatory processes, cell signaling, regulation of cell growth and differentiation, apoptosis, membrane fusion, exocytosis, and interaction with cytoskeletal proteins [1–3]. Although there have been recent advances in understanding the molecular mechanisms by which ANXs play a role in these cellular processes, the regulatory mechanism in cell proliferation and differenti- ation remains to be characterized. It has been reported that ANX-1 is involved in regulation of the mitogenic signal transduction pathways including the mitogen-activated protein (MAP) kinase-, the epidermal growth factor receptor (EGFR)-, and the hepatocyte growth factor receptor (HGFR) kinase-mediated signaling pathways [4–6]. The expression level of ANX-1 increases in response to phorbol 12-myristate 13-acetate (PMA) and interleukin (IL)-6 [7,8], and dys-regulation of ANX-1 results in development of various cancers [9,10]. Mitogens such as EGF and PMA induce phosphorylation, cleavage, and translocation of ANX-1 to the membrane [11–13] and this phosphorylation event is mediated by protein kinase C (PKC) [14]. ANX-1 mainly exists in the cytosol, but also exists in the membrane or the nucleus [15]. Recent reports suggest that subcellular localization of ANX-1 can be redistributed by treatment with specific stimuli. ANX-1 translocates to the membrane and is secreted to the extracellular surface of the cell membrane in response to glucocorticoid and PMA [16,17]. Although we have previously proposed that EGF, oxidative stress and heat shock induce translocation of ANX-1 to the nucleus [18,19], the mechanism for nuclear translocation of ANX-1 is still unknown. PKCs are serine-threonine kinases that are activated by diverse stimuli including mitogens and participate in a variety of cellular processes such as cell proliferaction and differentiation, and apoptosis [20,21]. The PKC family consists of 12 isoforms that are grouped into three subfamilies: the classical PKCs (a, b1, b2, v), the novel PKCs (d, e, g, h), and the atypical PKCs (n, k/i). PKCd,a member of the novel PKC subfamily, is activated by diacylglycerol (DAG) and phorbol esters in a calcium- independent manner and plays a critical role in the control of cell growth and apoptosis [22]. In this study, we aimed to elucidate whether PMA induces the translocation of ANX-1 to the nucleus in human embryonic kidney (HEK) cells and the roles of PKCd in the nuclear translocation of ANX-1. We propose a mechanism for the nuclear translocation of ANX-1 in response to PMA that may be involved in cellular processes such as cell proliferation and differentiation. Correspondence to D. S. Na, Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, 388–1 Poongnap-dong, Songpa-gu, Seoul 138–736, Korea. Fax: + 82 2 477 9715, Tel.: + 82 2 3010 4275, E-mail: dsna@amc.seoul.kr Abbreviations: ANX, annexin; PMA, phorbol 12-myristate 13-acetate; HEK, human embryonic kidney; PKC, protein kinase C; MAP kinase, mitogen-activated protein kinase; EGFR, epidermal growth factor receptor; HGFR, hepatocyte growth factor receptor; DAG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium. *Note: the first two authors contributed equally to this work. (Received 15 July 2003, revised 17 August 2003, accepted 21 August 2003) Eur. J. Biochem. 270, 4089–4094 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03800.x Materials and methods Materials Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Life Technolo- gies, Inc. (Gaithersburg, MD, USA). Rottlerin, Ro-31-8425, PD98059, Ly294002, SB202190 were from Calbiochem (San Diego, CA, USA). PMA and goat anti-(mouse IgG) Ig conjugated with tetramethylrhodamine isothiocyanate (TRITC) were products of Sigma (St Louis, MO, USA). Anti-ANX-1 monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY, USA). Cell culture HEK 293 cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 UÆmL )1 ), and streptomycin (100 lgÆmL )1 )at37°Cunder5%CO 2 atmosphere. For Western blot analysis, cells were seeded into 60 mm dishes at 1 · 10 6 cells per dish. After 18–24 h, cells were further grown in the same medium supplemented without FBS for 24 h. Serum-starved cells were treated with PMA for the indicated times. For immunostaining, 2 · 10 5 cells grown on cover slides (22 · 22 mm) were starved for 24 h before stimulation with PMA. Immunocytochemistry HEK 293 cells grown on cover slides were fixed with 3.7% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 in NaCl/KCl/P i (NaCl/P i , 137 m M NaCl, 2.7 m M KCl, 8 m M Na 2 HPO 4 ,1.5m M KH 2 PO 4 ) for 5 min. After washing the cells with NaCl/ KCl/P i three times, the cells were blocked for 30 min in NaCl/P i containing 1% bovine serum albumin. Immuno- staining was performed by incubation with anti-ANX-1 monoclonal antibody (0.05 lgÆmL )1 )for2h.After washing the cells with NaCl/P i three times, the cells were incubated with TRITC-conjugated goat anti-(mouse IgG) Ig for 1 h. Cover slides were washed with NaCl/P i , mounted, and examined using a Leica TCS SP2 Confo- cal microscope (Leica Microsystems, Wetzlar GmBH, Germany). Cell fractionation HEK 293 cells were seeded into 60 mm dishes at 1 · 10 6 cells/dish and cultured in DMEM for 18–24 h. The cells were starved for 24 h in serum-free media. After treatment with PMA for a given time, the cells were harvested and washed with ice-cold NaCl/P i .Thecells were then resuspended in 100 lL of lysis buffer (10 m M Hepes, 10 m M NaCl, 0.1 m M EDTA, 0.1 m M EGTA, 1% NP-40, 0.5 m M phenylmethylsulfonyl fluoride, 0.1 m M dithiothreitol, 0.1 m M sodium orthovanadate, and prote- ase inhibitors) and incubated on ice for 10 min. The nuclei were collected by centrifugation at 2000 g for 5min at 4°C. The supernatant was collected as a cytosolic fraction. Protein concentration of each sample was determined. Western blot analysis For preparing whole cell lysates, 1 · 10 6 cells were lysed in 1 · SDS gel-loading buffer (50 m M Tris/HCl, pH 6.8, 100 m M dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol). Protein samples were separated on 12% SDS/polyacrylamide gels and transferred to nitrocellulose filters. The blots were incubated with anti-ANX-1 mono- clonal antibody for 1 h. After washing three times with NaCl/P i containing 0.05% Tween 20, the blots were incubated with goat anti-(mouse IgG) Ig conjugated with horseradish peroxidase (HRP) for 1 h. The blots were washed three times with NaCl/P i containing 0.05% Tween 20 and developed with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech., Piscata- way, NJ, USA). Results PMA induces the cleavage of ANX-1 As ANX-1 is cleaved by treatment with PMA in epithelial A549 cells [7], we first examined whether PMA induces the cleavage of ANX-1 in HEK 293 cells. Cells were treated with 10 n M PMA for 30 min and cell lysates were subjected to Western blot analysis using ANX-1 antibody. As shown in Fig. 1, the cleaved form of ANX-1 was detected in PMA- stimulated HEK 293 cells, whereas only the intact form of ANX-1 was found in control cells. ANX-1 was partially cleaved by the treatment of PMA and most of ANX-1 remained in the intact form. This result indicates that PMA induces the cleavage of ANX-1 in HEK 293 cells. ANX-1 translocates to the nucleus by PMA stimulation There have been reports that the cleaved form of ANX-1 is secreted and presents mainly on the outer surface of cell membrane [17]. To examine the subcellular localization of ANX-1 in response to PMA in HEK 293 cells, cells were incubated in the absence or presence of 10 n M PMA for 30 min and immunostained with anti-ANX-1 monoclonal antibody. In unstimulated cells, ANX-1 was evenly detected throughout the cells including cytosol and nucleus (Fig. 2). However, the level of ANX-1 significantly increased in the nucleus in about 20–30% of PMA-treated cells observed (Fig. 2), indicating that ANX-1 translocates to the nucleus by PMA stimulation. Fig. 1. PMA induces the cleavage of ANX-1. Serum-starved HEK 293 cells were incubated in the absence or presence of 10 n M PMA for 30minandlysedwith1· SDS-loading dye. Whole cell lysates were separated in a 12% SDS/polyacrylamide gel and transferred to nitro- cellulose membrane. Cleavage of ANX-1 was detected by Western blotting with anti-ANX-1 monoclonal antibody. 4090 Y. S. Kim et al.(Eur. J. Biochem. 270) Ó FEBS 2003 PMA induces the nuclear translocation of the cleaved form of ANX-1 As PMA induced the cleavage of ANX-1 and accumulation of ANX-1 in the nucleus, we wondered if the cleaved ANX-1 is responsible for the increase of ANX-1 in the nucleus. To determine this possibility, HEK 293 cells were treated with 10 n M PMA for 30 min, and fractionated into cytosolic and nuclear fractions. As shown in Fig. 3A, the cleaved ANX-1 was detected in the nuclear fraction of PMA-treated cells and the amount of the intact ANX-1 level was not changed between the nuclear fractions of treated and untreated cells. These results indicate that accumulation of ANX-1 in the nucleus is a result of translocation of the cleaved ANX-1 to the nucleus. We next examined dose- and time-dependency of the nuclear translocation of the cleaved ANX-1. HEK 293 cells were treated with the indicated concentrations of PMA for 30 min and subjected to Western blot analysis. The cleavage and translocation of ANX-1 was induced at a concentration of more than 1 n M (Fig. 3B). Figure 3C shows that the cleaved ANX-1 in the nuclear fraction began to be found after 15 min of exposure to PMA. These results indicate that the cleavage and translocation of ANX-1 to the nucleus in response to PMA are immediate early events. PMA-induced nuclear translocation of ANX-1 is mediated via PKCd It has been reported that PKC induces phosphorylation and cleavage of ANX-1 [12]. Therefore, we examined whether PKC is involved in the cleavage and nuclear translocation of ANX-1. HEK 293 cells were preincubated in the presence of rottlerin or Ro-31-8425, and were stimulated with PMA. The cleavage and nuclear translocation of ANX-1 were inhibited by a PKCd-specific inhibitor rottlerin, but not by Ro-31-8425 (Fig. 4A). To investigate whether other signa- ling molecules are involved in the cleavage and nuclear translocation of ANX-1, Western blot analysis was con- ducted in the absence or presence of inhibitors of ERK (PD98059), p38 (SB202190), or PI-3 kinase (LY294002). Figure 4A shows that the PMA-induced cleavage and Fig. 2. ANX-1 accumulates in the nucleus by PMA stimulation. Serum- starved HEK 293 cells were incubated in the absence or presence of 10 n M PMA for 30 min and processed for immunocytochemical detection of endogenous ANX-1 using anti-ANX-1 monoclonal anti- body as described in Materials and methods. Confocal images of untreated (A) and PMA-treated (B) cells are representatives of the cells observed in three independent experiments. 2 Fig. 3. PMA induces the nuclear translocation of the cleaved ANX-1. (A) Serum-starved HEK 293 cells were incubated in the absence or presence of 10 n M PMA for 30 min. Cells were then fractionated into cytosol and nucleus, and separated in a 12% SDS/polyacrylamide gel and subjected to immunoblotting using anti-ANX-1 monoclonal antibody. (B) HEK 293 cells were incubated in serum-free DMEM for 24 h and stimulated with PMA at indicated concentrations for 30 min. Cells were harvested and separated into cytosolic and nuclear frac- tions, then samples were resolved by 12% SDS/polyacrylamide gels and transferred to nitrocellulose membrane. Translocation of ANX-1 was detected by Western blotting. (C) Serum-starved HEK 293 cells were treated with 10 n M PMA for indicated times, then harvested and fractionated into cytosol and nucleus. Samples were separated in 12% SDS/polyacrylamide gels. Time-dependent translocation of ANX-1 to the nucleus was detected by Western blotting with anti-ANX-1 monoclonal antibody. Ó FEBS 2003 PMA-induced nuclear translocation of annexin A1 (Eur. J. Biochem. 270) 4091 nuclear translocation of ANX-1 were not inhibited by the addition of these inhibitors suggesting that these molecules are not involved in the PMA-induced cleavage and nuclear translocation of ANX-1. We next confirmed the inhibitory effect of rottlerin on the cleavage and nuclear translocation of ANX-1 using immunocytochemical analysis (Fig. 4B). In cells preincu- bated with rotterin, PMA was not able to induce the increase of ANX-1 in the nucleus. These results indicate that PKCd plays a role in the PMA-induced cleavage and nuclear translocation of ANX-1. Discussion Despite recent advances in our understanding of the roles of ANX-1 in mitogenic signal transduction, the exact mechanism through which ANX-1 functions in response to mitogenic stimuli remains unclear. In this work, we attempted to elucidate the regulatory mechanism of ANX-1 in the PMA signaling pathway in HEK 293 cells. We have demonstrated that (a) PMA induces the cleavage of ANX-1. (b) the cleaved ANX-1 translocates to the nucleus in a time- dependent manner, and (c) the cleavage and nuclear translocation of ANX-1 is mediated by a PKCd-dependent mechanism. Several lines of evidences suggest that ANX-1 is cleaved in response to IL-6 and PMA in different cell lines [7,12] and that the cleaved ANX-1 is translocated to the extracellular surface of the cell membrane [16,17]. To investigate the role of ANX-1 in the PMA-induced signal transduction path- way, we first examined whether PMA induces the cleavage of ANX-1 in HEK 293 cells. Our data showed that PMA induced the cleavage of ANX-1 and the cleavage is comparable to that reported previously in other cell lines [7,12]. It has been reported that truncated ANX-1 that is missing the first 29 N-terminal amino acids is secreted from the prostate cancer cells [23]. Whether the same type of cleavage occurs in PMA treated cells is not clear. Never- theless it is reasonable to assume that the cleavage site of ANX-1 in response to PMA is at the N-terminal region of ANX-1. The cleavage site of ANX-1 and the enzyme that is responsible for the cleavage is under investigation in this laboratory. Interestingly, data from confocal microscopy show that ANX-1 is accumulated in the nucleus. We have previously reported that ANX-1 translocates to the nucleus by EGF, oxidative stress, and heat shock [18,19]. Our results suggest that PMA also induces the translocation of ANX-1 to the nucleus. However, the nuclear translocation of ANX-1 was observed in about 20–30% of PMA-treated cells indicating that this may be cell cycle-dependent event. When PMA- treated HEK 293 cells were fractionated into cytosolic and nuclear fractions, the cleaved form of ANX-1 was found in the nuclear fraction, indicating that the cleaved form but not the intact form of ANX-1 translocates to the nucleus. The cleaved ANX-1 was detected in the cytosolic fraction with a longer exposure, indicating that ANX-1 is cleaved in the cytosol, then the cleaved ANX-1 translocates to the nucleus. However, results from the Western blotting with a longer exposure and confocal microscopy demonstrate that the minimal level of the cleaved ANX-1 was also found in the nuclear fraction of control cells (data not shown). Therefore, it cannot be ruled out the possibility that the cleavage of ANX-1 occurs both in the cytosol and the nucleus. Fig. 4. PMA-induced nuclear translocation of ANX-1 is mediated via PKCd. (A) Serum- starved HEK 293 cells were preincubated in the absence or presence of rottlerin (5 l M ), Ro-31-8425 (50 l M ), PD98059 (50 l M ), LY294002 (10 l M ), and SB202190 (20 l M )for 30 min and stimulated with 10 n M PMA for 30 min. Cells were fractionated into cytosolic and nuclear fractions, and analyzed by 12% SDS/polyacrylamide gels and transferred to nitrocellulose membrane. Translocation of ANX-1 was probed with anti-ANX-1 mono- clonal antibody. (B) HEK 293 cells were incubatedinserum-freeDMEMfor24h. Serum-starved cells were preincubated in the absence or presence of PKCd-specific inhibitor rottlerin (5 l M ) for 30 min and treated with 10 n M PMA for 30 min. Cells were then immunostained with anti-ANX-1 monoclonal antibody as described in Materials and methods. The micrographs are representatives of the cells observed in three independent experiments. 3 4092 Y. S. Kim et al.(Eur. J. Biochem. 270) Ó FEBS 2003 PMA induced the cleavage and nuclear translocation of ANX-1 in a time-dependent manner. The cleaved ANX-1 began to be detected in the cytosolic fraction at 10 min of PMA treatment. After 15 min of exposure to PMA, the cleaved ANX-1 began to translocate to the nucleus. This result confirms the data that ANX-1 is cleaved first in the cytosol, then translocates to the nucleus. The nuclear translocation of ANX-1 seems to be an immediate early process and is comparable to that of other signaling molecules such as MAP kinase and NF-jB [24,25]. As ANX-1 binds both DNA and RNA [26], there is a possibility that the cleaved ANX-1 translocates to the nucleus and participates in cell proliferation and differen- tiation processes by regulating transcription. Taken together, the cleavage and translocation of ANX-1 to the nucleus by PMA may be a physiological process involved in cell proliferation and differentiation. The PKC family, which comprises several isoforms, plays an important role in cell proliferation and differentiation [20,21]. PKC is known to be involved in the cleavage and secretion of ANX-1 [12]. However, it is not clear which isoform of PKC mediates the cleavage of ANX-1. PKCd is a member of the novel PKC subfamily and is known to play a critical role in the regulation of cell proliferation and apoptosis [22]. Our data indicate that PKCd is responsible for the cleavage and nuclear translocation of ANX-1. It has been known that the major phosphorylation sites of ANX-1 by PKC are Ser-27 and Thr-41, and the phosphorylation of ANX-1 at Ser-27, Ser-28, and Thr-24 has also been identified [27]. PKCd probably phosphorylates ANX-1 at one or several of these sites directly or indirectly and induces the cleavage and nuclear translocation of ANX-1. We also investigated the involvement of other signaling molecules such as MEK, PI-3 kinase, and p38 in PMA-induced translocation of ANX-1. Inhibition of these molecules did not affect the cleavage and nuclear translocation of ANX-1 in response to PMA, indicating that these molecules are not required for the PMA-induced cleavage and nuclear trans- location of ANX-1. ANX-1 is involved in the regulation of cell proliferation and differentiation [28,29]. Recently, ANX-1 –/– mice were generated and partially characterized [30] and it has been confirmed that ANX-1 is a mediator of glucocorticoid- induced growth inhibition [31]. However, the exact mech- anism by which ANX-1 plays a role in cell growth is not known. In the present study, we propose the mechanism by which PMA induces the translocation of ANX-1 to the nucleus. We have demonstrated that PMA induces the cleavage of ANX-1 leading to the nuclear translocation of ANX-1, and that PKCd plays a critical role in this process. While further studies are required to characterize the exact functions of ANX-1 in the nucleus, from this study we can begin to understand the role of ANX-1 in the PMA-induced signal transduction, which may provide an important clue for understanding the molecular mechanism of cell proliferation and differentiation. Acknowledgements This work was supported by a Korea Research Foundation Grant (KRF-2002-042-C00076) (to D. S. N and J. K). References 1. 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