The Subcellular Localization of Cyclin Dependent Kinase 2 Determines the Fate of Mesangial Cells: Role in Apoptosis and Proliferation

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The subcellular localization of cyclin dependent kinase 2 determines the fate of mesangial cells: role in apoptosis and proliferation

K Hiromura1, JW Pippin1, MJ Blonski1, JM Roberts2 and SJ Shankland*,1

1Department of Medicine, Division of Nephrology, University of Washington School of Medicine, Seattle, Washington, WA 98195- 6521, USA; 2Department of Basic Sciences and Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington, WA 98104, USA

Apoptosis is closely linked to proliferation. In this study we Korsmeyer, 1998; Miller and Marx, 1998; Minn et al., showed that inducing apoptosis in mouse mesangial cells 1998). Proliferation requires that cells progress through with ultraviolet (UV) irradiation was associated with the cell cycle, which is governed by speci®c cell cycle increased cyclin A-cyclin dependent kinase (CDK) 2 regulatory proteins. Proliferation requires the activa- activity. Inhibiting CDK2 activity with Roscovitine or tion of cyclin-dependent kinases (CDKs) by their dominant negative mutant reduced apoptosis. Because partner cyclins during each phase of the cell cycle apoptosis typically begins in the cytoplasm, we tested the (Pines, 1994; Roussel, 1998; Shankland and Wolf, hypothesis that the subcellular localization of CDK2 2000; Sherr and Roberts, 1999). CDK4 and CDK6 are determines the proliferative or apoptotic fate of the cell. activated by D-type cyclins during early G1, CDK2 is Our results showed that cyclin A-CDK2 was nuclear in activated by cyclin E in G1/S and by cyclin A during S proliferating cells. However, inducing apoptosis in pro- and G2/M, and cyclin B1 activates cdc2 during mitosis. liferating cells with UV irradiation was associated with a The traditional view is that the cell cycle is restricted decrease in nuclear cyclin A and CDK2 protein levels. This to proliferation. However, recent studies have also coincided with an increase in protein and kinase activity for shown a link between the cell cycle and apoptosis. cyclin A-CDK2 in the cytoplasm. Translocation of cyclin There is a growing body of evidence showing that A-CDK2 also occurred in p537/7 mesangial cells. speci®c cyclin-CDKs participate in apoptosis. The Finally, we showed that caspase-3 activity was signi®- activity of cdc2 (Shi et al., 1994; Yao et al., 1996), cantly reduced by inhibiting CDK2 activity with Roscov- CDK4 and CDK6 (Dobashi et al., 1998; Park et al., itine. In summary, our results show that apoptosis is 1998) are increased in certain forms of apoptosis. associated with an increase in cytoplasmic cyclin A-CDK2 Overexpressing CDK2 accelerates thymocyte cell death activity, which is p53 independent and upstream of (Gil-Gomez et al., 1998). Cyclin A overexpression caspase-3. We propose that the subcellular localization induces apoptosis in rat ®broblasts exposed to low of CDK2 determines the proliferative or apoptotic fate of serum (Hoang et al., 1994), and also circumvents the the cell. anti-apoptotic activity of Bcl-2 in HeLa cells (Meik- Oncogene (2002) 21, 1750 ± 1758. DOI: 10.1038/sj/ rantz and Schlegel, 1996). Recent studies have shown onc/1205238 that cyclin A-CDK2 is active during apoptosis in the absence of DNA synthesis, and that inhibiting CDK2 Keywords: apoptosis; cell cycle; cyclin dependent kinase; activity reduces apoptosis (Adachi et al., 2001; Levkau mesangial cell et al., 1998; Meikrantz et al., 1994). The association of speci®c cyclin-CDKs and apoptosis has also been shown in vivo. Mice transgenic for both cyclin A and CDK2 show a greater incidence of apoptosis (Bortner Introduction and Rosenberg, 1995), and increased cyclin A-CDK2 activity coincides with a marked increase in apoptosis Apoptosis is a highly orchestrated form of pro- in in¯ammatory injury (Ophascharoensuk et al., 1998). grammed cell death required to maintain cell number. Taken together, these studies demonstrate that speci®c Under certain pathological states characterized by cell cyclin-CDK complexes are involved in both the proliferation, such as in¯ammation and neoplasia, proliferative and apoptotic pathways following injury. apoptosis is required to normalize cell number in order However, the precise mechanisms that determine this to maintain a normal tissue homeostasis (Chao and have not been clearly delineated. Although DNA synthesis and mitosis are nuclear events, apoptosis typically begins in the cytoplasm (Green and Reed, 1998; Minn et al., 1998). Accord- *Correspondence: SJ Shankland, Division of Nephrology, University ingly, we tested the hypothesis that the subcellular of Washington School of Medicine, Box 356521, Seattle, Washington, localization of active CDK2 determines if cells enter a WA 98195-6521, USA; E-mail: [email protected] Received 9 July 2001; revised 30 November 2001; accepted 6 proliferative or apoptotic pathway. Our results show December 2001 that nuclear CDK2 is associated with proliferation and CDK2 and apoptosis K Hiromura et al 1751 that active cytoplasmic CDK2 is associated with apoptosis. This ®nding supports the notion that cyclin A-CDK2 is involved in both growth and death pathways, and that the fate of the cell is determined in part by the subcellular localization of active cyclin A-CDK2.

Results

Cells with increased CDK2 activity are more vulnerable to UV induced apoptosis and reducing CDK2 activity decreases apoptosis Previous studies have shown that apoptosis is increased in proliferating cell populations in certain forms of apoptosis (Danno and Horio, 1982; Nishioka and Welsh, 1994; Syljuasen and McBride, 1999) (Baker et al., 1994; Ophascharoensuk et al., 1998). However, the mechanisms linking these processes are not fully elucidated. To test the hypothesis that the susceptibility of proliferating cells to apoptosis was due to increased CDK2 activity, apoptosis was induced by ultraviolet (UV) B irradiation in quiescent mouse mesangial cells (serum starved for 48 h) when CDK2 was inactive, and in proliferating mesangial cells (serum starved for 48 h, then add medium containing 20% FCS for 24 h) when CDK2 is active. Following UV exposure, apoptosis was increased in the proliferating cell population compared to UV exposure in a quiescent cell population (51.1+1.3% vs 27.7+2.1%, P50.01, 12 h after UV irradiation). These results demonstrate that when CDK2 is active in proliferating cells (data not shown), cells are more vulnerable to UV induced apoptosis. To determine if apoptosis was associated with increased CDK2 activity, proliferating mesangial cells were serum starved, and then exposed to UV irradiation. In the absence of UV, apoptosis was detected 1.3+0.7%, 2.0+1.3%, 2.3+0.5% at 6, 9, 12 h, respectively. Following UV irradiation, apoptosis was detected 4.7+1.2%, 28.5+3.7%, 53.9+4.9% at 6, Figure 1 CDK2 is active during apoptosis and decreasing 9, 12 h respectively. We harvested total cell extracts at CDK2 activity with Roscovitine or transfection of dominant each time point and determined the CDK2 activity by negative CDK2 mutant reduces apoptosis. (a) Upper panel: histone H1 kinase assay. As expected, CDK2 activity CDK2 activity, measured by a histone H1 kinase assay, is increased in proliferating mesangial cells (lane 1). CDK2 activity decreased in control serum starved cells not exposed to decreases progressively following the withdrawal of serum, in the UV (Figure 1a). In contrast, there was a marked absence of exposure to UV irradiation (designated 7). In increase in CDK2 activity in serum starved cells contrast, CDK2 remained active following UV irradiation exposed to UV irradiation (Figure 1a). (designated +), despite the withdrawal of serum. Lower panel: To con®rm that increased CDK2 activity partici- The changes in CDK2 activity were independent of total protein content, which remained unchanged, measured by Western blot pated in UV induced apoptosis, we inhibited CDK2 analysis. (b) Exposing proliferating cells to the CDK2 inhibitor, activity with Roscovitine and by transiently transfect- Roscovitine (+; light bars), decreased apoptosis induced by UV ing cells with a dominant negative CDK2 mutant. We irradiation at each time point compared to control cells without and others have previously shown that the purine Roscovitine (7; dark bars). *P50.01 vs no Roscovitine. (c) (a) Cells transfected with a dominant negative CDK2 plasmid analogue Roscovitine reduces CDK2 activity (Meijer et (CDK2-dn) were identi®ed by co-transfection with GFP plasmids. al., 1997; Pippin et al., 1997). Reducing CDK2 activity (b) Apoptosis induced by UV irradiation was detected in non- with Roscovitine signi®cantly decreased apoptosis at transfected cells (measured by Hoechst staining, represented by each time point following UV irradiation (Figure 1b). arrow heads). In contrast, CDK2-dn transfected cells had intact We next determined the eect of dominant negative nuclei (thick arrows). (c) Cells transfected with wild-type CDK2 plasmids (CDK2-wt) were identi®ed by co-transfection with GFP CDK2 mutant on apoptosis. As shown in Figure 1c, plasmids. (d) CDK2-wt did not protect cells from apoptosis (thin transfection of a dominant negative mutant CDK2 arrows)

Oncogene CDK2 and apoptosis K Hiromura et al 1752 plasmid (CDK2-dn) signi®cantly inhibited UV-induced apoptosis compared to control cells transfected with wild type CDK2 plasmid (CDK2-wt) (27.1+3.1% vs 42.4+5.9%, CDK2-dn vs CDK2-wt, 12 h after UV- irradiation, P50.01). These data demonstrated that active CDK2 participates in mesangial cell apoptosis induced by UV irradiation, and that UV-induced apoptosis is in part CDK2-dependent. To ensure that the increase in CDK2 activity following UV irradiation was not due to increased DNA synthesis, BrdU incorporation was measured. In control cells not injured by UV, strong BrdU incorporation was detected in 49.6%+5.7 cells. In contrast, intensity of BrdU incorporation was mark- edly reduced in UV-treated cells as shown in Figure 2. Taken together, these data demonstrated that following UV induced injury, CDK2 activity was increased in association with apoptosis and not DNA synthesis, and that reducing activity signi®cantly decreased apoptosis.

Cyclin A-CDK2 but not cyclin E-CDK2 nor cyclin B1-cdc2 activity is increased during apoptosis CDK2 is activated by cyclin E and cyclin A during G1 and S phases respectively. To examine which cyclin- CDK complex is activated during apoptosis, total cell lysates were immunoprecipitated with antibodies to CDK2, cdc2, cyclin A, cyclin E and cyclin B1, and their activities measured by histone-H1 kinase assay (Figure 3a). To determine the total protein levels, Figure 3 Cyclin A-CDK2 activity is selectively active in Western blot analysis was performed for each protein apoptotic cells. (a) Histone H1 kinase assay. Total cell lysates (Figure 3b). As expected, CDK2 and cdc2 activity were from proliferating (UV 0h; lane 1), control (UV untreated 12 h; increased in proliferating cells, and were barely lane 2) and apoptotic (UV treated 12 h; lane 3) cell populations detected in control cells that were serum starved and were immunoprecipitated with antibodies to CDK2, cdc2, cyclin A, E and B1, followed by a histone-H1 kinase assay. There was a not exposed to UV irradiation. In contrast to the decrease in cyclin A-CDK2, cyclin E-CDK2 and cyclin B1-cdc2 increase in CDK2 activity following UV irradiation, activity in control cells not exposed to UV (lane 2). The activity cdc2 activity was not increased. Our results also of cyclin B1-cdc2 and cyclin E-CDK2 was also decreased in showed that the increase in CDK2 activity was due apoptotic UV treated cells (lane 3). In contrast, cyclin A-CDK2 activity remained increased in cells exposed to UV (lane 3). (b) Western blot. Total protein levels of CDK2 and CDC2 remained unchanged. The protein levels of cyclin A, cyclin E and cyclin B1 were decreased in control cells not exposed to UV (lane 2). The protein levels of cyclin E and cyclin B1 were also decreased in apoptotic UV treated cells (lane 3). In contrast, the protein level of cyclin A protein remained increased in cells exposed to UV (lane 3)

to cyclin A, and not cyclin E. These results demonstrate that cyclin A-CDK2 was selectively active in apoptotic cells.

CDK2 localizes to the cytoplasm during apoptosis In contrast to DNA synthesis which occurs in the nucleus, apoptosis typically begins in the cytoplasm. Figure 2 CDK2 activity is independent from DNA synthesis During quiescence, CDK2 is inactive and the protein is during apoptosis. Proliferating cells were treated with or without abundant in the cytoplasm. However, in order for UV and incubated with bromodeoxyuridine (BrdU) for another DNA synthesis to occur, CDK2 protein moves, by yet 3 h. UV untreated cells (designated 7) showed strong BrdU incorporation. In contrast, UV treated cells (designated +) had poorly de®ned mechanisms, to the nucleus where it is faint BrdU incorporation activated by cyclins (Baptist et al., 1996; Bresnahan et

Oncogene CDK2 and apoptosis K Hiromura et al 1753 al., 1996; Moore et al., 1999; Pagano et al., 1993). To ing cells stained positive for CDK2 in the nucleus test the hypothesis that the subcellular localization of (Figure 4b). CDK2 determines if cells undergo either proliferation A major ®nding in this study was that nuclear or apoptosis, we examined CDK2 protein localization. staining for CDK2 was reduced signi®cantly following As shown in Figure 4a, less than 5% of quiescent cells the induction of apoptosis by UV irradiation in stain positive for CDK2 in the nucleus. Moreover, the proliferating cells (Figure 4c). Moreover, this coincided majority of quiescent cells displayed weak cytoplasmic with an increase in cytoplasmic staining for CDK2. staining for CDK2. In contrast, 70 ± 80% of proliferat- The change in the nuclear-cytoplasmic pattern of

Figure 4 Cyclin A and CDK2 translocates to the cytoplasm during apoptosis. CDK2 (a) In mesangial cells rendered quiescent by serum deprived for 48 h, CDK2 immunostaining is positive in the cytoplasm. (b) In the absence of UV irradiation, CDK2 immunostaining is nuclear in proliferating and viable cells grown in medium containing 20% FCS. (c) When proliferating cells were exposed to UV irradiation, there was a marked decrease in nuclear staining for CDK2, and an increase in cytoplasmic staining 6 h after UV irradiation. (d) The cytoplasmic (C) and nuclear (N) localization of CDK2 was con®rmed by Western blot analysis performed on these protein fractions. Cyclin A.(e) Cyclin A immunostaining was not detected in quiescent mesangial cells, and was nuclear in proliferating cells grown in serum (f). (g) Inducing apoptosis in proliferating cells by UV irradiation was associated with a decrease in nuclear intensity for cyclin A and an increase in cytoplasmic staining 6 h after UV irradiation. (h) The cytoplasmic (C) and nuclear (N) localization of cyclin A was con®rmed by Western blot analysis

Oncogene CDK2 and apoptosis K Hiromura et al 1754 CDK2 staining occurred within 6 h, and preceded the CDK2 was cytoplasmic (Figure 6). Similar results were classical nuclear condensation of apoptosis. To con®rm obtained when we stained and blotted for heterogeneous the cytoplasmic subcellular localization of CDK2 nuclear ribonucleoprotein (hnRNP) K-protein (Bomsz- following an apoptotic injury, Western blot analysis tyk et al., 1997). Second, inhibiting apoptosis with the was also performed for CDK2 on separate nuclear and caspase inhibitor Z-VAD-FMK signi®cantly reduced cytoplasmic protein fractions in control and UV UV induced apoptosis compare to control cells irradiated cells. As expected, Western blot analysis (53.9+4.9% vs 8.4+1.3%, P50.01), but did not showed that CDK2 protein was predominantly nuclear interfere with the translocation of CDK2 (data not in proliferating cells not exposed to UV (Figure 4d). In contrast, CDK2 protein was predominantly cytoplasmic in proliferating cells exposed to the apoptotic trigger, UV irradiation. The nuclear-cytoplasmic shift in CDK2 protein was not associated with any obvious dierences in total CDK2 protein abundance, because CDK2 levels remained unchanged in protein obtained from total cell lysates isolated in both proliferating and apoptotic cells (Figure 1a).

Cytoplasmic CDK2 is activated by cyclin A We showed earlier that cyclin A-CDK2, but not cyclin Figure 5 Cytoplasmic cyclin A-CDK2 activity is increased E-CDK2 activity increased during apoptosis. Accord- during apoptosis. CDK2 activity was measured by histone H1 ingly, we measured cyclin A protein expression kinase assay in cytoplasmic (C) and nuclear (N) protein fractions following the induction of apoptosis in proliferating following serum withdrawal in cells 6 h after exposure to UV (+) cells. Cyclin A staining was barely detected in quiescent or control cells not exposed to UV (7). Cyclin A-CDK2 activity was detected predominantly in the nuclear fraction in control cells cells (Figure 4e), but as expected, increased in the not exposed to UV irradiation. In contrast, cytoplasmic cyclin A- nucleus following serum stimulation (Figure 4f). Six CDK2 activity was more abundant in UV treated cells compared hours after the induction of apoptosis by UV to the nuclear fraction irradiation, the intensity of nuclear cyclin A staining decreased signi®cantly in proliferating cells, and this coincided with an increase in cyclin A staining in the cytoplasm (Figure 4g). Finally, the subcellular localization of cyclin A was con®rmed by Western blot (Figure 4h). Taken together, these data demonstrate that both cyclin A and CDK2 proteins translocate from the nucleus to the cytoplasm following the induction of apoptosis by UV irradiation in a proliferating cell population. Monomeric CDK2 protein is inactive and requires the activation by a partner cyclin to function as a serine- threonine kinase. To ensure that cytoplasmic CDK2 was active following an apoptotic trigger, a histone H1- kinase assay was performed on separate nuclear and cytoplasmic protein fractions in control (no exposure to UV) and apoptotic (UV treated) cells. Similar to the protein, there was an increase in cyclin A-CDK2 activity in the cytoplasm of cells undergoing apoptosis (Figure 5). Thus, these results show that cyclin A and CDK2 protein were present in the cytoplasm following UV, and the complex remains active.

CDK2 translocation is not due to nuclear membrane breakdown We were concerned that the translocation of CDK2 and cyclin A to the cytoplasm may have been simply due to disruption of the nuclear membrane. Two methods were Figure 6 Cyclin E remains in the nucleus during apoptosis. (a) used to show that the movement of cyclin A-CDK2 was In contrast to cyclin A and CDK2, immunostaining showed that cyclin E localized to the nucleus 6 h after UV-irradiation, and was indeed speci®c. First, immunostaining and Western blot not detected in the cytoplasm. (b) The nuclear (N) localization of analysis for the nuclear protein cyclin E, remained cyclin E was con®rmed by Western blot analysis. B-tubulin was con®ned to the nucleus during apoptosis at a time when used as a cytoplasmic marker

Oncogene CDK2 and apoptosis K Hiromura et al 1755 shown). Taken together, these results showed that UV irradiation caused a speci®c movement of cyclin A- CDK2 from the nucleus to the cytoplasm in proliferating cells, and that this was not due to a generalized leakage of nuclear proteins. To determine if cytoplasmic cyclin A-CDK2 required new protein synthesis, cells were exposed to the protein synthesis inhibitor cycloheximide following UV irradiation. Cycloheximide did not aect the increase in cyclin A and CDK2 (data not shown), indicating that cytoplasmic CDK2 and cyclin A were preformed proteins, and that cytoplasmic cyclin A and CDK2 were not due to new protein synthesis.

CDK2 translocation does not require p53 Recent studies have shown that following DNA damage, the nuclear-cytoplasmic movement of cdc2 is sequestered in the cytoplasm by a p53-dependent and 14-3-3d mediated mechanism (Chan et al., 1999). To Figure 7 Cyclin A-CDK2 is upstream of caspase-3 in the determine if the translocation of CDK2 was p53- apoptotic pathway. (a) Exposing mesangial cells to the CDK2 dependent, we studied mesangial cells derived from p53 inhibitor Roscovitine (+; light bars) signi®cantly decreased caspase-3 activity at each time point after exposure to UV null mice. Although p53 null mesangial cells undergo irradiation compared to control cells without Roscovitine (7; UV induced apoptosis, the translocation of cyclin A dark bars). Caspase-3 activity was expressed as the per cent and CDK2 following UV irradiation was also increase compared to a pre-treated cells. *P50.01 vs Roscovitine recognized in p537/7 mesangial cells, and was (7). (b) The cleaved caspase-3 (active form) with (+) or without comparable to p53+/+ cells (data not shown). These (7) Roscovitine was con®rmed by Western blot analysis results demonstrate that nuclear-cytoplasmic CDK2 translocation is p53-independent.

cell cycle proteins regulate apoptosis. Our data shows Cyclin A-CDK2 is upstream of caspase-3 in the apoptotic that when active CDK2 is nuclear, cells proliferate. In pathway contrast, we showed that when UV was applied to The caspase cascade is a critical eector in mediating proliferating cells, there is a translocation of active many forms of apoptosis, and recent studies have CDK2 to the cytoplasm, which is associated with shown that caspase-3 is one of the ®nal downstream apoptosis. These data show that the subcellular events required for UV induced cell death (Woo et al., localization of active CDK2 determines the cell's 1998). A previous study has shown that caspase-3 is growth or death fate. upstream of CDK2 (Hakem et al., 1999), yet others Cyclin A-CDK2 is required for DNA synthesis have shown that caspase-3 is downstream of CDK2 during the S phase of the cell cycle (Pines, 1994; Reed, (Harvey et al., 2000; Levkau et al., 1998). To 1997; Shankland and Wolf, 2000) and is also rate determine if active cytoplasmic CDK2 was proximal limiting for the entry into mitosis (Furuno et al., 1999). to the activation of caspase-3, caspase-3 activity was Although apoptosis is typically increased in proliferat- measured in UV irradiated cells in the presence and ing cell populations in in¯ammatory diseases, such as absence of the CDK2 inhibitor, Roscovitine. Caspase- occurs in mesangial cells in renal disease (Baker et al., 3 activity was signi®cantly increased in UV irradiated 1994; Ophascharoensuk et al., 1998), the mechanisms cells compared to the non-irradiated control cells. In underlying the link between proliferation and apoptosis contrast, Roscovitine signi®cantly reduced caspase-3 following injury are poorly de®ned. There is a growing activity (Figure 7a). Moreover, the cleaved active body of evidence showing that both proliferation and caspase-3 protein induced by UV was con®rmed by apoptosis are closely linked at the level of the cell cycle. Western blot analysis, and this cleavage product was A coordinated activation of speci®c CDKs by partner reduced when CDK2 activity was decreased (Figure cyclins is necessary for proliferation. However, we and 7b). These data demonstrate that CDK2 is upstream others have more recently shown that certain CDKs, of caspase-3 in the apoptotic pathway in mesangial including CDK2 are also activated during apoptosis, cells. and that inhibiting the kinase activity reduces apoptosis (Adachi et al., 2001; Dobashi et al., 1998; Gil-Gomez et al., 1998; Hakem et al., 1999; Hiromura Discussion et al., 1999; Hoang et al., 1994; Levkau et al., 1998; Meikrantz et al., 1994; Meikrantz and Schlegel, 1996; In this study we show that the subcellular localization Park et al., 1998; Shi et al., 1994; Yao et al., 1996). In of CDK2 provides a novel mechanism whereby speci®c the current study we reasoned that because apoptosis

Oncogene CDK2 and apoptosis K Hiromura et al 1756 typically begins in the cytoplasm, and that proliferation Materials and methods is governed within the nucleus, that the subcellular localization of CDK2 may determine the fate of the Cell culture cell. Our results showed that CDK2 protein translocates Mouse mesangial cells were isolated from normal mice and from p537/7 mice (obtained from Chris Kemp, Fred from the nucleus to the cytoplasm following exposure Hutchinson Cancer Research Center, Seattle, WA, USA) as to an apoptotic trigger in proliferating cells. Moreover, previously described (Hiromura et al., 1999). Cells were CDK2 remains active by binding to cyclin A, but not passaged in growth medium of DMEM (Irvine Scienti®c, cyclin E. Reducing CDK2 activity with Roscovitine or Santa Ana, CA, USA), 20% (v/v) Ham's-F12 (Irvine a dominant negative CDK2 mutant signi®cantly Scienti®c, Santa Ana, CA), 20% (v/v) FCS (Summit decreased UV-induced apoptosis, but does not com- Biotechnology, Ft Collins, CO, USA), 2 mM glutamine pletely abrogate apoptosis. This result suggests that (Sigma, St. Louis, MO, USA), 10 mM HEPES (Sigma), there are CDK2-dependent and CDK2-independent 100 U/ml penicillin - 100 mg/ml streptomycin (Irvine Scien- pathways that mediate apoptosis. The movement of ti®c) and 16trace element (Bio¯uids, Rockville, MD, USA) CDK2 to the cytoplasm was not a generalized at 378C in humidi®ed 5% CO2 in air. phenomenon, because other nuclear speci®c proteins, cyclin E and K-protein, did not translocate to the Inducing apoptosis cytoplasm following the same injury. Our results also Mesangial cells were plated (density 2736104 cells/cm2)in showed that the cytoplasmic translocation of CDK2 growth media. To synchronize cells in G0/G1, growing cells was not p53-dependent, as translocation of CDK2 were washed three times with HBSS (without calcium and during UV induced apoptosis also occurred in p53 null magnesium salts; Irvine Scienti®c), and then incubated in mesangial cells. serum-free media (growth media without FCS) for 48 h. Studies have shown that nuclear-cytoplasmic track- Apoptosis was induced by UV-B irradiation exposed to a ing of certain cell cycle proteins requires a nuclear 302 nM UV transilluminator source (LM-20e; VWR Scien- export signal (NES), while others are under the control ti®c, Seattle, WA, USA) for 10 min as described by Martin and Cotter (1991). This dose of UV was determined by a of a nuclear localization signal (NLS). Leptomycin B dose that induces 50% apoptosis at 12 h, in proliferating inhibits the export of proteins containing a NES, mesangial cells. The same dose eect is observed in Rat-1 including cyclin B1 (Toyoshima et al., 1998; Yang et ®broblasts (supplied by DM Hockenbery, Fred Hutchinson al., 1998). Leptomycin B (gift from Minoru Yoshida, Cancer Research Center, Seattle, WA, USA). In selected Tokyo University, Tokyo, Japan) (Yoshida and experiments, CDK2 activity was reduced by adding Roscov- Horinouchi, 1999) did not inhibit the translocation of itine (50 mM, Calbiochem, La Jolla, CA, USA) to the CDK2 to the cytoplasm (data not shown). The medium, and caspases were inhibited with Z-VAD-FMK physiological shuttling of CDK2 from the cytoplasm (100 mM, Enzyme Systems Products; Livermore, CA, USA). to the nucleus during proliferation is poorly de®ned, To inhibit protein synthesis, cycloheximide (100 mM, Sigma) and the movement of CDK2 from the nucleus to the was used. cytoplasm described in this study during apoptosis also requires further investigation. Measuring apoptosis We attempted to elucidate potential cytoplasmic Apoptosis was measured by Hoechst 33342 (Molecular substrates whereby CDK2, a serine-threonine kinase, probes, Eugene, OR, USA) staining and by careful induces apoptosis. In the current study, we demon- morphological analysis as previously reported (Hiromura et strated that CDK2 activity is proximal to caspase-3 al., 1999; Mooney et al., 1997). The percentage of apoptotic activation. A previous study has shown that CDK2 is cells were measured in six randomly selected ®elds, and upstream of caspases (Hakem et al., 1999). Others have counted in triplicate in each experiment. shown that CDK2 is downstream of caspases (Harvey et al., 2000; Levkau et al., 1998). Thus, the role of Immunohistochemical staining CDK2 may depend on the cell type and/or the nature To measure the expression of speci®c cell cycle proteins by of the apoptotic stimulus. In order to determine if immunostaining, cells were grown on 8-well permanox CDK2 binds to and phosphorylates candidate members chamber slides (Nalge Nunc, Naperrville, IL, USA) and of the Bcl-2 family, we immunoprecipitated CDK2 ®xed in methanol and acetone (1 : 1) at 7208C for 15 min. with Bcl-2, BAD and BAX. We were unable to show Cells were incubated overnight at 48C with primary that these proteins colocalized with CDK2, nor could antibodies to CDK2 (M2; rabbit polyclonal; Santa Cruz we demonstrate that these proteins were phosphory- Biotechnology, Santa Cruz, CA, USA), cyclin A (C19; rabbit lated (data not shown). polyclonal; Santa Cruz Biotechnology), cyclin E (rabbit In summary, our study provides a novel role for polyclonal; Upstate Biotechnology, Lake Placid, NY, USA), cyclin A-CDK2 in mediating both growth and death cyclin B1 (H433; rabbit polyclonal; Santa Cruz Biotechnol- ogy) and hnRNP K-protein (rabbit polyclonal; gift from pathways. We propose that active nuclear cyclin A- Karol Bomsztyk, University of Washington, Seattle, WA, CDK2 is required for cell proliferation, whereas active USA) diluted in 1% bovine serum albumin ± phosphate buer cytoplasmic cyclin A-CDK2 causes apoptosis and is saline (PBS). A secondary biotinylated anti-rabbit IgG upstream of caspase-3. The targets of CDK2 and the (Vector, Burlingame, CA, USA) was incubated at room mechanisms underlying its translocation remain to be temperature for 60 min, and immuno¯uorescent staining was elucidated. detected by streptavidin-¯uorescein (Amersham Pharmacia

Oncogene CDK2 and apoptosis K Hiromura et al 1757 Biotech, Piscataway, NJ, USA). Nuclei were counterstained BrdU incorporation with Hoechst 33342. DNA synthesis was measured by BrdU incorporation as reported elsewhere (Hiromura et al., 1999). Brie¯y, cells were Histone H1 kinase assay and Western blot analysis incubated with BrdU (Amersham Pharmacia Biotech) for 3 h Total protein was extracted from mesangial cells (Primaria and incorporated BrdU was detected by anti-BrdU antibody Tissue Culture Dish 100620 mm; Becton Dickinson Lab- (Amersham Pharmacia Biotech) followed by biotinylated ware, Franklin Lakes, NJ, USA), using a buer containing anti-mouse IgG (Vector) and streptavidin-¯uorescein. 1% Triton, 10% glycerol, 20 mM HEPES, 100 mM NaCl (Sigma) and proteinase inhibitors (Boehringer Mannheim, Transfection and analysis of apoptosis on transfected cells Indianapolis, IN, USA) as previously reported (Hiromura et al., 1999). To determine the subcellular localization and To determine the role of CDK2 activity in apoptosis induced activity of speci®c cell cycle proteins, nuclear and cytoplasmic by UV irradiation, proliferating mesangial cells grown in 24- protein fractions were isolated using a modi®ed method by well plates were co-transfected with 0.8 mg of a dominant- Orend et al. (1998). In brief, cells were harvested by trypsin negative mutant CDK2 plasmid (pCMV-CDK2-dn) and digestion, rinsed with cold PBS and placed on ice for 15 min 0.2 mg pEGFP-N1 plasmids (Clontech Laboratories, Palo in hypotonic buer A (10 mM HEPES pH 7.9, 10 mM KCl, Alto, CA, USA) or 0.8 mg wild-type CDK2 plasmid (pCMV- 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) CDK2-wt) and 0.2 mg pEGFP-N1, using GenePORTERTM and proteinase inhibitors. Nonidet P-40 (NP-40; ®nal Transfection Reagent (Gene therapy systems. San Diego, CA, concentration 0.1%; Sigma) was added, and suspensions USA), according to the manufacturer's instruction. Twenty- were vortexed for 10 s. The cytoplasmic fraction was four hours after transfection, cells were exposed to UV-B separated from the nuclear fraction by centrifugation for irradiation. Twelve hours after UV-irradiation, double- 5 min at 1400 g. The nuclear protein was extracted with immunostaining was performed with Hoechst 33342 to buer C (20 mM HEPES pH 7.9, 0.4 mM,1mM EDTA, determine the percentage of transfected (green) cells under- 1mM EGTA, 1 mM DTT, 1 mM PMSF). After 15 min going apoptosis (nuclear condensation), and apoptosis was rocking at 4 degrees, the supernatant was collected by measured in 200 consecutive cells/well in triplicate. centrifugation for 10 min at 17 000 g. Protein concentration was determined by the BCA protein assay (Pierce, Rockford, Caspase-3 assay IL, USA) according to the manufacturer's directions. The activities of CDK2 and cdc2 were measured by the To measure caspase-3 activity, 100 mg whole cell lysates were histone H1 kinase assay as previously described (Hiromura et incubated with 100 mM Ac-DEVD-pNA (Calbiochem) in al., 1999). Brie¯y, protein extracts (50 mg) were precipitated 200 ml of a buer (100 mM HEPES pH 7.4, 20% vol/vol with antibodies to CDK2 (M2; Santa Cruz), cyclin A (C-19; glycerol, 5 mM DTT) in a 96-well plate. The plate was Santa Cruz), cyclin E (Upstate Biotechnology), cyclin B1 incubated at 378C and the release of free pNA, which absorbs (H433; Santa Cruz) and cdc2 (H297; rabbit polyclonal; Santa at 405 nm, was monitored using plate reader, SpectraCount Cruz Biotechnology) using protein A sepharose (Amersham (Packard, Meriden, CT, USA). Caspase-3 activity in Pharmacia Biotech). The immunoprecipitation was incubated experimental cells was expressed as the fold increase with histone H1 (Boehringer Mannheim) and 32P-ATP (NEN compared to control cells. Life Science Products, Boston, MA, USA), and resolved on a 15% SDS-polyacrylamide gel under reduced condition. The Statistical analysis gel was exposed to autoradiographic ®lm (Amersham Pharmacia Biotech). Statistical analysis was performed using StatView 5.0.1 For Western blot analysis, 10 mg of protein extract (40 mg software program (Abacus Concepts, Berkeley, CA, USA); for caspase-3) was separated under reduced conditions on a statistical signi®cance was evaluated using the Student's t- 15% or 8% SDS-polyacrylamide gel and transferred to test. PVDF membrane (Immobilon-P; Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies to CDK2, cyclin A, cyclin E, K-protein, b-tubulin (mouse monoclonal; Sigma) and caspase-3 (H277; rabbit Acknowledgments polyclonal; Santa Cruz Biotechnology) overnight at 48C, This work was supported by Public Health Service grants followed by incubation with an alkaline phosphatase- (DK34198, DK52121, DK51096, DK56799) and a George conjugated secondary antibody (Promega, Madison, WI, M O'Brien Kidney Center Grant (DK47659). JM Roberts USA) at room temperature for 60 min. Detection was is an Investigator of the Howard Hughes Medical Institute. determined with the chromagen 5-bromo-4-chloro-3-indolyl K Hiromura was supported by the Uehara Memorial phosphate/nitro blue tetrazolium (Sigma). Foundation.

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