Lats2, a Putative Tumor Suppressor, Inhibits G1/S Transition

Lats2, a putative tumor suppressor, inhibits G1/S transition

Yunfang Li1,3, Jing Pei1,3, Hong Xia1, Hengning Ke1, Hongyan Wang1 and Wufan Tao*,1,2

1Stem Cell Institute, Division of Hematology, Oncology and Transplantation, 2Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455, USA

Lats2 is a new member of the Lats tumor suppressor (or warts) results in tumor development in mosaic flies family. The human LATS2 gene is located at chromosome and causes embryonic or pupae lethality in lats 13q11–12, which has been shown to be a hot spot (67%) homozygotes (Justice et al., 1995; Xu et al., 1995). for LOH in nonsmall cell lung cancer. In order to LATS1 (or WARTS1), a human homolog of Drosophila understand the function of LATS2 in the control of tumor lats, is able to rescue all developmental defects in lats development, we ectopically expressed mouse Lats2 via homozygous mutants and suppress tumor formation in retroviral infection in NIH3T3/v-ras cells to examine lats mosaic flies (Tao et al., 1999). Lats1-deficient female whether Lats2 plays a role in suppressing tumor develop- mice spontaneously develop soft tissue sarcomas and ment and regulating cell proliferation. We have found that ovarian stromal cell tumors, demonstrating that the ectopic expression of Lats2 in NIH3T3/v-ras cells Lats1 gene functions as a tumor suppressor in mice (St suppresses development of tumors in athymic nude mice John et al., 1999). No somatic mutations in the LATS1 and inhibits proliferation of NIH3T3/v-ras cells in an in coding region have been identified by RT–PCR SSCP vitro assay. Cell cycle profile analysis demonstrated that analysis of 25breast cancer samples, although this also ectopic expression of Lats2 inhibited the G1/S transition. does not rule out the possibility that LATS1 may be Further mechanistic studies revealed that cyclin E/CDK2 inactivated in human breast tumors by other epigenetic kinase activity was downregulated in Lats2-transduced mechanisms such as methylation (Morinaga et al., NIH3T3/v-ras cells, while other cell cycle regulators 2000). controlling the G1/S transition were not affected. We Mechanistic studies on LATS1 revealed that it might have also shown that LATS2kinase activity and two control tumorigenesis by negatively regulating the cell LATS conserved domains (LCDs) are required for Lats2 cycle. The LATS1 protein associates with a major to suppress tumorigenicity and to inhibit cell growth. In mitotic kinase, CDC2, at G2/M transition or in early addition, the LATS2protein is cytoplasmic during mitosis in HeLa cells. CDC2, when associated with interphase in NIH3T3 cells, while it becomes localized LATS1, was found to be inactive for its kinase activity to the mitotic apparatus during mitosis. Finally, we (Tao et al., 1999). Loss of function of lats in Drosophila propose a model in which a combination of mammalian resulted in the upregulation of Cyclin A (Tao et al., Lats2 and Lats1 control cell proliferation by negatively 1999). Ectopic expression of Lats1 in human cancer cell regulating different cell cycle check points. lines leads to the downregulation of Cyclin A and Cyclin Oncogene (2003) 22, 4398–4405. doi:10.1038/sj.onc.1206603 B at the protein level (Xia et al., 2002) and/or inactivation of CDC2 kinase, thereby blocking cells at Keywords: Lats2; tumor suppressor; G1/S transition; G2/M and preventing tumor development in nude mice cell cycle (Yang et al., 2001; Xia et al., 2002). Microinjection of a truncated human LATS1 protein (a.a 136–700) inter- fered with the localization of zyxin to the mitotic apparatus and lengthened mitosis (Hirota et al., 2000). Introduction Ectopic expression of LATS1 in human tumor cell lines has also been shown to induce apoptosis by upregulat- The Lats tumor suppressor family has been shown to ing the level of BAX protein (Xia et al., 2002) or play important roles in the control of tumor develop- upregulating Caspase-3 activity (Yang et al., 2001), ment and the cell cycles from flies (Justice et al., 1995; indicating that LATS1 may also control tumorigenesis Xu et al., 1995) to human (Nishiyama et al., 1999; by inducing apoptosis. St John et al., 1999; Tao et al., 1999; Yang et al., 2001; Lats2 (Yabuta et al., 2000), also known as KPM Xia et al., 2002). Loss of function of Drosophila lats (Hori et al., 2000), is the second mammalian member of the Lats tumor suppressor gene family. Human LATS2 *Correspondence: W Tao, MMC716, 420 Delaware Street SE, has been mapped onto human chromosome 13q11–12 University of Minnesota, Minneapolis, MN 55455, USA; (Yabuta et al., 2000), a hot spot (67%) for LOH in E-mail: [email protected] nonsmall cell lung cancer (Girard et al., 2000). Lats2 3These two authors contribute equally Received 11 October 2002; revised 5March 2003; accepted 21 March encodes a putative Ser/Thr protein kinase. The LATS2 2003 protein shares 85% and 75% sequence identity to Lats 2, a putative tumor suppressor YLiet al 4399 human LATS1 and Drosophila lats proteins in the kinase domain (Hori et al., 2000; Yabuta et al., 2000). Although the overall sequence similarity in the amino terminus between mammalian LATS1 and LATS2 proteins is low, two stretches of highly conserved sequence (B50% identical amino acids) are identifiable (Hori et al., 2000 and Figure 4d). The function of these two conserved domains remains to be unveiled. Although the human LATS2 protein is claimed to be phosphorylated in mitosis, transient transfection of wild-type Lats2 and mutant Lats2K697A (kinase-inactive) Figure 1 Lats2 suppresses v-ras-mediated tumorigenicity of into 293 and HeLa cells revealed no information about NIH3T3 cells and inhibits cell proliferation. (a) Expression of the role of Lats2 in the control of cell proliferation and/ exogenous LATS2 protein in NIH3T3/v-ras cells. Exogenous or the cell cycle (Hori et al., 2000). Therefore, it is LATS2 proteins detected by Western blot in NIH3T3/v-ras cells unknown if the LATS2 gene functions as a tumor with anti-MYC antibodies: (1) in NIH3T3/v-ras cells without virus infection, (2) in MIGR1-Lats2-transduced NIH3T3/v-ras cells suppressor. It is also unclear if LATS1 and LATS2 play without enrichment and (3) in MIGR1-Lats2-transduced different roles in the control of tumor development and NIH3T3/v-ras cells enriched by FACS. Equal amounts of proteins the cell cycle. Here, we present evidence that Lats2 has a were loaded. (b) Exogenous Lats2 suppresses anchorage-indepen- tumor suppressor function by showing that ectopic dent growth of NIH3T3/v-ras cells in soft agar. Colonies derived expression of Lats2 suppresses tumorigenicity of from 500 cells that were infected by MIGR1 (left) or MIGR1-Lats2 (right) were grown for 3 weeks in the soft agar. (c) Growth NIH3T3/v-ras cells and inhibits cell growth. Interest- inhibition assay. In total, 7000 of the Lats2-transduced NIH3T3/v- ingly, we found that Lats2 controls cell proliferation by ras cells or control cells were plated in a 6 cm dish. Total cell negatively regulating the G1/S transition, which is numbers were counted daily for 7 days. Experiments were repeated different from the G2/M blockage imposed by LATS1. more than three times Our studies reveal that ectopic expression of Lats2 results in a downregulation of Cyclin E/CDK2 kinase activity. We also demonstrate that LATS2 kinase Lats2 was measured in soft agar, and tumor develop- activity and two LATS conserved domains (LCDs) are ment in athymic nude mice was examined. We found required for its ability to inhibit cell proliferation and that expression of Lats2 in NIH3T3/v-ras cells drama- tumorigenicity. Finally, we show that the LATS2 tically suppressed colony growth of NIH3T3/v-ras cells protein is cytoplasmic during interphase, while it also in soft agar as compared to the control (Figure 1b). locates to the mitotic apparatus during mitosis. Ectopic expression of LATS2 in NIH3T3/v-ras cells also suppressed v-ras-mediated tumorigenesis in athymic nude mice. Our results showed that Lats2-transduced NIH3T3/v-ras cells failed to develop tumors in nude Results mice (six out of six) 2 weeks after inoculation, while control virus-transduced cells developed large tumors in Expression of exogenous Lats2 gene in NIH3T3 cells mice (six out of six). These studies demonstrate that In order to study the role of Lats2 in the control of ectopic expression of Lats2 suppressed the anchorage- tumorigenesis and cell proliferation, a replication- independent growth of NIH3T3/v-ras cells in soft agar defective retrovirus, MIGR1-Lats2, was generated (see and the tumorigenicity of NIH3T3/v-ras cells in vivo, Materials and methods), in which the LATS2 protein indicating that Lats2 may play a role in preventing and EGFP are coexpressed as a bicistron. The expres- tumor development. sion of endogenous LATS2 protein cannot be detected by Western blot in either NIH3T3/v-ras cells (data not Lats2 inhibits cell proliferation by blocking G1/S shown) or its parental cell, NIH3T3 (Yabuta et al., transition 2000). The expression of exogenous Lats2 was confirmed by Western blot in Lats2-transduced NIH3T3/ To understand the mechanism(s) by which the expres- v-ras cells (Figure 1a, lanes 2 and 3). Using EGFP as a sion of Lats2 suppresses the tumorigenicity of NIH3T3/ marker, retrovirus-transduced cells can be enriched by v-ras cells, we examined the effects of ectopic expression FACS for further analysis (Figure 1a). All retrovirus- of Lats2 on cell proliferation. As shown in Figure 1c, infected cells used in the following experiments were growth of Lats2-transduced NIH3T3/v-ras cells was enriched by FACS. strongly inhibited compared to the control virus- transduced cells. Over a 7-day period, the total cell Lats2 /v-ras Lats2 suppresses tumorigenicity of NIH3T3/v-ras cells number of -transduced NIH3T3 cells only increased 30-fold, while the control cells increased 210- To examine whether expression of Lats2 is able to fold. These results suggest that Lats2, like the LATS1 suppress the tumorigenicity of tumor cells, mouse tumor suppressor, may be another negative cell cycle NIH3T3/v-ras cells were infected with either MIGR1- regulator. To further study the role of Lats2 in cell cycle Lats2 or the control virus MIGR1. Anchorage-indepen- control, the effects of ectopic expression of Lats2 on the dent growth of NIH3T3/v-ras cells expressing exogenous cell cycle progression of NIH3T3/v-ras cells were

Oncogene Lats 2, a putative tumor suppressor YLiet al 4400 examined. NIH3T3/v-ras cells infected with MIGR1- in the control of initiation of S phase or the control of Lats2 or the MIGR1 control virus were first synchro- the DNA synthesis rate, DNA synthesis was measured nized at G1/S transition by aphidicolin treatment (see in the synchronized Lats2-transduced NIH3T3/v-ras Materials and Methods). The cell cycle progression of cells and the control cells. First, cells were blocked at the cells released from a G1/S block was examined by G0/G1 by serum starvation. After cells reentered the cell DNA content FACS analysis (Figure 2a). Results cycle by serum stimulation, they were pulse-labeled by showed that the control virus-transduced cells entered BrdU for 1.5h at the indicated time points (Figure 2b), S phase 1.5h after being released from the G1/S block, and then harvested for measuring the BrdU-positive while Lats2-transduced cells had just started DNA cells by FACS (see Materials and methods). As shown in synthesis at 2.5h after the removal of aphidicolin. These Figure 2b, B25% of control cells were labeled by BrdU data suggest that ectopic expression of Lats2 may block 8 h after serum stimulation, indicating they had entered or retard initiation of DNA synthesis. However, data S phase. However, o1% of the LATS2-transduced cells from the DNA content FACS analysis (Figure 2a) did were BrdU positive. At 12.5h after serum stimulation, not rule out the possibility that Lats2 reduced the DNA 58% of the control cells and B30% of the Lats2- synthesis rate. To determine whether Lats2 plays a role transduced cells were in S phase. However, 25% of the BrdU-positive control cells were already at late S phase or G2/M (DNA content r4N), while o5%ofLats2- transduced cells were at late S phase or G2/M (Figure 2c). These results further confirmed that initiation of DNA synthesis in Lats2-transduced cells is partially blocked or retarded. About 16 h after serum stimulation, an almost equal amount of Lats2-transduced and control cells were labeled by BrdU (Figure 2b). DNA synthesis rate can be measured by the fluorescence intensity of BrdU incorporated into newly synthesized DNA. The intensity of BrdU staining of Lats2-transduced cells will be lower than that of the control cells if ectopic expression of Lats2 reduces the DNA synthesis rate. However, the intensity of the BrdU staining in the control or Lats2-transduced cells was not significantly different (Figure 2c), suggesting that the rate of DNA synthesis is very similar between control cells and Lats2-transduced cells. These observations indicate that ectopically expressed LATS2 protein has little or no effect on the DNA synthesis rate. Therefore, we conclude that Lats2 negatively regulates the initiation of DNA synthesis.

Lats2 regulates G1/S transition by modulating the kinase activity of Cyclin E/CDK2 Figure 2 Lats2 inhibits G1/S transition. (a) Cell cycle analysis of steady-state NIH3T3/v-ras cells infected by MIGR1-Lats2 or the To further investigate the molecular mechanisms by control virus. The infected cells were synchronized at the G1/S which Lats2 regulates G1/S transition, we examined the transition by serum starvation and aphidicolin treatment (see protein levels of cell cycle regulators that have been details in Materials and methods). The cells were then harvested for DNA content analysis at indicated time points after release from implicated in the control of G1/S transition. While aphidicolin-mediated G1/S blockage. At 0 h, when aphidicolin was ectopic expression of LATS1 in MCF-7 cells resulted in removed, cells had 2n DNA content. As they progressed into the degradation of Cyclins A and B (Xia et al., 2002), S phase, the ﬂuorescence intensity per cell increased (peak shift to ectopic expression of Lats2 in NIH3T3/v-ras cells caused the right) to give 42n but o4n. (b) Cell cycle progress of the no signiﬁcant changes at the protein level of G1/S BrdU-pulse-labeled NIH3T3/v-ras cells infected by MIGR1-Lats2 or the control cells. The cells were blocked at G./G1 by serum regulators such as Cyclin E, Cyclin A and CDK2, nor of WARF1/CIP1 starvation. The cells were pulse-labeled by BrdU for 1.5h at the negative cell cycle regulators such as P21 , indicated time points after serum stimulation and harvested at the p27KIP1 and p57KIP2 (Figure 3a). Further in vitro kinase end of labeling for FACS analysis (see Materials and methods). assays revealed that the kinase activity of Cyclin E/ The cell cycle progress from G1 into S phase was monitored by Lats2 measuring the percentage of BrdU-positive cells at each time point. CDK2 was downregulated in -transduced Values are means 7 s.d. for triplicates. (c) DNA content analysis NIH3T3/v-ras cells as compared to control virus- of the BrdU-positive cells from two time points (0 and 12.5h) in transduced cells (Figure 3b). However, the kinase (b), showing the difference of cell cycle progression between Lats2- activities of Cyclin D/CDK4/6, which are also involved transduced cells and the control cells. To simplify DNA content in the control of G1 progression (Sherr, 1996; Sherr and analysis, total BrdU-positive cells were divided into two parts, early S phase and late S phase /G2/M (boxed in (c)). The percentage of Roberts, 1999), were not affected (data not shown). K655M the BrdU-positive cells (average of three experiments) is indicated Lats2 encodes an active kinase and Lats2 is a for each cell population (above the box) kinase-inactive mutant (Hori et al.). The expression of

Oncogene Lats 2, a putative tumor suppressor YLiet al 4401

Figure 3 Lats2 downregulates Cyclin E/CDK2 kinase activity. (a) Figure 4 LATS2 kinase activity and its LCDs are required to Western blot analysis revealed no significant change at the protein suppress tumorigenicity and inhibit the growth of NIH3T3/v-ras level for all cell cycle regulators examined in Lats2-transduced cells. NIH3T3/v-ras cells were infected with retroviruses expressing NIH3T3/v-ras cells. Lats2-transduced NIH3T3/v-ras cells or wild-type Lats2 or its mutants, Lats2K655M, Lats2S830A, control cells were first enriched by FACS 36–48 h after infection. Lats2DLCD1, and Lats2DLCD2, as well as the control virus The enriched cells were allowed to recover for another 24 h, then MIGR1. Expression of LATS2 protein and its mutants were lysed for direct Western blot with the various antibodies indicated. confirmed by Western blot using anti-MYC antibody (a). The (b) Lats2 downregulates Cyclin E/CDK2 kinase activity and an methods for soft agar colony formation assay (b) and growth active kinase is responsible for inhibiting the activity of Cyclin E/ inhibition assay (c) are the same as described in Figure 1. Cell CDK2. Anti-Cyclin E antibodies were used to immunoprecipitate numbers seeded at day 1 and total cell numbers at day 5are shown CyclinE/CDK2 complex for the histone H1 kinase assay from cell in panel (c). All experiments were repeated three times. Alignment lysate synchronized at G1/S (see Materials and methods) of the amino acid sequence of LCD1 and LCD2 from human LATS1, mouse LATS1, human LATS2 and mouse LATS2 (d). Identical amino acids are shaded in gray. The amino-acid sequence of mouse LATS1 protein was composed of two stretches of

K655M sequences from GenBank, a.a. 1–168 was deduced from a mouse mouse Lats2 mutant protein (equivalent to human genome sequence (Accession NW_000023) according to sequence LATS2K697A, see Hori, et al.) failed to down regulate the homology to human LATS1, and the rest was from a mouse cDNA kinase activity of Cyclin E/CDK2 (Figure 3b), demon- (Accession AAD16883) strating that an active kinase of LATS2 is responsible for inhibiting the activity of Cyclin E/CDK2. All of these results suggest that Lats2 negatively regulates G1/ S transition by directly or indirectly modulating the in subdomain eight of the kinase domain (Hanks et al., kinase activity of Cyclin E/CDK2. 1988). This conserved amino-acid residue is usually phosphorylated to regulate its kinase activity. To test whether the kinase activity of the LATS2 protein is Requirement of kinase activity of LATS2 and LCD necessary for its tumor suppressor function, two point domains mutants, Lats2K655M and Lats2S830A, were created and Lats2 encodes a Ser/Thr protein kinase (Hori et al., expressed in NIH3T3/v-ras cells using the same retro- 2000; Yabuta et al., 2000), and the LATS2 protein is viral vector, MIGR1 (Figure 4a). Expression of the two phosphorylated in a cell cycle-dependent manner (Hori LATS2 mutant proteins, LATS2K655M and LATS2S830A, et al., 2000). Lys 655 of LATS2 is conserved in the failed to suppress NIH3T3/v-ras cells from forming kinase domain of all Ser/Thr kinases (equivalent to colonies in soft agar (Figure 4b). The growth inhibition Lys72 deﬁned in Hanks et al., 1988). This conserved Lys assay also demonstrates that LATS2K655M and LATS2- residue is essential for the recognition of the phosphate S830A lose their ability to inhibit cell growth (Figure 4c). group of Mg-ATP (Hanks et al., 1988). Mutating this These data reveal that the kinase activity of the LATS2 Lys residue usually results in a catalytically inactive protein is required for suppressing tumorigenicity and protein. Ser 830 of LATS2 is another residue conserved inhibiting cell proliferation.

Oncogene Lats 2, a putative tumor suppressor YLiet al 4402 There are two stretches of highly conserved sequence (B50% identical amino acids) in the amino-terminus between LATS1and LATS2 (Hori et al., 2000; Yabuta et al., 2000). For the convenience of description, these two conserved sequences are named as LATS conserved domain 1 (LCD1) and LATS conserved domain 2 (LCD2). The alignment of LCD1 and LCD2 in four mammalian LATS proteins is shown in Figure 4d. LCD2 is also conserved among mammalian LATS and Drosophila lats proteins (data not shown). To examine the necessity of LCD1 and LCD2 for the function of the Lats2 tumor suppressor, two Lats2 mutant retroviruses, MIGR1-Lats2DLCD1 and MIGR1-Lats2DLCD2, were generated, in which either LCD1 or LCD2 was deleted (see Materials and methods). The results shown in Figure 4b and c demonstrate that deleting LCD1 or LCD2 leads to the inability of LATS2 to suppress the tumorigenicity of NIH3T3/v-ras cells and to inhibit cell growth. This indicates that the two LATS conserved domains, LCD1 and LCD2, are required for the normal function of Lats2 in the suppression of tumor development.

The LATS2 proteins are associated with mitotic apparatus in mitotic cells

Human LATS1 protein was shown to be associated Figure 5 Cellular localization of LATS2 proteins. (A) Exogenous with the mitotic apparatus, suggesting a potential role MYC-tagged LATS2 proteins at different phases of the cell cycle. of LATS1 in mitosis (Hirota et al., 2000). To explore NIH3T3 cells were infected by a RT-Lats2-3myc retrovirus. After two days, the cells were fixed and incubated with rabbit anti-MYC if Lats2 also plays a role in mitosis, we examined cellular antibody only (a), or with rabbit anti-MYC antibody/mouse anti- localization of ectopically expressed LATS2. Surpris- tubulin antibody. Immunofluorescent staining was then performed ingly, the majority of exogenous LATS2 was localized with Rhodamine-conjugated anti-rabbit IgG antibody (a, d, g, j) in the cytoplasm, while there was only very faint and FITC-conjugated anti-mouse IgG antibody (e, h, k) or DAPI (b). Merged pictures are shown in the panels on the right side (c, f, LATS2 staining in the nucleus of interphase cells i, l). The majority of exogenous LATS2 protein is located in the (a–c in Figure 5A). This result was further confirmed cytoplasm during interphase (a–c). However, exogenous LATS2 by subcellular fractionation experiments showing that proteins are also associated with the mitotic apparatus in mitotic endogenous LATS2 proteins in human lung cancer cells, cells (d–l). Representative Lats2-transduced NIH3T3 cells in H661 and H460, are mainly cytoplasmic (Figure 5B). metaphase (d–f), anaphase (g–i) and telophase (j–l). The cells were examined in an inverted fluorescent microscope under a 60 Â oil We also observed that a fraction of exogenous LATS2 immersion objective. (b) Subcellular localization of endogenous protein was associated with the mitotic apparatus in LATS2 proteins. The asynchronized human lung cancer cells, H661 mitosis, in addition to cytoplasmic localization (d–l in and H460, were fragmentated into cytoplasmic (cyto) and nuclear Figure 5A). This result suggests that Lats2 may also (nuc) fractions using the methods described by Yabuta et al. (2000). The same volume of cellular and nuclear extracts were loaded for play a role in the regulation of mitosis. More detailed Western blot with anti-LATS2, anti-a-tubulin or anti-GATA2 studies are needed to further understand the roles of the antibodies LATS1 and LATS2 in mitosis.

Discussion to form colonies in soft agar. These results provide the first experimental evidence suggesting that Lats2, Drosophila lats and mouse Lats1 genes have been like other members of the Lats family, may function shown to play important roles in suppressing tumor- as a tumor suppressor in the control of mammalian igenesis in flies and mice (Justice et al., 1995; Xu et al., tumorigenesis. 1995; St John et al., 1999; Tao et al., 1999). The human Drosophila lats genetically interacts with cyclin A and LATS2 gene is located at chromosome 13q11–12 cdc2 in flies. Inactivation of Drosophila lats resulted in (Yabuta et al., 2000), a hot spot (67%) of LOH the upregulation of cyclin A, without affecting cyclin B for nonsmall cell lung cancer (Girard et al., 2000), (Tao et al., 1999). Ectopic expression of the human raising the possibility that LATS2 may play LATS1 gene in several human tumor cell lines down- an important role in the control of human tumor regulates the protein levels of Cyclins A and B (Xia et al., development. Here, we have demonstrated that ectopic 2002) and CDC2 kinase activity, thereby arresting cells expression of Lats2 suppressed the ability of NIH3T3/v- at G2/M and preventing tumor development (Yang ras cells to develop tumors in athymic nude mice and et al., 2001; Xia et al., 2002). Here, we have shown that

Oncogene Lats 2, a putative tumor suppressor YLiet al 4403 the ectopic expression of Lats2 also inhibits proliferation of NIH3T3/v-ras cells. Cell cycle profile analysis and BrdU-pulse-labeling experiments (Figure 2a-c) revealed that, different from that of LATS1-mediated G2/M arrest (Xia et al., 2002), ectopic expression of Lats2 inhibited G1/S transition (Figure 2) in addition to its potential role in mitosis (Figure 5). Currently, we cannot rule out the possibility that the different effects of LATS1 and Lats2 on the cell cycle are due to the different cell lines used for the studies. However, the possibility seems unlikely because we have also observed that the human lung cancer cell line, H460, displays similar growth inhibition when it is transduced by MIGR1-Lats2 retrovirus (data not shown), while expression of Lats1 in H460 resulted in a G2/M block and induced apoptosis (Xia et al., 2002). Furthermore, we have found that, unlike LATS1-mediated downregulation of Cyclins A and B, ectopic expression of Lats2 does not affect the protein levels of the cell cycle regulators affecting G1/S transition, but downregulates Cyclin E/CDK2 kinase activity (Figure 3). The down- Figure 6 Models for mammalian LATS1 and LATS2 tumor suppressors negatively regulating the cell cycle. Lats1 controls G2/ regulation of Cyclin E/CDK2 kinase activity, without M transition by negatively regulating Cyclin A/CDC2 and/or affecting Cyclin D/CDK4/6 kinase activities (data not Cyclin B/CDC2 activities. Lats2 affects G1/S transition by shown) that are required for G1 progression (Sherr, modulating Cyclin E/CDK2 activity 1996), further supports the notion that Lats2 negatively regulates G1/S transition without affecting G1 progression. However, it is not yet clear whether ectopic LATS2 negatively regulates Cyclin E/CDK2 kinase expression of Lats2 directly or indirectly affects Cyclin activity. E/CDK2 kinase activity. Our results from both immunostaining of exogenous The activation of the Cyclin E/CDK2 kinase is LATS2 protein in NIH3T3 cells (Figure 5A) and necessary for the initiation of DNA replication (Ekholm subcellular fractionation of endogenous LATS2 protein & Reed, 2000; Bartek & Lukas, 2001; Blow, 2001). in human lung cancer cells (Figure 5B) showed that the Recently, Cyclin E/CDK2 has also been shown to be majority of LATS2 protein is located in the cytoplasm, involved in the regulation of an intra-S phase check while a very small amount is in the nucleus. Our finding point in response to DNA damage (Sherr, 1996; Sanchez differs from the report that human LATS2 protein is a et al., 1997; Molinari et al., 2000; Falck et al., 2001). nuclear protein (Yabuta et al., 2000). We have tried Therefore, we propose that the two mammalian subcellular fractionation experiment using the same cell members of the Lats tumor suppressor family, LATS1 line (HeLa) and methods described by Yabuta et al. and Lats2, negatively regulate different cell cycle check Owing to the crossreaction of monoclonal anti-LATS2 points by modulating different cell cycle kinases antibody to the proteins from HeLa cells, we failed to (Figure 6). The LATS1 protein is phosphorylated at repeat the reported results by direct Western blot using G2/M transition, and forms a complex with CDC2 and our anti-LATS2 antibody, although we were able to downregulates the protein level of Cyclins A and B, as detect a single band of endogenous LATS2 protein in well as CDC2 kinase activity (Yang et al., 2001; Xia HeLa cells at low level by immunoprecipitation followed et al., 2002). Thus, LATS1 negatively regulates G2/M by Western blot using the same antibody (data not transition by downregulating CDC2 kinase activity. In shown). Therefore, it is still possible that the discrepancy contrast, Lats2 inhibits G1/S transition by down- results from the difference among the different cell lines regulating Cyclin E/CDK2 activity through some used for the experiments. unknown mechanism(s) (Figure 6). The activity of In summary, the data presented here indicate that waf1/ Cyclin E/CDK2 can be inhibited by binding to P21 Lats2, a new member of the Lats tumor suppressor cip1 kip1 or P27 (Sherr and Roberts, 1999; Ekholm and family, is a negative cell cycle regulator that inhibits G1/ Reed, 2000) and by phosphorylation at Tyr15and S transition and may be involved in the control of tumor Thr14 (Gu et al., 1993; McGowan and Russell, 1993; development. Morgan, 1995). Rapid inactivation of Cyclin E/CDK2 through the CHK2–CDC25A–CDK2 pathway has been found to play a critical role in both the G1/S check point (Sanchez et al., 1997; Blomberg and Hoffmann, 1999) Materials and methods and the intra-S phase check point response upon genotoxic stress (Molinari et al., 2000; Falck et al., Cell cultures 2001, 2002). However, a more detailed investigation is The human embryonic kidney cell line, 293 (high passage), was needed to fully characterize the mechanisms by which cultured in Dulbecco’s modified eagle’s medium (DMEM)

Oncogene Lats 2, a putative tumor suppressor YLiet al 4404 with 10% fetal bovine serum (FBS) at 371C, 5% CO2. The v- iodide (PI) for DNA content analysis by FACS (Becton ras transformed mouse fibroblast, NIH3T3, was a gift from Dr Dickinson). All of the data from FACS were analyzed with Richard Jove (University of South Florida) and grown in Cellquest software (Becton Dickinson). DMEM with 10% calf bovine serum. Human lung cancer cell lines, H661 and H460, were gifts from Dr Robert Kratzke and BrdU labeling were cultured in RPMI1640 with 10% calf bovine serum. The enrichment of Lats2 or control virus-transduced NIH3T3/ v-ras cells and the serum starvation were the same as described Production of the replication-defective recombinant retrovirus above. However, after releasing from G0/G1 block, the cells A mouse Lats2 cDNA fragment containing the whole coding were pulse-labeled by BrdU for 1.5h at various time points. region (a gift from Dr Tian Xu at Yale University) was tagged The harvested cells were stained with PE-conjugated mono- by an MYC epitope and cloned into an amphotropic retro- clonal anti-BrdU antibody (BD Pharmingen) and analyzed by virus expression vector MigR1 (Pear et al., 1998). The resulting FACS for the BrdU-positive cells following the manufacturer’s plasmid, MigR1-Lats2, and the retrovirus genomic plasmid instruction (BrdU Flow Kit, BD Pharmingen). pCL were cotransfected into high-passage 293 cells using the calcium phosphate precipitation method to produce the Western blot analysis and kinase assay replication-defective recombinant retrovirus MIGR1-Lats2. In MIGR1-Lats2K655M and MIGR1-Lats2S830A, two residues in Lats2 or control virus-transduced NIH3T3/v-ras cells enriched the LATS1 kinase domain, Lys 655 and Ser 830, which are by FACS were allowed to recover for 24 h. Then, cells were conserved in the kinase domains of all Ser/Thr kinases from either harvested for Western blot analysis or synchronized at yeast to human, were mutated into Met and Ala, respectively, G1/S as described previously for an in vitro kinase assay. The using site-directed mutagenesis (Promega). The plasmid pRT- harvested cells were lysed in TG buffer (Tao et al., 1999) with m Lats2, a derivative of the plasmid MigR1-Lats2 without IRES- 1m PMSF and 1 Â complete proteinase inhibitors (Roche EGFP elements, was used to generate a replication-defective Molecular Biochemicals). Cell lysate proteins (30–120 mg) were Lats2 retrovirus without EGFP, which was used for immu- used for Western blot. Anti-MYC antibody (A-14), anti-mouse nostaining to determine the cellular localization of exogenous CDK2(M20), CDK4, Cyclin A, Cyclin E, Cyclin D, p21, p27, LATS2 protein. p57, and b-actin were purchased from Santa Cruz. An anti- LATS2 monoclonal antibody was a generous gift from Dr Tian Xu at Yale University. For the H1 kinase assay, CDK2 Growth inhibition assay proteins were immunoprecipitated with anti-Cyclin E (M20) NIH3T3/v-ras cells were infected separately by MIGR1-Lats2, from the lysates of cells synchronized at G1/S. CDK4 or LATS2 mutant retrovirus, or MIGR1 for 4–5h. The virus CDK6 were immunoprecipitated from nonsynchronized cell infection was repeated once 20–24 h after the first infection. lysate with anti-CDK4 or anti-CDK6 antibodies and used to Approximately 60–70% of cells were infected 48 h after the access their kinase activities with Rb–C fusion protein (Cell second infection, as determined by flow cytometry using EGFP Signaling) as a substrate. The kinase assay was performed as as a marker. Then, EGFP-positive cells were enriched by described by Tao et al. (1999). FACS. In all, 7000 transduced NIH3T3/v-ras cells were replated in 60 mm dishes and the total number of cells was Immunofluorescence staining counted daily using a hemacytometer. NIH3T3 cells were transduced by a retrovirus, RT-Lats2-myc. After 48 h later, the cells were fixed in ice-cold 100% methanol Soft agar and nude mice studies for 20 min at À201C and permeabilized for 5min in 0.1% In total, 500 of the Lats2 or control virus-transduced NIH3T3/ Triton X-100. The coverslips were washed and blocked in 1% v-ras cells enriched by FACS were plated in 4 ml of 0.4% normal goat serum in PBS and incubated with rabbit anti- agarose (SeaPlaque, BioProducts) in DMEM with 10% CBS. MYC antibody (10 mg/ml) and/or monoclonal anti-tubulin The cells grew for 3 weeks at 371C, 5% CO2. The colonies were antibodies for 1 h at room temperature. Then, immunofluor- stained with MTT (Aftab et al., 1997) for photographing and escent staining was performed with Rodamine-conjugated colony counting. To access the tumorigenicity of Lats2- anti-rabbit IgG antibody and/or FITC-conjugated anti-mouse transduced NIH3T3/v-ras cells in vivo, 2.75 Â 105 of Lats2- IgG antibody and DAPI (1 mg/ml). The coverslips were washed transduced cells (in 50 ml of PBS) were injected into the flank of six times with PBS and mounted. Control staining was athymic mice. Control virus-transduced cells were injected in performed without primary antibodies (data not shown). separate mice as the control. Tumors were allowed to develop for 2 weeks. Subcellular fractionation The methods described by Yabuta et al. for subcellular Cell synchronization and cell cycle profile analysis fractionation were exactly followed. The same volume of To synchronize the cells, the method described by Wright et al. cellular and nuclear extracts was used for Western blot. The (1999) was followed with minor modification. Briefly, Lats2 or same membrane was blotted with anti-LATS2 antibody first, control virus-transduced NIH3T3/v-ras cells were first en- then stripped repeatedly for blotting cellular and nuclear riched by FACS 48 h after infection, then 1.25 Â 105 cells were protein markers. reseeded in 60 mm dishes coated by 0.01% polylysine for 24 h for recovery. The cells were then serum-starved for 36 h in Acknowledgements DMEM. Next, cells were stimulated with 20% FBS, and at the We are grateful to Dr Catherine Verfaillie for her support and same time, 1 mg/ml of aphidicolin (ICN Biomedicals) was helpful advice for this research. We thank Dr Tian Xu at Yale added to block the cells at G1/S. After 16 h, the cells were University for Lats2 cDNA clone and anti-LATS2 antibody. washed three times with 1 Â PBS to remove excess aphidicolin We thank Dr Robert Sheaff for critical manuscript readings and 10% CBS DMEM was added. The cells were harvested at and antibodies, Dr Richard Jove for the NIH3T/v-ras cell line various time points (Figure 2a) and stained with propidium and Dr Robert Kratzke for the human lung cancer cell lines,

Oncogene Lats 2, a putative tumor suppressor YLiet al 4405 H661 and H460. We also thank Huilin Qi for a lot of valuable of Medicine and Stem Cell Institute, a Medical School Faculty discussion and technical help and Tishonna Woods for editing Grant from Minnesota Medical Foundation, an American of the manuscript, and Julie Prebyl and Janet Peller for their Cancer Society Institutional Research Grant (IRG-58-001-40- assistance with FACS and data analyses. This work is IRG40) and Research Scholar Grant from the American supported by the startup fund for W Tao from the Department Cancer Society (RSG-02-065-01-MGO).

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