Human LATS1 Is a Mitotic Exit Network Kinase
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Research Article Human LATS1 Is a Mitotic Exit Network Kinase John Bothos,1,2 Robyn L. Tuttle,1 Michelle Ottey,3 Francis C. Luca,3 and Thanos D. Halazonetis1,4 1The Wistar Institute; 2Program in Cell and Molecular Biology, Biomedical Graduate Studies; 3Department of Animal Biology, School of Veterinary Medicine; and 4Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Abstract cerevisiae, there are three members in the NDR family. Dbf2 and The kinase LATS/WARTS is a tumor suppressor protein its nearly identical and redundant Dbf20 kinase are key components of the mitotic exit network (MEN), a signaling pathway that conserved in evolution, but its function at the molecular level is not well understood. We report here that human LATS1 coordinates cyclin-dependent kinase (CDK) inactivation, sister interacts with MOB1A, a protein whose homologue in budding chromatid decondensation, and cytokinesis at the end of mitosis yeast associates with kinases involved in mitotic exit. This (13–15); on the other hand, Cbk1, the third member, promotes suggested that LATS1 may be a component of the previously polarized growth and activates daughter-specific gene transcription uncharacterized mitotic exit network in higher eukaryotes. after cytokinesis (16–19). In Schizosaccharomyces pombe, there Indeed, moderate overexpression of human LATS1 in cells are two members in the NDR family. Sid2, the orthologue of exposed to microtubule poisons facilitated mitotic exit, and Dbf2/Dbf20, functions in the septation initiation network (SIN), this activity required MOB1A. Reciprocally, small interfering which is analogous to the S. cerevisiae MEN (13–15); on the other RNA–mediated suppression of LATS1 or MOB1A prolonged hand, Orb6, like its orthologue Cbk1, is required for maintenance of cell polarity after completion of mitosis (20). telophase, but had no effect on the length of the earlier phases of mitosis. A role of LATS1 in mitotic exit may explain its The yeast NDR kinases require regulatory subunits for catalytic previously described abilities to induce G arrest and promote activity. In S. cerevisiae, there are two such subunits, Mob1 and 2 Mob2. Mob1 interacts with Dbf2 and Dbf20, whereas Mob2 cytokinesis. (Cancer Res 2005; 65(15): 6568-75) interacts with Cbk1. These interactions are conserved in S. pombe, such that S. pombe Mob1 and Mob2 interact with Sid2 and Orb6, Introduction respectively (18, 19, 21–25). LATS (large tumor suppressor, also known as WARTS) was The assignment of LATS1 to the NDR protein kinase family originally identified in a screen for genes whose inactivation leads to raises the possibility that LATS1 is an orthologue of yeast NDR overproliferation of cells in Drosophila (1, 2). Two homologues of kinases (either the Mob1- or the Mob2-interacting kinases). Indeed, LATS have been identified in mammals and are called LATS1/ we report here that in human cells, LATS1 interacts with MOB1A WARTS and LATS2/KPM (3–6). Inactivation of Lats1 in mice leads and has functional similarities to the yeast NDR kinases that to development of various tumors, including sarcomas and ovarian interact with Mob1 (S. cerevisiae Dbf2/Dbf20 and S. pombe Sid2). cancer (4). Further, promoter methylation, loss of heterozygosity, Thus, LATS1 is a component of the previously uncharacterized and missense mutations targeting LATS1 have been reported in mammalian MEN. human sarcomas and ovarian carcinomas (7, 8), suggesting that LATS functions as a tumor suppressor across evolution from Materials and Methods Drosophila to humans. At the molecular level, the function of LATS1 is not well Expression plasmids and cell transfections. The coding sequences of understood. LATS1 has been reported to inhibit the CDC2 kinase human NDR1, MOB1A, and MOB2 were amplified from expressed sequence tags by PCR and subcloned into a pCDNAzeo3.1 vector, which was modified by associating with CDC2 and displacing its cyclin subunit (3). to contain either hemagglutinin (HA) or FLAG tags within its polylinker. A More recently, LATS1 has been reported to inhibit the LIMK1 pCDNA3.1 plasmid expressing myc-tagged human LATS1 was provided by kinase again by directly interacting with LIMK1 (9). Inhibition of Dr. Wufan Tao (The Stem Cell Institute, University of Minnesota Medi- CDC2 may explain why LATS1 overexpression inhibits the cal School, Minneapolis, MN). These plasmids were transfected in U2OS transition from G2 to mitosis (3, 10, 11), whereas inhibition of osteosarcoma cells by calcium phosphate precipitation and their protein LIMK1 may explain the ability of LATS1 to promote cytokinesis products were analyzed 72 hours after transfection. For stable expression, because LIMK1 inhibits actin polymerization (9). the inserts encoding myc-tagged LATS1 and HA-tagged MOB1A were sub- Interestingly, LATS1 itself is a protein kinase, but its kinase cloned in the pIRESN2 vector. U2OS cells transfected with these vectors activity is not required for inhibition of LIMK1 and possibly of CDC2 or with a pIRESN2 vector that had no insert (neo control) were selected (3, 9). In turn, this suggests that LATS1 has functions that require its with 0.5 mg/mL G418 for 4 weeks. G418-resistant colonies were then pooled by trypsinization and propagated in tissue culture media containing 0.25 kinase activity and are independent of CDC2 and LIMK1. Based on mg/mL G418. its amino acid sequence, LATS1 has been assigned to the nuclear Small interfering RNA transfection. U2OS cells (150,000) were seeded Dbf2-related (NDR) family of protein kinases (12). In yeast, NDR in 60 mm tissue culture dishes and the following day were transfected with kinases are involved in cell cycle control. In Saccharomyces small interfering RNA (siRNA) specific for human MOB1A (AAUGCAGAAG- CAACUCUAGGA) or LATS1 (9) or luciferase (Dharmacon, Lafayette, CO) as a control. For analysis of cell cycle progression, the cells were synchronized 1 day later with a single thymidine block as described below. Requests for reprints: Thanos D. Halazonetis, The Wistar Institute, 3601 Spruce Cell cycle synchronization. The cells were synchronized by adding 1.3 Street, Philadelphia, PA 19104-4268. Phone: 215-898-3789; Fax: 215-573-9271; E-mail: [email protected]. mmol/L thymidine in the tissue culture media. After 20 hours, the cells were I2005 American Association for Cancer Research. washed twice with PBS and released into media containing 250 Amol/L Cancer Res 2005; 65: (15). August 1, 2005 6568 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Human Mitotic Exit Network thymidine and 250 Amol/L deoxycytidine. Where indicated, 8 hours after similarity to yeast Mob1; whereas MOB2 (also known as MMh) has release into S phase, the cells were exposed to 1 Amol/L nocodazole. high sequence similarity to yeast Mob2 (26, 27). Interactions Live cell videomicroscopy. Cells that had been cultured on 12 mm between LATS1 and human MOB proteins were assayed by autoclaved glass slides were synchronized and placed on the microscope transiently expressing in cells myc-tagged LATS1 together with stage 8 hours after release from the G -S block. The cells were observed 1 either HA-tagged MOB1A or HA-tagged MOB2 and performing using a phase contrast lens and maintained at 37jC using a stage heater (Bioptechs, Butler, PA). Images were acquired every 5 minutes using an coimmunoprecipitations. As controls, we also examined the ORCA ER digital camera (Hamamatsu, Hamamatsu City, Japan). interaction of MOB1A and MOB2 with human FLAG-tagged NDR1 Antibodies. A monoclonal antibody against human MOB1A was because NDR1 was recently reported to associate with human prepared using as antigen full-length MOB1A fused to the COOH terminus MOB2 (28). Myc-tagged LATS1 associated preferentially with HA- of glutathione S-transferase. The other antibodies were obtained commer- tagged MOB1A, whereas FLAG-tagged NDR1 associated preferen- cially: g-tubulin polyclonal (Sigma, St. Louis, MO), LATS1 polyclonal (Santa tially with HA-tagged MOB2 (Fig. 1A). Cruz Biotechnologies, Santa Cruz, CA), HA monoclonal (Covance, Princeton, The interaction between human LATS1 and MOB1A was further NJ), FLAG monoclonal (Sigma), 9E10-Myc monoclonal (Upstate, Charlottes- 10 examined by studying endogenous MOB1A. We prepared U2OS ville, VA), and histone H3 Ser phosphospecific polyclonal (Upstate). cells that stably express myc-tagged LATS1 (clones 1 and 2; Fig. 1B) Flow cytometry. For flow cytometry analysis, 1 Â 106 million cells were and used these cells (clone 1) to show that a monoclonal antibody trypsinized, washed once in PBS, and fixed in 70% ethanol at 4jC for at least 15 minutes. The cells were then washed in PBS, incubated in PBS that recognizes human MOB1A, but not a control antibody, supplemented with 0.25% Triton X-100 on ice for 5 minutes and then in coprecipitated myc-tagged LATS1 (Fig. 1C). We could not show the 200 AL 1% bovine serum albumin (BSA)/PBS containing 1 Ag anti–phospho- reciprocal interaction, because untagged human endogenous Ser10 histone H3 antibody at room temperature for 1 hour. After washing, MOB1A comigrates with immunoglobulin light chain (see Fig. 1A the cells were incubated in 200 AL 1% BSA/PBS containing 1 Ag Alexafluor showing HA-tagged MOB1A migrating just slightly slower than the 488–conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR) immunoglobulin light chain). Finally, we could also detect an for 30 minutes