Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1. hBUB1 and hBUBR1 bind to kinetochores where they are postulated to be components of the mitotic checkpoint that monitors kinetochore activities to determine if chromosomes have achieved alignment at the spindle equator (Jablonski, S.A., G.K.T. Chan, C.A. Cooke, W.C. Earnshaw, and T.J. Yen. 1998. Chromosoma. 107:386–396). In support of this, hBUB1 and the homologous mouse BUB1 have been shown to be important for the mitotic checkpoint (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Willson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300–303; Taylor, S.S., and F. McKeon. 1997. Cell. 89:727–735). We now demonstrate that hBUBR1 is also an essential component of the mitotic checkpoint. hBUBR1 is required by cells that are exposed to microtubule inhibitors to arrest in mitosis. Additionally, hBUBR1 is essential for normal mitotic progression as it prevents cells from prematurely entering anaphase. We establish that one of hBUBR1's checkpoint functions is to monitor kinetochore activities that depend on the kinetochore motor CENP-E. hBUBR1 is expressed throughout the cell cycle, but its kinase activity is detected after cells have entered mitosis. hBUBR1 kinase activity was rapidly stimulated when the spindle was disrupted in mitotic cells. Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells. The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E–dependent activities at the kinetochore and regulating cyclosome/APC activity.

To investigate possible involvement of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, we have used indirect immunofluorescence with a highly specific phospho-MAP kinase antibody and found that a portion of the active MAP kinase is localized at kinetochores, asters, and the midbody during mitosis. Although the aster labeling was constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase, started to fade during chromosome congression, and then disappeared at midanaphase. At telophase, active MAP kinase localized at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substrate for MAP kinase. CENP-E was phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules.

We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase. Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint. Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores. In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores. hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E. The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension. hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores. We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner. Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore. INTRODUCTIONThe mitotic checkpoint is a fail-safe mechanism that ensures accurate chromosome segregation by preventing cells from prematurely exiting mitosis in the presence of unaligned chromosomes (Nicklas, 1997;Rieder and Salmon, 1998;Amon, 1999). This checkpoint system is highly sensitive, because even a single unaligned chromosome is sufficient to block cells from entering anaphase (Rieder et al., 1994;Li and Nicklas, 1997). The mitotic checkpoint has been shown to monitor both microtubule attachment and tension generated across sister kinetochores by poleward forces (Rieder et al., 1994;Li and Nicklas, 1997;Waters et al., 1998). Failure of the mitotic checkpoint causes cells to exit mitosis in the presence of unaligned chromosomes and is a major mechanism responsible for aneuploidy (Jallepalli and Lengauer, 2001). Seven mitotic checkpoint genes, BUB1, BUB2, BUB3, MAD1, MAD2, MAD3, and MPS1, were originally identified via genetic screens in Saccharomyces cerevisiae (Hoyt et al., 1991;Li and Murray, 1991;Weiss and Winey, 1996). These genes act along two separate mitotic checkpoint pathways (Clarke and Gimenez-Abian, 2000;Daum et al., 2000;Gardner and Burke, 2000). MPS1, BUB1, BUB3, MAD1, MAD2, and MAD3 monitor kinetochore microtubule attachments and prevent premature chromosome segregation by inhibiting degradation of securin/Pds1 and mitotic cyclins (Wassmann and Benezra, 2001;Peters, 2002). BUB2 acts along a different pathway that monitors spindle integrity and orientation and prevents premature cytokinesis by inhibiting the degradation of the mitotic cyclin Clb2 (Alexandru et al., 1999;Fesquet et al., 1999;Fraschini et al., 1999;Li, 1999;Bardin et al., 2000;Bloecher et al., 2000;Pereira et al., 2000).Many of the mitotic checkpoint genes in yeast are evolutionarily conserved, because orthologues of M...

Ataxia telangiectasia-mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNAdependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA-dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATMassociated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway. INTRODUCTIONCells respond to DNA damage by activating checkpoint pathways that delay progression through the cell cycle. This cell cycle delay provides the necessary time for the cell to assess and repair the damage before reentering the cell cycle. If the damage is determined to be beyond repair, the cell may undergo apoptosis to prevent mutations from being propagated. When mammalian cells are exposed to ionizing radiation (IR) or radiomimetic drugs, a signal transduction pathway is activated that arrests cells in G 1 , S, and/or G 2 phases of the cell cycle. The G 1 arrest is the best characterized and is dependent on a functional p53 response that leads to transcriptional activation of the G 1 -specific cyclin-dependent kinase inhibitor p21/ WAF1/CIP (Kastan et al., 1991). The S and G 2 checkpoints seem to be p53 independent because p53-defective cells retain these checkpoints (El-Deiry et al., 1993). Although the existence of DNA damage-dependent checkpoint pathways has been known for some time, the molecular mechanism(s) by which the cell senses the DNA double-strand breaks and converts this information into a growth arrest signal remains unclear.Ataxia telangiectasia (AT) 1 is an autosomal recessive disorder characterized by cerebellar ataxia, dilated blood vessels in the eyes and skin (oculocutaneous telangiectasias), immunodeficiency, hypersensitivity to IR, and a 100-fold increase in the risk of some types * Corresponding author. E-mail address: TJYen@fccc.edu. 1 Abbreviations used: AT, ataxia telangiectasia; ATM, ataxia telangiectasia-mutated gene; ATR, ATM-related protein kinase...