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The mitotic checkpoint protein kinase BUB1 is an engine in the TGF-β signaling apparatus

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Science Signaling  13 Jan 2015:
Vol. 8, Issue 359, pp. fs1
DOI: 10.1126/scisignal.aaa4636

Abstract

The transforming growth factor–β (TGF-β) pathway mediates critical events in cell behavior that contribute to development and disease. The mitotic checkpoint guarantees faithful chromosomal segregation during cell division. In the 6 January 2015 issue of Science Signaling, Nyati et al. reported that the mitotic checkpoint kinase BUB1 promotes the activity of TGF-β receptors, which adds new molecular links between these fundamental biological processes.

The evolutionarily conserved protein trio of the BUB1 and BUB1-related protein (BUBR1) protein kinases and associated adaptor BUB3 adorn the centromeres (Fig. 1) before microtubule association, to aid in the assembly of the supramolecular kinetochore apparatus. This apparatus comprises more than a dozen proteins, including the adaptors Mad1 and Mad2; the protein kinases Mps1 and Aurora-B; the motors centromere protein E (CENP-E), kinesin and dynein; and the ubiquitin ligase anaphase-promoting complex (APC) with its molecular switch, Cdc20 (1). These protein assemblies organize centromere attachment to one end of a microtubule of the mitotic spindle and thus secure proper chromosomal segregation during mitosis. This mechanism is known as the spindle assembly checkpoint (SAC) or mitotic checkpoint.

Fig. 1 Shared molecular players between the SAC and TGF-β signaling.

In a cell preparing to divide (left), BUB1 functions at the centromere to recruit a multiprotein complex called the kinetochore, which provides a “wait” signal to ensure that chromosomes are properly connected to microtubules before segregation into daughter cells. BUB1 also binds the TGF-β receptor complex and promotes downstream signaling (right). BUB1 may traffic in and out of the nucleus and be transported to TGF-β receptors along microtubules, similar to the manner in which dephosphorylated Smad2/3 returns. Other proteins that are common to both the mitotic checkpoint and TGF-β signaling—including the E3 ubiquitin ligases, APC and Smurf2, and the kinase Mps1—indicate crosstalk regulation between these critical regulatory mechanisms in cellular and organismal physiology. TF, transcription factor.

CREDIT: H. MCDONALD/SCIENCE SIGNALING

In brief, during prometaphase, BUB3-BUB1 and BUB3-BUBR1 complexes bound to unattached centromeres sequester Cdc20 and secure silencing of the APC-proteasome system (1). When all centromeres attach to spindle microtubules, forming proper bipolar associations of sister chromatids, BUBR1 and Mps1 mediate release of Cdc20, which activates APC and leads to ubiquitination of securin and cyclin B. Securin, an inhibitor of the protease separase, becomes inactivated, and separase is free to degrade the chromatin factor cohesin that “glues” sister chromatids together, which initiates sister chromatid segregation during anaphase (1). Concomitantly, the APC ubiquitylates cyclin B to promote its degradation, which provides the signal for exit from mitosis and preparation for entry into the G1 phase of the next cell cycle (1). When misregulated, this leads to chromosomal aneuploidy, a feature of degenerative diseases, including cancer.

BUB1 stabilizes the assembly of many of the kinetochore protein complexes before microtubule tethering and phosphorylates Cdc20, which is required for association with the kinetochore and indirect inactivation of the APC ligase (1). Nyati and colleagues (2) now show a place for BUB1 away from the centromere and close to the plasma membrane receptors of the transforming growth factor–β (TGF-β) signaling pathway. TGF-β is important during embryonic development, contributes to tissue homeostasis in adult organisms, and is misregulated in various diseases (3). By binding to the type II and type I receptor kinases that phosphorylate Smad proteins and activate mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K), extracellular TGF-β induces gene expression that mediates physiological changes, including cell cycle arrest at the G1 phase, epithelial-mesenchymal transition (EMT), extracellular matrix deposition, and cell migration (Fig. 1). Using a kinome-wide RNA interference screen, Nyati and colleagues identified BUB1 as a kinase that positively contributes to TGF-β signaling (Fig. 1) (2). BUB1 associated with both TGF-β receptors and promoted their heteromeric assembly, and a micromolecular inhibitor of the kinase activity of BUB1 (2OH-BNPP1) abrogated all aspects of TGF-β signaling tested (2). Despite the necessity of the kinase activity of BUB1 during TGF-β signaling, a substrate of BUB1 has not yet been identified in this pathway, and the endogenous BUB1–TGF-β receptor complex was not observed. This concern, together with the as-yet-uncharacterized mechanism that mediates shuttling of BUB1 from centromeres to the plasma membrane, provides open challenges to understanding this exciting new signaling mechanism. Relevant to this is the cell cycle–dependent regulation of TGF-β signaling that deserves further analysis.

There are interesting corollaries based on crosstalk between SAC components and the TGF-β pathway. Along with Smad7, the E3 ubiquitin ligase Smurf  2 shuttles from the nucleus to the plasma membrane to associate with and promote the degradation of the TGF-β receptor complex (3). Smurf  2 can also associate with centromeres (Fig. 1) where it stabilizes Mad2; the absence of Smurf  2 causes premature anaphase and mitotic defects (4). Is the Smad7-Smurf  2 module relevant to BUB1 translocation to TGF-β receptors? In addition, Smad2/3 mediates the cooperation of nuclear Smurf  2 with APC to direct the degradation of SnoN (Fig. 1), a key Smad transcriptional corepressor (5, 6). Notably, Nyati et al. show that BUB1 can form complexes with Smad2; thus, Smads may mediate BUB1 shuttling. Furthermore, the kinase Mps1 is required for Mad1/2 localization to kinetochores (1) and can also directly phosphorylate Smad2/3 at the same C-terminal di-serine motif where Smads are phosphorylated by the TGF-β receptor (7). Thus, like BUB1, Mps1 promotes the response to TGF-β; it will be interesting to test whether BUB1 and Mps1 cooperate to promote TGF-β signaling. The ability of these centromere-tethered kinases to shuttle within the cell and traffic toward membrane receptors is appealing and compatible with the knowledge that Smads traffic between the nucleus and plasma membrane using dynein and kinesin motors along microtubules [reviewed in (8)]. Given that kinetochore-localized BUB1 controls chromosome-microtubule association, it is possible that BUB1 could also slide along microtubules, possibly together with Smads and associated kinesins, to reach the TGF-β receptors (Fig. 1).

Finally, is the crosstalk between SAC components and TGF-β signaling of physiological importance, and is it related to the regulation of the cell cycle? Nyati et al. provide examples of EMT and cancer cell invasion as parameters for which BUB1 function is required during TGF-β signaling (2). In bone marrow stromal cells, TGF-β regulates the activity of the APC ligase through Smad3 and thus controls separase activity and mitotic chromosome segregation (9). BUB1 might contribute to the regulation of this process by acting on both centromeres and TGF-β receptors. Experimental mitotic arrest leads to Smad3 association with the mitotic spindle while TGF-β receptor endocytosis is impaired, a process observed in cancer cells (10) and in fly embryos in which the whole endocytic apparatus with TGF-β receptors segregates to daughter cells with the help of the mitotic spindle [reviewed in (8)]. It is therefore evident that the study from Nyati and colleagues opens new fertile ground for investigation. Future studies on the crosstalk of the mitotic checkpoint with TGF-β signaling promise new cellular mechanisms and signaling events that are relevant to both normal and malignant cell biology.

References and Notes

Funding: Research in the Moustakas laboratory is supported by the Ludwig Institute for Cancer Research, the Swedish Cancer Society, the Swedish Research Council, and the Marie Curie Initial Training Network “IT-Liver” under the European Union FP7 program. Competing interests: The author declares that he has no competing interests.
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