Protein Interaction Domain Mapping of Human Kinetochore Protein Blinkin Reveals a Consensus Motif for Binding of Spindle Assembly Checkpoint Proteins Bub1 and BubR1

Abstract

The kinetochore is a supramolecular structure essential for microtubule attachment and the mitotic checkpoint. Human blinkin/human Spc105 (hSpc105)/hKNL1 was identified originally as a mixed-lineage leukemia (MLL) fusion partner and later as a kinetochore component. Blinkin directly binds to several structural and regulatory proteins, but the precise binding sites have not been defined. Here, we report distinct and essential binding domains for Bub1 and BubR1 (here designated Bubs) at the N terminus of blinkin and for Zwint-1 and hMis14/hNsl1 at the C terminus. The minimal binding sites for Bub1 and BubR1 are separate but contain a consensus KI motif, KI(D/N)XXXF(L/I)XXLK. RNA interference (RNAi)-mediated replacement with mutant blinkin reveals that the Bubs-binding domain is functionally important for chromosome alignment and segregation. We also provide evidence that hMis14 mediates hNdc80 binding to blinkin at the kinetochore. The C-terminal fragment of blinkin locates at kinetochores in a dominant-negative fashion by displacing endogenous blinkin from kinetochores. This negative dominance is relieved by mutations of the hMis14 binding PPSS motif on the C terminus of blinkin or by fusion of the N sequence that binds to Bub1 and BubR1. Taken together, these results indicate that blinkin functions to connect Bub1 and BubR1 with the hMis12, Ndc80, and Zwint-1 complexes, and disruption of this connection may lead to tumorigenesis.The kinetochore is formed on centromeric DNA and is essential for microtubule attachment and mitotic checkpoint signaling during mitosis (3,6,23,27,34). Recent genetic and mass spectrometric analyses have identified more than 80 kinetochore components in fungi, nematodes, insects, and mammalian cells and have revealed both conserved core components and species-specific kinetochore proteins (4,14,22,25,30). These proteins form several subcomplexes and assemble and disassemble in an ordered fashion to build functional kinetochores during mitosis.The Mis12 complex is a key conserved kinetochore subcomplex which consists of four proteins, Mis12, Mis13/Dsn1, Mis14/Nsl1, and Nnf1/PMF1 (4,10,11,18,25). The human Mis12 (hMis12) complex interacts with heterochromatin protein 1 (HP1) during interphase and dissociates from HP1 during mitosis to form the inner centromere structure between sister kinetochores (16). During mitosis, the hMis12 complex associates with the microtubule-binding protein blinkin (alternatively called hSpc105, hKNL1, CASC5, and D40) and the Ndc80/Hec1 complex (2, 5, 33), as well as the mitotic checkpoint-related proteins Zwint-1 and Bub1 and BubR1 (Bub1-related protein) (17). Thus, the hMis12 complex plays a key role in inner centromere and kinetochore assembly as well as kinetochore-microtubule attachment and mitotic checkpoint signaling in human cells.Yeast two-hybrid (Y2H) mapping, localization dependency, and RNA interference (RNAi) rescue experiments have suggested that the hMis12 complex directly interacts with the C terminus of blinkin and that the N terminus of blinki...

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…Mps1 copurifies with Ndc80 and interacts with its N-terminal domain in vitro (Kemmler et al 2009), suggesting that Ndc80 may be the kinetochore receptor for Mps1. Phosphorylation of the Spc105 protein on conserved MELT motifs by Mps1 recruits the Bub1 and Bub3 proteins to the kinetochore (Kiyomitsu et al 2007(Kiyomitsu et al , 2011Krenn et al 2012;London et al 2012;Shepperd et al 2012;Yamagishi et al 2012). Mps1, Bub1, and Bub3 are all required for the recruitment of the Mad1 and Mad2 proteins to kinetochores, although the Mad1 receptor has not yet been identified (Gillett et al 2004;Heinrich et al 2012).…”

“…In addition to contributing to KMN function, Spc105 also appears to be a scaffold for other outer kinetochore proteins. It recruits the Bub1 and Bub3 proteins to the kinetochore, and it may be a regulatory subunit for PP1 at the kinetochore (Kiyomitsu et al 2007(Kiyomitsu et al , 2011Liu et al 2010;Rosenberg et al 2011) (discussed below, The Spindle Checkpoint). The Ndc80 complex has two globular head domains that are connected by a long rod (Wei et al 2005;Ciferri et al 2008).…”

Mph1 kinetochore localization is crucial and upstream in the hierarchy of spindle assembly checkpoint protein recruitment to kinetochores

“…(Kemmler et al, 2009;London et al, 2012;Shepperd et al, 2012;Yamagishi et al, 2012) or SAC proteins (Hardwick et al, 1996). Recruitment of Bub1 and Bub3, probably to the outer kinetochore protein Spc7 (KNL-1, Blinkin) (Kiyomitsu et al, 2007;BolanosGarcia et al, 2011;Kiyomitsu et al, 2011;Krenn et al, 2012), may initiate a feedback loop with Ark1 (Aurora B) to strengthen the Windecker et al, 2009;§, Vanoosthuyse et al, 2004;#, Kadura et al, 2005;*, Millband and Hardwick, 2002). n.d., not determined.…”

The dynamic protein Knl1 – a kinetochore rendezvous

“…Knl1, the largest subunit of the KMN network, is required for accurate chromosome segregation during mitosis (Desai et al, 2003), and for both activating and inactivating the spindle assembly checkpoint (SAC), a conserved signaling cascade that delays anaphase onset in the presence of unattached or improperly attached chromosomes (Kiyomitsu et al, 2007;Meadows et al, 2011;Rosenberg et al, 2011;Espeut et al, 2012). Defects in Knl1 function have been implicated in genome instability, leukemia, microcephaly and neurological disorders (Kiyomitsu et al, 2007;Kiyomitsu et al, 2011;Yang et al, 2014;Genin et al, 2012). Interestingly, the phenotype observed in cells in which Knl1 is depleted is similar to that associated with the suppressed expression of the mitotic checkpoint proteins Bub1 and BubR1 (Kiyomitsu et al, 2007;Kiyomitsu et al, 2011;Cheeseman et al, 2006;Cheeseman et al, 2008), suggesting that a major function of Knl1 is to coordinate Bub1 and BubR1 signaling, a notion that has seen significant support from a number of recent studies in yeasts, Caenorhabditis elegans, Drosophila melanogaster and cultured human cells (Desai et al, 2003;Cheeseman et al, 2008;Schittenhelm et al, 2009;Venkei et al, 2012;Varma et al, 2013; reviewed by .…”

“…Defects in Knl1 function have been implicated in genome instability, leukemia, microcephaly and neurological disorders (Kiyomitsu et al, 2007;Kiyomitsu et al, 2011;Yang et al, 2014;Genin et al, 2012). Interestingly, the phenotype observed in cells in which Knl1 is depleted is similar to that associated with the suppressed expression of the mitotic checkpoint proteins Bub1 and BubR1 (Kiyomitsu et al, 2007;Kiyomitsu et al, 2011;Cheeseman et al, 2006;Cheeseman et al, 2008), suggesting that a major function of Knl1 is to coordinate Bub1 and BubR1 signaling, a notion that has seen significant support from a number of recent studies in yeasts, Caenorhabditis elegans, Drosophila melanogaster and cultured human cells (Desai et al, 2003;Cheeseman et al, 2008;Schittenhelm et al, 2009;Venkei et al, 2012;Varma et al, 2013; reviewed by . Knl1 has also emerged as the central 'switchboard' for Aurora B activity.…”