The INCENP Coil is a Single Alpha Helical ( SAH ) Domain that Binds Directly to Microtubules and is Important for CPC Localization and Function in Mitosis

The chromosome passenger complex (CPC) is a master regulator of mitosis. INCENP acts as a scaffold regulating CPC localisation and activity. During early mitosis the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN-box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled-coil domain acting as a spacer between the N and C terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ~32 nm long single alpha helical (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ~80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible dogleash allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via a SAH domain, the CPC avoids a need for dimerization (required for coiled-coil formation), which would greatly complicate regulation of the proximityinduced trans-phosphorylation that is critical for Aurora B activation. JBC C o n f i d e n t i a l INCENP SAH domain regulates the localisation of CPC complex 2 INCENP is the scaffolding protein upon which the chromosomal passenger complex (CPC) assembles (1-3). The Nterminal region of INCENP assembles a threehelix bundle with survivin and borealin(4) that contributes to targeting the CPC to inner centromeres via haspin-mediated phosphorylation of histone H3 (5-7). This region of INCENP also contributes to CPC localisation by binding to HP1 and to microtubules. The IN-box, a conserved motif near the C-terminus of INCENP is responsible for binding and activating Aurora B kinase (3,8). The central portion of INCENP is predicted to form a coiled-coil spacer between the localisation and activation modules (3), and has been proposed to act like a “dog leash” allowing Aurora B tethered to chromatin to phosphorylate substrates within a constrained region (9). This tethering of Aurora B is critical for the regulation of chromosome alignment and the correction of kinetochore attachment errors. Spindle tension causes the elastic chromatin of the inner centromere to stretch, allowing sister kinetochores to move away from the inner centromere, where INCENP is concentrated during prometaphase and metaphase. As first proposed for budding yeast and later confirmed in mammalian cells, this centromere stretch moves kinetochore targets of Aurora B away from regions of high kinase concentration and decreases their phosphorylation, thereby stabilising kinetochore-microtubule interactions(10-12). In mis-attached chromosomes, which do not exhibit comparable centromere-stretch, Aurora B remains in closer proximity to the outer kinetochore. This allows the kinase to phosphorylate key kinetochore components, causing them to release microtubules (13-15). This correction mechanism is now well accepted, but what is less clear is how exactly INCENP achieves the dynamic flexibility to allow Aurora B to extend into the outer kinetochore and to track with kinetochore components at the dynamic-microtubule interface. For example, since the Ndc80 and Ska complexes are composed of relatively rigid helical bundles (16,17), it is not clear how Aurora B is able to associate with them if they undergo conformational changes on the dynamically growing and shrinking kinetochore-associated microtubules. Here we show that the central region of INCENP is not a coiled-coil, but instead is a single alpha-helix (SAH), similar to that found in myosin 10 and many other proteins (18-21). The N-terminal portion of this SAH is capable of binding directly to microtubules. Furthermore, SAH domains are highly extensible, and by analogy with the myosin SAH domain (20), it is likely that extension of the relatively lengthy INCENP SAH might allow the IN-Box with its bound Aurora B to undergo excursions of up to ~80 nm under relatively light loads. These data support the suggestion that the INCENP coil functions as a “dog leash” that allows Aurora B to “wander” across a substantial target area to reach its substrates (9). By using a SAH rather than a coiled-coil to achieve this flexibility, INCENP avoids the requirement for dimerization, which would significantly complicate the currently accepted mechanism of proximity/clustering-induced activation of the CPC (12,22-24). EXPERIMENTAL PROCEDURES Protein expression and purificationAll proteins were expressed in E. coli BL21 Rosetta 2 (Novagen) and purified using a NiNTA affinity chromatography column. Proteins were dialysed against 150 mM NaCl, 20 mM Tris, 1 mM DTT, pH 8.0 and proteolysed for 2 h at room temperature, using ULP1 recombinant SUMO protease in a substrate to enzyme ratio 100:1. Next, proteins were purified on ion-exchange columns using an AKTA system. The purest fractions were combined and concentrated resulting in a 1–2 mg/ml protein solution. Purified protein was dialyzed against 100 mM NaCl, 10 mM JBC C o n f i d e n t i a l INCENP SAH domain regulates the localisation of CPC complex 3 sodium phosphate, pH 7.4, and snap-frozen in liquid nitrogen for long term storage at -80 °C. Mass spectrometry-Protein samples (~0.2 ml; 20 μM) were dialyzed (GBiosciences dialyzers 2 kDa mwco) overnight against 50 mM ammonium acetate, pH 7.4 and analyzed by TOF MS analysis (The University of Leeds Mass Spectrometry Facility). CD spectroscopyCD measurements were performed on an Applied Photo Physics Chirascan CD spectropolarimeter with a 0.1 cm path length quartz cuvette in 0.1M NaCl, 10 mM sodium phosphate, pH 7.4 buffer. Data were collected every 1 nm with 30 s averaging time, each measurement being an average of two repeated scans. Data presented are averaged from at least two separate measurements of different protein preps. Thermal measurements were performed in a temperature range from 10 to 85 °C with a 0.7 °C/min heating rate, data acquisition every 1 °C and 20 s averaging time. The sample cooling rate prior to measurement of refolded protein was ~ 2 °C/min. The mean residue molar ellipticity of proteins was calculated as described (25). The helical content of proteins was calculated from values of the amide nπ* transition at 222 nm ([MRE222]), as previously described (25). Protein concentration was measured by absorption at 280 nm. Absorption coefficients were obtained from ProtParam software. Standard concentrations were in the range 10–20 μM. In the salt dependence experiments, stock buffer (5 M NaCl, 10 mM sodium phosphate, pH 7.4) was mixed with stock protein solution to obtain desired salt and protein (10 μM) concentration. cDNA constructsSequences encoding putative full length INCENP SAH domain (UniProt id: P53352, Gallus gallus, residues: 503–715) and its N-terminal (residues 503597) and C-terminal (598-715) fragments were subcloned into the pET28a SUMO vector (26) to introduce an N-terminal His-tag and SUMO protein for increased expression and solubility. For all constructs, a tryptophan residue was added to the C-terminus to enable A280 concentration measurements. INCENP SAH mutant constructs were based on Triple affinity purification (TrAP)–tagged INCENP class I under control of an SV40 promoter that is insensitive to doxycycline repression (27). The TrAP tag incorporates His, streptavidin-binding peptide (SBP), and S tags and can be monitored by immunoblotting and immunofluorescence using a monoclonal antibody recognizing the SBP tag (28,29). GFP was inserted in front of the TrAP tag to visualize the mutants. Silent mutations were introduced into INCENP cDNA to create Bam HI, Eco RI, Hind III sites around SAH domain so that SAH region can be easily modified. Wild type SAH, half SAH, double SAH, double MyoM SAH (30) cassettes were synthesized at Geneart (Life technologies) and cloned into the GFP-TrAP-INCENP constructs. Cell cultureDT40 cells were grown in RPMI 1640 medium supplemented with 10% FBS, 1% chicken serum, and maintained in 5% CO2 at 39°C. Doxycycline at a final concentration of 500 ng/ml was added to the culture medium to repress transcription of the promoter-hijacked endogenous INCENP locus (29). HeLa Kyoto were grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% foetal calf serum, 0.2 mM l-Glutamine, 100 U /ml penicillin and 100 μg/ml streptomycin. ImmunoblottingWhole cell lysates were prepared, and the equivalent to 0.51x10 cells were loaded onto a polyacrylamide gel. SDS-PAGE and immunoblotting were performed following standard procedures. Donkey anti mouse or rabbit IRdye 800CW were used for Li-Cor Odyssey Quantitative fluorescence Imager analysis. Indirect immunofluorescence microscopyAll fixation, permeabilisation and immunostaining were performed at room temperature, as previously described (31). JBC C o n f i d e n t i a l INCENP SAH domain regulates the localisation of CPC complex 4 Cells attached on poly-Lysine coated coverslips were fixed in a 3.7% formaldehyde/PBS solution for 10 min and permeabilised in PBS-0.15% Triton X-100 for 4 min. Cells were blocked in 10% normal donkey serum for 1hr at RT prior to antibody incubations. Antibodies used were α-tubulin antibody (B512 or DMIA; Sigma-Aldrich), anti-H3S10ph (Millipore), anti-GFP (Life Science technologies), anti-HEC1 mouse monoclonal (Abcam), anti-Dsn1ph (32), antiH3Ser28ph (33), rabbit polyclonal (WCE1186), anti-INCENP (3D3), anti-Aurora B, anti-CENP-T were previously described (1,27,34), All affinity purified donkey secondary antibodies (labelled ei