Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro

Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.


Introduction
For the accurate transmission of genomic information in eukaryotic cells, multiple mitotic events, such as nuclear envelope breakdown, chromosome condensation, and spindle assembly, must be completed in a tightly regulated manner.Such dynamic rearrangements of intracellular structures are under the control of cyclin-dependent kinase 1 (Cdk1), which is complexed with cyclin B [1,2].A rapid increase in Cdk1's activity from mitotic prophase to metaphase results in large-scale phosphorylation of a variety of protein substrates, often at multiple sites on individual substrates [3].
The activity of cyclin B-Cdk1 is regulated by multilayered mechanisms.By late G2 phase, cyclin B-Cdk1 is maintained in an inactive form in which the T14 and Y15 residues of Cdk1 are phosphorylated by the kinases Wee1 and Myt1, respectively.Upon entry into mitosis, these inhibitory modifications are removed by the protein phosphatase Cdc25, thereby converting cyclin B-Cdk1 into an active form [4,5].A CDK-activating kinase (CAK) is also responsible for full activation of cyclinB-Cdk1 by phosphorylating T161 in the T-loop of Cdk1, which is located close to its catalytic center [6][7][8].Finally, Cks1, a small adaptor protein that bridges Cdk1 and phosphorylated threonine-containing substrates, is known to accelerate multi-site phosphorylation reactions observed in vivo [3,[9][10][11][12].
Although the cell cycle regulation of Cdk1 has been extensively studied over the past decades, much less is known about how the structure and functions of Cdk1's substrates might be regulated by phosphorylation.To address this fundamental question, there is a high demand for technically simple purification of a pure and active fraction of cyclin B-Cdk1 and for recapitulation of physiologically relevant, multi-site phosphorylation reactions in vitro.Several protocols for the preparation of native and recombinant versions of cyclin B-Cdk1 have been published so far [8,[13][14][15][16][17][18][19][20][21].However, they are not necessarily technically straightforward, and the starting materials, if they are of native origin, may not be readily available to many researchers.In this paper, we report a modified protocol for the production of recombinant cyclin B-Cdk1 that can efficiently and specifically phosphorylate cyclin-dependent kinase consensus motifs (SP and TP sites) of substrates [22].Furthermore, our in vitro assays demonstrate that mitosis-specific multi-site phosphorylation of the condensin I complex, an essential player in chromosome condensation [23][24][25], can be recapitulated using recombinant cyclin B-Cdk1 and Suc1, a fission yeast homolog of Cks1 [26].

Preparation of recombinant X. tropicalis and human M-CDKs
A cDNA fragment for X. tropicalis Cdk1 carrying T14A/Y15F mutations and flanked with a Cterminal hexahistidine (His) tag was codon-optimized for Trichoplusia ni and synthesized using commercial services provided by Eurofins Genomics.The fragment for X. tropicalis cyclin B1 (amino acids 131-397) carrying C133S/C142S/C316S mutations and flanked with an N-terminal 3×FLAG tag and a C-terminal Twin-Strep (TS) tag was synthesized in the same way.These fragments are individually cloned into between a polyhedrin promotor and terminator sequences on the plasmid vector pLIB (Addgene, Catalog # 80610) to create the gene expression cassettes (GECs).Next, a pair of the GECs are cloned into a pBIG1D vector (Addgene, 80614) according to the biGBac assembly methods [27,28].Using the resultant vector, the Escherichia coli strain DH10EMBacY (Geneva Biotech) was transformed to generate a recombinant bacmid DNA via glycerol) supplemented with 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), and concentrated with an Amicon Ultra 30K-device (Merck, UFC503024).The concentrated protein sample was dispensed into small aliquots, snap-frozen in liquid nitrogen, and stored at -80ºC until use.Once an aliquot was thawed, it was stored on ice and used within 2 weeks.To evaluate the homogeneity of every preparation, an aliquot was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining.The concentration of the final preparation of M-CDK was determined by measuring the absorbance at 280 nm.For an analytical purpose, the M-CDK preparation purified from a Strep-Tactin-conjugated column was further fractionated by sizeexclusion chromatography (SEC) using a Superose 6 increase 10/300 GL column (Cytiva, 29091596).To validate the presence of pT161-positive Cdk1 in the final preparation, immunoblotting was carried out using anti-pT161 and anti-PSTAIR antibodies, and images were acquired by using an Image analyzer (Odyssey XF, LI-COR Biosciences [for fluorescence detection] or Amersham Imager 680, Cytiva [for chemiluminescence detection]).Human M-CDK was expressed and purified using almost the same protocol.The only exception was that the pFastBac Dual vector (Thermo Fisher Scientific, 10712024) was used instead of pBIG1D.A yield of the cyclin B-Cdk1 complex from 1 g insect cells was as follows: X. tropicalis M-CDK, 10~30 µg at the concentration of 1~2 µM; human M-CDK, 50~100 µg at the concentration of 2~10 µM.

Preparation of recombinant proteins other than M-CDK
Linker histones.cDNAs encoding of X. laevis H1.1 and H1.8 (provided by Kiyoe Ura [Chiba University, Japan] and Keita Ohsumi [Nagoya University, Japan], respectively) were cloned into a pET28-3C vector (a lab-made derivative of pET28a [Novagen,69864], in which the original thrombin protease recognition site was replaced with the 3C protease recognition site).Both H1.1 and H1.8 were expressed and purified by the same procedure.The plasmid DNA was introduced into the E. coli strain BL21 (DE3), and the transformant was grown in LB at 37ºC until OD600 reached 0.8.IPTG was then added at a final concentration of 200 µM.After an 1-hr incubation at 37ºC, the culture was transferred to a temperature of 20ºC, and incubation was continued for another 16 hr to allow the expression of the recombinant proteins.The cells were harvested, suspended in buffer HisL supplemented with EDTA-free Complete Protease Inhibitor Cocktail (Roche), and lysed by sonication.The lysate was clarified by centrifugation and loaded on a Ni 2+ -charged Chelating Sepharose Fast Flow column.After washing the column with buffer HisW, the bead-binding proteins were eluted with buffer HisE.The eluate was dialyzed against buffer S100 (20 mM HEPES-NaOH [pH 7.2], 100 mM NaCl) along with lab-made human rhinovirus 3C protease to liberate the His tag and applied to a HiTrap SP HP column (Cytiva, 17115101).The column was developed with a linear gradient of NaCl (100-600 mM).
Peak fractions were pooled and dialyzed against buffer NHG150/10 (20 mM Hepes-NaOH [pH 7.7], 150 mM NaCl, 10% glycerol).Typically, approximately 1 mg of linker histones were obtained from a 1-liter culture and the concentration of stock aliquots ranged from 200 µM to 500 µM.XD2-C.cDNA fragments encoding XD2-C WT and 3A were codon-optimized for E. coli and synthesized (Eurofins Genomics) and cloned into the pMAL-c6T vector (New England Biolabs, N0378).Protein expression in the E. coli host was performed as described above.The resultant XD2-C proteins fused with a His-MBP tandem tag at their N-termini were purified using a Ni 2+charged Chelating Sepharose Fast Flow column.Peak fractions were pooled and dialyzed against buffer KHG150/10.Typically, 300 µg of XD2-C were obtained from a 1-liter culture and the concentration of stock aliquots ranged from 50 µM to 100 µM.

Suc1. A cDNA encoding Schizosaccharomyces pombe Suc1 (provided by Eiichi Okumura
[Tokyo Institute of Technology, Japan]) was cloned into the pET28-3C vector.Protein expression in the E. coli host and coarse purification using a Ni 2+ -charged column were performed as described above.The resultant suc1 fused with a His tag at their N-terminus was purified by using a Ni 2+ -charged Chelating Sepharose Fast Flow column.Peak fractions were pooled, further purified with a HiTrap Q HP column (Cytiva, 17115301), and dialyzed against buffer buffer KHG150/10.Typically, 10 mg protein was obtained from a 1-liter culture and the concentration of stock aliquots ranged from 200 µM to 500 µM.Condensin I holocomplexes.A baculovirus vector for the expression of X. laevis condensin I holocomplex was made according to the biGBac assembly protocol [27,28].Briefly, cDNA fragments encoding five subunits of condensin I (XCAP-C/Smc4, -D2, -E/Smc2, -G, -H [fused with a Twin-Strep tag at its C-terminus]) were codon-optimized for Trichoplusia ni and synthesized (GeneArt, Thermo Fisher Scientific).They were inserted stepwise into intermediate plasmid vectors (namely, pLIB and pBIG1 series vectors) and finally combined into a single pBIG2ABC vector (Addgene, 80617).Baculovirus production and protein expression were carried out as described for M-CDKs.The resultant condensin I holocomplex was first purified with a Strep-tactin-conjugated column and further purified by SEC using a Superose 6 increase column.Peak fractions were pooled and dialyzed against buffer KHG150/10.Typically, 20 µg of the protein complex was obtained from 1 g insect cell pellet at a concentration of 2 µM.A mammalian version of the condensin I holocomplex (composed of mouse Smc2 and Smc4, and human CAP-D2, -G, and -H) was prepared as previously described [29,30].

Phosphorylation assays using recombinant M-CDKs
A protein mixture containing a substrate (linker histone, XD2-C, or condensin I), M-CDK, and Suc1 (in a volume ranging from 30 to 100 µl) was dialyzed against kinase buffer (20 mM HEPES-KOH [pH 7.7], 80 mM KCl and 5 mM MgCl2) using an Xpress Micro Dialyzer MD100 (Scienova, 40078) at 4ºC.The dialysate was supplemented with ATP at a concentration of 2 mM and incubated at 25ºC.The concentrations of protein components in each assay are listed in Table 1.At the indicated time points, aliquots were taken and mixed with an equal volume of 2×SDS sample buffer (125 mM Tris-HCl [pH 6.8], 10% 2-mercaptoethanol, 4% SDS, 20% glycerol, 0.2% Βromophenol blue).The resultant samples were subjected to SDS-PAGE.For analyzing either H1.1 or XD2-C by Phos-tag SDS-PAGE, gels containing 10% or 7.5% acrylamide, 50 µM Phos-tag acrylamide (Fujifilm Wako Pure Chemical, AAL-107), and 100 µM MnCl2 were used, respectively.For visualizing phosphorylated proteins prior to CBB staining, the gels were stained with Pro-Q Diamond phosphoprotein stain gel solution (Thermo Fisher Scientific, P33300) according to the manufacturer's instruction with the exception that 1,2-propanediol was used instead of acetonitrile in the destaining step.After staining the gel, images were acquired using an image analyzer (Amersham Imager 680, Cytiva).Semiquantitative analyses of the phosphorylation on H1.1 or XD2-C by M-CDK were performed as follows: First, average numbers of phosphorylated residues per each substrate molecule were calculated at each time point by quantifying band density in Phos-tag gels and plotted against the time (Fig 2E and 3E, kinetics [left panels]); Next, the slope of the abovementioned kinetics graph for the first 10 minutes was calculated, and the value multiplied by the molar ratio of the substrate to M-CDK was approximated as the phosphorylation rate (Fig 2E and 3E, rate [right panels]).

Material availability
All unique reagents generated in this study are available from the corresponding author on request.

Results and Discussion
Preparation of recombinant cyclin B-Cdk1 complexes expressed in insect cells.We wished to produce a homogeneous and enzymatically active preparation composed of recombinant cyclin B and Cdk1 for the functional and structural characterization of Cdk1's substrates.To this end, we designed a baculoviral vector by taking into account the following points: (1) to stabilize the cyclin B moiety in host insect cells, the N-terminal region of Xenopus tropicalis cyclin B1 (amino acids 1-130), which is required for ubiquitin-mediated proteolysis [33], was deleted, and three non-conserved cysteines (i.e., C133, C142, and C316) were replaced with serines as previously shown [34] We first set up a reaction mixture, in which recombinant H1.1 or H1.8 (10 µM) was mixed with M-CDK (50 nM) along with ATP (2 mM) (Table 1).Aliquots of the reaction mixture were taken at various time points and subjected to conventional SDS-PAGE.In the resulting gel, phosphorylated proteins were visualized with the Pro-Q Diamond solution, and then total proteins were stained with CBB.The result clearly demonstrated that H1.1 was rapidly phosphorylated by purified M-CDK whereas H1.8 was hardly phosphorylated under the same condition (Fig 2B).We also confirmed that the phosphorylation of H1.1 was blocked when the Cdk1-specific inhibitor Ro-3306 was added to the reaction mixture [35] (Fig 2C).
We next investigated the kinetics of H1.1 phosphorylation in more detail by using a Phostag acrylamide-containing gel that magnifies the mobility shift of phosphorylated proteins [36].
When a relatively low concentration of M-CDK (10 nM) was added to the assay (Table 1), phosphorylation of H1.Condensin I is a pentameric protein complex that plays an indispensable role in mitotic chromosome assembly [37,38].A native condensin I comeplex purified from egg extracts has been shown to act as one of the six protein factors required for chromosome reconstitution in vitro, but it does so only when it is phosphorylated by cyclin B-Cdk1 [25].A number of Cdk1 phosphorylation sites in the condensin I subunits have been identified so far.Among them are three threonine residues (T1314, T1348, and T1353) in the C-terminal intrinsically disordered region (IDR) of the XCAP-D2 subunit in X. laevis [24](Fig 3A).We tested whether these residues could be phosphorylated using our recombinant M-CDK.To this end, the C-terminal IDR of XCAP-D2 (referred to as XD2-C WT) was expressed as a polypeptide fused to the maltose-binding protein, purified and incubated with M-CDK as a substrate.We found that M-CDK efficiently phosphorylated this fusion protein, causing mobility shifts on a Phos-tag gel Our recent work has shown that several SP and TP motifs in a recombinant mammalian condensin I complex are phosphorylated in mitotic egg extracts [30].We confirmed that our M-CDK can phosphorylate at least two of them (T1339 and T1353 of human CAP-D2) in vitro , thereby extending our above-mentioned observation using X. laevis condensin I as a substrate.Taken together, it is reasonable to conclude that the multi-site phosphorylation of condensin I subunits observed in mitotic egg extracts can be recapitulated with the recombinant proteins in vitro.

Conclusions and perspectives.
In the current study, we describe a simplified protocol for the production of frog and human versions of the recombinant cyclin B-Cdk1 complex (M-CDK).It should be emphasized that our protocol is superior to previously reported ones in the following three points: (1) the recombinant M-CDKs can be easily and reproducibly purified with a hitherto unprecedented degree of homogeneity by two chromatography steps; (2) the phosphorylation at T161 of Cdk1, which is required for kinase activation, can be achieved in the host cells without the co-expression of exogenous CAK; (3) all procedures are easy to implement for standard molecular biology laboratories, and the expression vectors described in the current study are available upon request.Furthermore, we provide a compelling set of data that the recombinant M-CDKs specifically phosphorylate SP and TP motifs.Importantly, multisubunit and multi-site phosphorylation of the pentameric condensin I complex can be recapitulated by combining recombinant M-CDK with its cofactor Suc1.Thus, recombinant M-CDKs reported in the current study will be of great help in the in-vitro reconstitution of elaborate protein machineries that govern critical processes in mitosis.
;(2) to prevent Cdk1 from receiving inhibitory phosphorylations in the host cells, T14 and Y15 in X. tropicalis Cdk1 were mutated to alanine and phenylalanine, respectively; (3) to maximize the yield and homogeneity of the purified protein complex, these mutant proteins (cyclin B1 (DN, CS) and Cdk1 (AF)) were conjugated with affinity tags (3×FLAG-tag and Twin-Strep [TS]-tag at the N-and C-termini of cyclin B1 and the hexahistidine [His]-tag at the C-terminus of Cdk1), and were expressed from a single vector rather than from a combination of vectors expressing individual proteins[27,28](Fig 1A).From High-Five insect cells infected with the recombinant virus, the cyclin B-Cdk1 complex was successfully purified by tandem affinity chromatography using Ni 2+ and Strep-Tactin columns (Fig 1Band 1C).Size-exclusion chromatography of the eluate from the second column resulted in a single major peak, demonstrating that our two-step purification protocol yielded a protein preparation mainly composed of one molecule each of cyclin B1 (DN, CS) and Cdk1 (AF) (Fig1D).We noticed that Cdk1 in the purified preparation was separated into two discrete bands as judged by SDS-PAGE.Fluorescence immunoblotting analysis, which allowed us to simultaneously detect pan-Cdk1 (recognized by anti-PSTAIR) and its active form (recognized by anti-pT161) in different channels, demonstrated that the faster migrating band corresponds to the active form of Cdk1 (Fig1E).It is reasonable to speculate that endogenous CAK phosphorylates T161 of the recombinant Cdk1 in the host insect cells.We also engineered human cyclin B1 and Cdk1 sequences in a similar strategy and succeeded in producing a homogeneous and pT161-positive recombinant complex (Fig1F and G).We hereafter refer to the purified cyclin B-Cdk1 complex as M-CDK, which stands for mitotic cyclin-dependent kinase, and show the results using X. tropicalis M-CDK unless otherwise indicated.Recombinant M-CDKs phosphorylate a substrate containing SP motifs.To clarify whether the recombinant M-CDKs are enzymatically active and, if so, to what extent, we decided to use two different variants of recombinant Xenopus laevis linker histones as substrates: one was H1.1, which has five SP motifs, and the other was H1.8, which has no SP or TP motifs (Fig 2A).
1 gradually proceeded up to 20 min (Fig 2D).Populations of H1.1 phosphorylated at 1-2 sites were dominant at early time points, whereas multiple (up to five) sites of H1.1 were phosphorylated sites at late points.Taken all together, it is reasonable to speculate that our recombinant M-CDK catalyzes phosphorylation selectively at the SP sites of H1.1 that match the cyclin-dependent kinase consensus sites [22].Semi-quantitative estimation showed that each M-CDK molecule catalyzes the phosphorylation of H1.1 at a rate of 394 ± 13 [phosphate/min] (Fig 2E).We also confirmed that our preparation of human M-CDK could phosphorylate H1.1 and that its activity was roughly equivalent to that of X. tropicalis M-CDK (Fig 2F).

(
Fig 3B) and generating phosphoepitopes (pT1314 and pT1353) as judged by immunoblotting (Fig 3C).In striking contrast, virtually no phosphorylation was detectable when a mutant fusion protein (XD2-C 3A), in which all three threonines (T1314, T1348, and T1353) were substituted with alanines, was used as a substrate (Fig3B).These results convincingly demonstrated that the three TP motifs in XD2-C WT are the major targets of M-CDK.The Cks1 family proteins are known to guide a substrate containing previously phosphorylated threonines to the active site of Cdk1, thereby promoting phosphorylation of other proximal sites[9].We next tested whether Suc1, a fission yeast homolog of Cks1[26], could facilitate the multi-site phosphorylation of XD2-C and, if so, to what extent the reaction was accelerated.We found that Suc1 indeed accelerated the phosphorylation of XD2-C in a dose-dependent manner (Fig 3D).Quantitation of Phos-tag SDS-PAGE revealed that Suc1 increased the rate of XD2-C phosphorylation approximately 2.4-fold under the assay conditions tested (Fig 3E).Multi-site phosphorylation of the condensin I holocomplex can be recapitulated by M-CDK and Suc1.We next sought to recapitulate multi-subunit and multi-site phosphorylation of the condensin I holocomplex by M-CDK in vitro.To this end, a recombinant holocomplex composed of X. laevis condensin I subunits was expressed in insect cells, purified, and incubated with M-CDK and ATP.The resulting phosphorylation of the condensin I subunits was analyzed by SDS-PAGE, followed by Pro-Q diamond staining.We found that M-CDK alone phosphorylated the XCAP-C, -D2, and -H subunits to a modest level.Remarkably, inclusion of Suc1 in the reaction mixture resulted in much more efficient phosphorylation of the same set of subunits (Fig4A).Notably, only in the presence of Suc1, XCAP-H displayed a massive mobility shift (Fig 4Aand 4B), a phenomenon that had also been observed in mitotic egg extracts[23].Moreover, Suc1 boosted the phosphorylation at T1314 and T1353 of XCAP-D2 in the holocomplex (Fig 4B), as well as that of the XD2-C substrate (Fig3D and E).

Fig 3 .
Fig 3. Suc1 accelerates M-CDK phosphorylation of a substrate containing multiple TP

Fig 4 .
Fig 4. Multi-site phosphorylation of the condensin I holocomplex can be recapitulated by Fig 1