Table 1.
Concentrations of the protein components in various phosphorylation assays.
Fig 1.
Preparation of recombinant cyclin B-Cdk1 complexes.
(A) cDNA fragments encoding engineered versions of Xenopus tropicalis Cdk1 and cyclin B were cloned into a plasmid vector. The DNA segment depicted in the cartoon was transposed into a single baculovirus vector, and the two polypeptides were simultaneously expressed in insect cells. (B) A lysate was prepared from the virus-infected cells and fractionated using a Ni2+-charged column. The loaded sample (L), the flow-through (F), and the eluates were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). (C) Eluates from the Ni2+-charged column were further fractionated using a Strep-Tactin-conjugated column. Samples were analyzed by SDS-PAGE followed by CBB staining (top). Selected samples were also analyzed by immunoblotting with the indicated antibodies (bottom). (D) The cyclin B-Cdk1 complex purified by tandem affinity chromatography was analyzed by size-exclusion chromatography (SEC). Its elution profile monitored by absorbance at 280 nm is shown. (E) The final preparation of X. tropicalis M-CDK was analyzed by SDS-PAGE followed by CBB staining (top). The same set of samples was analyzed by fluorescence immunoblotting (bottom) with anti-pT161 (to visualize an active form of Cdk1) and anti-PSTAIR (to visualize both active and inactive form of Cdk1). (F) The final preparation of human M-CDK was analyzed by SDS-PAGE. (G) X. tropicalis and human M-CDKs were analyzed side-by-side by immunoblotting with the indicated antibodies.
Fig 2.
Recombinant M-CDKs phosphorylate a substrate containing SP motifs.
(A) The Xenopus laevis linker histone variant H1.1 contains five SP and no TP motifs whereas another variant H1.8 contains neither SP nor TP motifs. (B) Recombinant H1.1 and H1.8 were incubated with X. tropicalis M-CDK and ATP at 25°C. At the indicated time points, the reactions were terminated and analyzed by SDS-PAGE. The gel was first stained with Pro-Q Diamond solution to visualize phosphorylated proteins and then stained with CBB to visualize total proteins. (C) An H1.1 phosphorylation assay was performed in the absence or presence of the Cdk1-specific inhibitor Ro-3306. After a 60-min incubation, the reactions were analyzed as above. (D) H1.1 was phosphorylated with a relatively low concentration of M-CDK (see Table 1). At the indicated time points, the reactions were analyzed by Phos-tag SDS-PAGE to separate proteins phosphorylated at different levels into discrete bands. (E) Kinetics (left) and rate (right) of H1.1 phosphorylation were quantified. The mean ± standard errors from three independent experiments are shown. (F) Increasing concentrations of H1.1 were phosphorylated with a fixed concentration of M-CDK. The initial velocities of phosphorylation in each reaction were plotted against the concentrations of SP sites (equivalent to five times the concentration of H1.1) and fitted to the Michalis-Menten equation (which is indicated by a regression curve). The estimated kinetic parameters are shown in Table 2. (G) H1.1 was phosphorylated with increasing concentrations of X. tropicalis and human M-CDKs. After a 10-min incubation, the reactions were terminated and analyzed by Phos-tag SDS-PAGE.
Table 2.
Kinetic parameters of X. tropicalis M-CDK and their comparison with previous studies.
Fig 3.
Suc1 accelerates M-CDK phosphorylation of a substrate containing multiple TP motifs.
(A) Primary structure of the C-terminal region of XCAP-D2 (amino acids 1285–1364, XD2-C) that was expressed in E. coli as a fusion with maltose-binding protein (MBP). XD2-C contains three TP motifs (shown in red). In XD2-C 3A mutant, all threonine residues in these motifs were replaced with alanines. (B) XD2-Cs (wild-type [WT] and 3A) were incubated with M-CDK and ATP. Aliquots were taken at the indicated time points and subjected to Phos-tag SDS-PAGE followed by CBB staining. The asterisk (*) indicates a degradation product of XD2-C. (C) Selected reactions from an XD2-C phosphorylation assay (shown in panel B) were subjected to Phos-tag SDS-PAGE followed by immunoblotting. (D) XD2-C was phosphorylated by M-CDK in the presence of different concentrations of Suc1 and analyzed as described in (B). (E) Kinetics (left) and rate (right) of XD2-C phosphorylation were quantified. The mean ± standard errors from three independent experiments are shown.
Fig 4.
Multi-site phosphorylation of the condensin I holocomplex can be recapitulated by M-CDK and Suc1.
(A and B) A recombinant Xenopus laevis condensin I holocomplex was phosphorylated by M-CDK with or without Suc1. The reaction mixtures were analyzed by SDS-PAGE. To visualize phosphorylated proteins, the gel was stained with Pro-Q Diamond solution (A). Mobility shifts of the XCAP-C and -H subunits and the generation of mitosis-specific phospho-epitopes (pT1314 and pT1353) on the XCAP-D2 subunit were examined by immunoblotting (B). (C) A recombinant mammalian condensin I holocomplex was phosphorylated by M-CDK with or without Suc1. The reaction mixtures were analyzed by immunoblotting with antibodies raised against mitosis-specific phospho-epitopes (pT1339 and pT1384) on the hCAP-D2 subunit.