Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests (original) (raw)
Fig 1
Mad3p phosphorylation.
A. Mad3p modification is Mps1Mph1-dependent. Wild-type and mph1-Δkinase strains expressing Mad3-TAP were arrested in mitosis by overproducing Mad2p (Mad2OP) from pREP1-Mad2. Yeast extracts were made, separated by 2D-PAGE and immunoblotted with peroxidase-conjugated anti-peroxidase antibody to detect Mad3-TAP isoforms. B. Mps1Mph1 kinase phosphorylates Mad3p in vitro. Mps1Mph1-SZZ and Mps1Mph1kinase-dead-SZZ were purified from yeast extracts then used to phosphorylate Mad3-MBP or MBP. C. Mad3p phosphorylation sites. Mad3p schematic, highlighting KEN boxes, the TPR region, and positions of the N9A and C9A alleles. Mass spectrometry identified many sites in Mad3p: those sites identified both in the in vitro Mps1Mph1 kinase assay and in vivo are highlighted in red. The 16th in vitro Mps1Mph1 site (T195) is not labelled on this schematic. There is a putative Ark1 site in green (T82) and CDK site (S289) in blue. Alignments of Mad3p KEN box regions from the four sequenced fission yeasts (Sp Schizosaccharomyces pombe, Soct Schizosaccharomyces octosporus, Sjap Schizosaccharomyces japonicus, _Scry Schizosaccharomyces cryophilus–_sequences available from the Broad Institute, USA) highlighting clusters of phosphorylation sites flanking the KEN boxes. D. Mps1Mph1 kinase phosphorylates Mad3p alanine mutants at reduced levels in vitro. The indicated recombinant Mad3-MBP fusion proteins were purified from bacteria and used as substrates in an Mps1Mph1-SZZ kinase assay. The kinase assay was performed 4 times, a representative coomassie gel and autoradiograph are shown here, and their quantitation plotted (mean with SEM).