Dual Inhibition of Sister Chromatid Separation at Metaphase

Abstract

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins

“…Figure 7b shows a product ion spectrum for the same concentration of phosphopeptide with a 1.5 s accumulation time in Q2 and a total acquisition time of 2 s. Remarkably, the Q2 CAD MS 2 spectra for this particular peptide exhibited many fragment ions bearing the intact allyl phosphoester and, thus, allowed unambiguous identification of the phosphorylation site. This phenomenon has also been observed for this peptide in a 3-D ion°trap°and°appears°to°be°sequence-specific° [48].…”

Anaphase-promoting complex-dependent proteolysis of cell cycle regulators and genomic instability of cancer cells

“…Expression of a nondegradable form of cyclin B1 can lead to an anaphase-like arrest in mammalian cells unable to undergo cytokinesis (Wheatley et al, 1997). However, recent results indicate that a nondegradable form of cyclin B1 may block already the metaphase to anaphase transition in human cells (Chang et al, 2003) and frog eggs (Stemmann et al, 2001). The exact phenotype depends on the amount of nondegradable cyclin B1 expressed in the cell (Stemmann et al, 2001;Hagting et al, 2002;Chang et al, 2003).…”

“…However, recent results indicate that a nondegradable form of cyclin B1 may block already the metaphase to anaphase transition in human cells (Chang et al, 2003) and frog eggs (Stemmann et al, 2001). The exact phenotype depends on the amount of nondegradable cyclin B1 expressed in the cell (Stemmann et al, 2001;Hagting et al, 2002;Chang et al, 2003). Similarly, as in yeast (Wa¨sch and Cross, 2002), more experiments with expression of the nondestructible cyclin B1 (as well as cyclin A2) from the endogenous promoter, mimicking more closely endogenous expression levels and promoter regulation, might clarify the role of cyclin B1 (cyclin A2) destruction in human cells more definitely.…”

High throughput genomic and proteomic technologies in the fight against infectious diseases

“…Furthermore, methods enabling quantitative analysis of proteomes by mass spectrometry have been developed. Among them, two main approaches can be distinguished, namely stable isotope labeling (including ICAT [20], iTRAQ [21], SILAC [22], AQUA [23]) and label-free quantitation (including spectral counting and peak intensity/area quantitation) [24].…”