SummaryThe widespread reorganisation of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, Protein Phosphatase 1 (PP1) and Protein Phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes1-6. We now describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 to the regulatory subunits of PP2A-B55 and PP2A-B56 holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because Cdk1-Cyclin B inhibits PP1 activity but declining Cyclin B levels later in mitosis permit PP1 to auto-reactivate1,7-10. PP1 first reactivates PP2A-B55; this enables PP2A-B55, in turn, to promote the reactivation of PP2A-B56 by dephosphorylating a PP1 docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic, PP2A holoenzymes and the sequences of these conserved PP1 docking motifs11,12 suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.

Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites of phosphate addition. An approach to selective mapping of protein phosphorylation sites on a specific target protein of interest using LC-MS is described here. In this approach multiple reaction monitoring is used as an extremely sensitive MS survey scan for potential phosphopeptides from a known protein. This is automatically followed by peptide sequencing and subsequent location of the phosphorylation site; both of these steps occur in a single LC-MS run, providing greater efficiency of sample use. The method is capable of detecting and sequencing phosphopeptides at low femtomole levels with high selectivity. As proof of the value of this approach in an experimental setting, a key Schizosaccharomyces pombe cell cycle regulatory protein, Cyclin B, was purified, and associated proteins were identified. Phosphorylation sites on these proteins were located. The technique, which we have called multiple reaction monitoring-initiated detection and sequencing (MIDAS), is shown to be a highly sensitive approach to the determination of protein phosphorylation.

Mitotic exit in

Activation of mitosis promoting factor (MPF) drives mitotic commitment1. In human cells active MPF appears first on centrosomes2. We show that local activation of MPF on the equivalent organelle of fission yeast, the spindle pole body (SPB), promotes Polo kinase activity at the SPBs long before global MPF activation drives mitotic commitment. Artificially promoting MPF or Polo activity at various locations revealed that this local control of Plo1 activity on G2 phase SPBs dictates the timing of mitotic commitment. Cytokinesis of the rod shaped fission yeast cell generates a naïve “new” cell end. Growth is restricted to the experienced old end until a point in G2 phase called “New End Take Off” (NETO) when bipolar growth is triggered3. NETO coincided with MPF activation of Plo1 on G2 phase SPBs4. Both MPF and Polo activities were required for NETO and both induced NETO when ectopically activated at interphase SPBs. NETO promotion by MPF required polo. Thus, local MPF activation on G2 SPBs directs polo kinase to control at least two distinct and temporally separated, cell cycle transitions at remote locations.

The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and a screen for molecules that antagonized EB1 function. The conditional peg1.1 mutant enabled us to identify key features of Peg1 function. First, Peg1 was required to form a spindle and astral microtubules, yet destabilized interphase microtubules. Second, Peg1 was required to slow the polymerization rate of interphase microtubules that establish end-on contact with the cortex at cell tips. Third, Peg1 antagonized the action of S. pombe CLIP170 (Tip1) and EB1 (Mal3). Fourth, although Peg1 resembled higher eukaryotic CLASPs by physically associating with both Mal3 and Tip1, neither Tip1 nor Mal3 was required for Peg1 to destabilize interphase microtubules or for it to associate with microtubules. Conversely, neither Mal3 nor Tip1 required Peg1 to associate with microtubules or cell tips. Consistently, while mal3.⌬ and tip1.⌬ disrupted linear growth, corrupting peg1 + did not. Fifth, peg1.1 phenotypes resembled those arising from deletion of the single heavy or both light chains of fission yeast dynein. Furthermore, all interphase phenotypes arising from peg1 + manipulation relied on dynein function. Thus, the impact of S. pombe CLASP on interphase microtubule behavior is more closely aligned to dynein than EB1 or CLIP170.[Keywords: CLASP; dynein; S. pombe; fission yeast; CLIP170; EB1] Supplemental material is available at http://www.genesdev.org.

The Aspergillus nidulans protein kinase NIMA regulates mitotic commitment, while the human and Xenopus equivalents in¯uence centrosome function. Two recessive, temperature-sensitive mutations in the Schizosaccharomyces pombe NIMA homologue, Fin1, blocked spindle formation at 37°C. One of the two spindle pole bodies (SPBs) failed to nucleate microtubules. This phenotype was reduced by accelerating mitotic commitment through genetic inhibition of Wee1 or activation of either Cdc25 or Cdc2. Polo kinase (Plo1) normally associates with the SPB of mitotic, but not interphase cells. cut12.s11 is a dominant mutation in an SPB component that both suppresses cdc25 mutants and promotes Plo1 association with the interphase SPB. Both cut12.s11 phenotypes were abolished by removing Fin1 function. Elevating Fin1 levels promoted Plo1 recruitment to the interphase SPB of wild-type cells and reduced the severity of the cdc25.22 phenotype. These data are consistent with Fin1 regulating Plo1 function during mitotic commitment. The ®n1 mitotic commitment and spindle phenotypes resemble distinct nimA phenotypes in different systems and suggest that the function of this family of kinases may be conserved across species.

Insertion into and release of the cytoplasmic domain of the Schizosaccharomyces pombe spindle pole body from a nuclear envelope fenestra during mitosis requires Brr6.

PP1 recruitment to Cut12 sets a threshold for Polo's feedback-loop activity that locks the cell in interphase until Cdc25 pushes MPF activity through this barrier to initiate mitosis. We propose that events on the SPB (and, by inference, the centrosome) integrate inputs from diverse signaling networks to generate a coherent decision to divide that is appropriate for the particular environmental context of each cell. PP1 recruitment sets one or more critical thresholds for single or multiple local events within this switch.