Extended Data Figure 1: PP1 phosphorylation by Cdk1–cyclin B on T316 represses activity and reduces abundance in a manner that is mimicked by mutation of the target site to an acidic residue in PP1Dis2.T316D. | Nature

Extended Data Figure 1: PP1 phosphorylation by Cdk1–cyclin B on T316 represses activity and reduces abundance in a manner that is mimicked by mutation of the target site to an acidic residue in PP1Dis2.T316D.

From: A PP1–PP2A phosphatase relay controls mitotic progression

Extended Data Figure 1

a, Clustal W alignments of the C-terminal residues of the indicated PP1 isoforms. The Cdk1–cyclin B phosphorylation site is highlighted by red shading. Red letters within this region of shading highlight the clear deviation from this consensus in the S. pombe PP1Sds21 isoform. PP1Sds21 is unable to bind to either B55Pab1 or B56Par1. The molecular basis for this inability of PP1Sds21 to bind the regulatory subunits of PP2A remains to be established. The initial threonine in the S. pombe PP1Dis2 shaded consensus sequence region is T316. b, c, Phosphatase assays of the indicated samples with activity detection by either EnzChek (b) or a second established PP1 activity assay34 histone H3 serine 10 dephosphorylation (c) . For each assay the graphs show arbitrary units. Error bars show s.d. Identical results were obtained with each assay. The similarity between the recombinant rabbit PP1γ and potato acid phosphatase (PAP) activities indicate that both reactions measure PP1 activity. The inhibition of the activity of PP1Dis2 precipitates by 1 μM, but not 10 nM okadaic acid (OA), suggests that it is the PP1Dis2 activity rather than any co-precipitating PP2A activity that is being monitored in these assays. This view is consolidated by the inhibition of activity in both assays by the PP1-specific inhibitor NIPP1 (2, 5, 10 and 15 nM; c and Fig. 1a). 5 nM is routinely used to inhibit mammalian PP1 (refs 34, 39). Manufacturer’s instructions were followed for EnzChek assays. For histone H3 serine 10 assays, histone H3 that had been phosphorylated by auroraArk1 kinase35 was used as a substrate. The dephosphorylation reaction was conducted in 20 mM HEPES, 100 mM NaCl, 1 mM EDTA, 0.1% NP40 at 30 °C for 1 h. The molarity of NaCl indicates the salt concentration of the buffer used for the immunoprecipitation, not the molarity within the phosphatase reaction. All phosphatase reactions for each substrate were conducted under identical conditions. A 1.2 M NaCl, buffer was used to isolate PP1Dis2 for the experiments in Fig. 3c, d and Extended Data Fig. 6d, g, h. The level of PP1 activity detected in either assay shown in b and c was the same irrespective of the NaCl concentration in the buffer used to isolate PP1Dis2. Phos-Stop is a universal phosphatase inhibitor. The key conclusion from all these assays is that the PP1Dis2.T316D protein has a similar level of activity as the Cdk1–cyclin B phosphorylated enzyme and so behaves as a ‘genetically inhibited’ phosphatase in the subsequent experiments described in the manuscript. d, Validation of the T316Phos antibody. PP1Dis2 immunoprecipitates from the indicated strains were treated as indicated and probed with either PP1Dis2 or T316Phos polyclonal antibodies. eg, Cultures synchronized with respect to cell cycle progression by size selection through elutrient centrifugation were probed with the PP1Dis2 antibodies at the high dilution that revealed the fluctuations in PP1Dis2 levels in Fig. 1c. Graphs show the septation index alongside the PP1Dis2 levels in each sample normalized to the level of the α-tubulin loading control and a sample from an asynchronous culture that was run on each gel. e, Samples from this Rpn12mts3.1 culture were split into three immediately after elution and one-third was maintained at the permissive temperature of 25 °C for the remainder of the experiment, another third was immediately shifted to 36 °C to inactivate this essential component of the proteasome 26S subunit40, while the inactivating temperature shift of the remaining third was done 200 min later, after cells had completed one round of division at 25 °C. Note that the mitotic decline in PP1Dis2 levels at the permissive temperature as cells progress from mitotic commitment to the metaphase anaphase transition is not seen at 36 °C when the proteasome function is inactivated, despite the fact that the chromosome condensation index indicates that over 60% of cells have arrested cell cycle progression with a metaphase spindle. f, g, PP1Dis2 levels fail to oscillate when the Cdk1–cyclin B phosphorylation site at position T316 is mutated. A switch to the phospho-mimetic glutamic acid results in persistently low protein levels (f) while levels are persistently high upon mutation to alanine to block phosphorylation (g). Because these levels remain constant throughout the cell cycle these normalized levels have been superimposed upon the data from a wild-type culture in Fig. 1c to show each level relative to the oscillating wild-type protein levels. The septation profile in Fig. 1c is the profile for that wild-type culture. The septation profile in Fig. 1c does not contain any data from either of the cultures shown here in Extended Data Fig. 1f, g. The septation profiles for each mutant culture are shown in f and g. Biological replicates: for b and c, n = 3; for d, n = 2; for eg, n = 1.

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