Supplementary Figure 1:

Serine 402 of Plo1 is phosphorylated in response to heat but not oxidative or osmotic stresses.

(a) A whole cell extract from cells that had been shifted from 25°C to 36°C for 90 minutes probed with the antibodies raised to phospho-peptides in which serine 402 was phosphorylated recognize a single band in western blots (WCE – whole cell extract, M – molecular weight marker). These antibodies are used throughout the study.

(b-e) Early-log phase wild type (b,c), sty1::ura4+ (d) cells which had been grown at 25°C in supplemented EMM2 were either shifted to 37°C (b,c) or had 1M sorbitol or 25mM H202 added to them (d) before samples were removed at the indicated times (minutes). (b) The shifted extract was blotted with anti-PS402 antibodies alone (left), polyclonal anti-Plo1 antibodies alone (right) or a mixture of the two (middle) to show that, consistent with the phosphorylation event, the band that is phosphorylated on serine 402 migrates much more slowly than the majority of Plo1. For c and d samples were processed for western blotting with S402P, HN184 anti-Plo1, TAT1 anti--tubulin and PN24 anti-Cdc2 antibodies as indicated. The lane marked wt in d is loaded with wild type extracts from pre (left) and post (right) heat shock to indicate that the distinction between sty1::ura4+and wild type is not a processing artifact.

Supplementary Figure 2:

Plo1 Serine 402 is phosphorylated during recovery from heat shock as it associates with Spc1/Sty1 after stimulation of this MAP kinase has peaked.

(a) Small G2 cells were isolated from a wild type culture grown at 25°C in supplemented EMM2 by centrifugal elutriation at time zero. The temperature was immediately shifted to 37°C and samples were taken for western blotting with S402P and antiCdc2 antibodies and the septation index was scored with calcofluor white. (b) Samples from an asynchronous YES cdc25.22 culture in which the temperature was shifted from 25°C to 37°C at t = 0 were blotted with S402P and nti-cdc2 antibodies. (c-e) spc1-12myc cells were grown to early log phase at 25°C and the temperature shifted to 37°C at t = 0. Sty1/Spc1.myc was immuno-precipitated with 9E10 antibodies that recognize the myc epitope at the indicated times and blots were probed with: antibodies that recognise the conserved regions activating regions of MAP kinases when they have been phosphorylated on the two activating residues (-P-TY, in the case of Sty1 T171 and Y173), antibodies that recognise the conserved regions activating regions of MAP kinases when they are only phosphorylated on the activating tyrosine (Y173 for Sty1) or Plo1 antibodies to identify any Plo1 that had been precipitated alongside Sty1/Spc1.myc. d and e are repeat experiments conducted under the same conditions.

Supplementary Figure 3:

Centrifugation promotes the disassociation of F-actin patches from the cell tip of wild type cells.

Early-log phase wild type cells which had been grown at 25°C in supplemented EMM2 were stained for F-actin either before or 5 minutes after centrifugation for three two minute pulses at 2900 g. The upper panel shows phalloidin staining of F-actin, middle, calcofluor staining of cell wall and lower panel a merge of the two with F-actin in red and cell wall in green. F-actin disassociates from the tips following centrifugation.

Supplementary Figure 4:

The sequences around serine 402 do not promote Plo1 dimerisation.

Introduction:

The phosphorylation site ARKSTDG contains an ST motif. ST motifs have recently been shown to be the optimal recognition sequences for recruitment of a polo box, the optimum consensus sequence being S-(pT/pS)-P/X)1. However, because Xenopus Plx can bind S-pT-Q, the proline at position +1 is not vital2. Thus, the sequences around serine 402 could constitute a polo box binding site. Structural studies show that the serine at –1 is absolutely essential for the association of the PBD with substrates3. Therefore were T403 to be a polo consensus binding site, then phosphorylation of S402 would be predicted to block the binding of a PBD to the site as it would disrupt the very strict criteria for the binding of PBD for its substrate. In other words phosphorylation of S402 would block pT403 mediated recruitment of Plo1 to itself or the recruitment of a Plo1 molecule in trans. If it works in trans we would expect to see evidence of dimerisation of Plo1 and that phosphorylation of S402 or mutation to anything other than serine would inhibit this. However, Plo1 does not interact with itself in the budding yeast two hybrid assay4 and it is not possible to co-precipitate non-tagged Plo1 alongside an epitope tagged protein5. The two proteins do not interact at any stage of the cell cycle in wild type strains and in strains that have been arrested at the G2/M boundary by inactivation of cdc25.22 or when these cdc25.22 arrested cells are released to generate a synchronous mitosis5. Thus it is unlikely that Plo1 forms a homo-dimer under unperturbed conditions. However, it is possible that Plo1 could dimerise during the initial stages of heat shock (1-20 mins) and the phosphorylation of S402 that follows blocks any more dimerisation. To test this hypothesis we subjected cells in which the HA tagged protein has been induced to a low level in an otherwise wild type cell5 to a heat shock from 25°C to 37°C and precipitated Plo1.HA and blotted with either a polyclonal antibody to Plo1 or the 12CA5 monoclonal antibody against the HA epitope. Legend:

Early-log phase IH1314 cells in which a version of the plo1+ gene which had been tagged at its N terminus with three copies of the HA epitope that is recognised by the 12CA5 antibody was induced for 17 hours before the experiment started was shifted from 25C to 37C and cell extracts were prepared at the times indicated. 12CA5 antibodies were used to precipitate Plo1 according to the conditions outlined in Tanaka et al5. The immunoprecipitates were probed with either 12CA5 to identify the epitope tagged molecule, or with poly-clonal HN184 antibodies to recognise both the tagged and the endogenous Plo1 molecules in the immunoprecipitate (lower panel).

Results/Discussion:

Despite the ability of the poly-clonal antibodies to detect both proteins in the whole cell lysates (left lanes – Total), they only detected a single band in the immunoprecipitates, indicating that the native molecule is not precipitated along with the epitope tagged protein. It is therefore unlikely that the sequences around serine 402 constitute a Plo1 docking site that works in trans.

The functional polo box unit includes a helical stretch 45 amino acids upstream of human Plk1 and budding yeast Cdc53. The gap between serine 402 and this inferred unit of association (polo box plus 45 amino acids) is 40 amino acids. As structural data for this region are lacking it is unclear whether these 40 amino acids could permit the molecule to fold back on itself and use S402 as an auto inhibitory site is beyond the scope of the present study. Such a mechanism could account for the inhibitory impact of the C terminus upon in vitro polo kinase activity6, but it awaits a more detailed analysis of the structure/function relationships of Plo1.

Supplementary Figure 5:

A Cartoon showing the two functions executed by Plo1 S402 phosphorylation. (A) SRP promotes phosphorylation of Plo1 on serine 402 in unperturbed cell cycles this phosphorylation promotes Plo1 recruitment to SPBs and thereafter commitment to mitosis. The exact mechanism by which Plo1 recruitment mediates commitment to mitosis awaits clarification. (B) A second role for serine 402 phosphorylation follows the Spc1/Sty1 stimulated response to heat shock or centrifugation. While stress mediated stimulation of Spc1/Sty1 arrests nuclear division and de-polarises the actin cytoskeleton, Plo1 S402 phosphorylation promotes the resumption of the cell cycle and actin mediated cell tip growth once the stimulation of Spc1/Sty1 has receded.

Supplementary Table 1:

Strains used in this study.

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2.Yoo, H. Y., Kumagai, A., Shevchenko, A. & Dunphy, W. G. Adaptation of a DNA replication checkpoint response depends upon inactivation of claspin by the Polo-like kinase. Cell117, 575-88 (2004).

3.Elia, A. E. et al. The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell115, 83-95 (2003).

4.MacIver, F. H., Tanaka, K., Robertson, A. M. & Hagan, I. M. Physical and functional interactions between polo kinase and the spindle pole component Cut12 regulate mitotic commitment in S. pombe.Genes Dev17, 1507-23 (2003).

5.Tanaka, K. et al. The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe.EMBO J.20, 1259-1270 (2001).

6.Mundt, K., Golsteyn, R., Lane, H. A. & Nigg, E. A. On the regulation and function of human polo-like kinase 1 (PLK1): Effects of overexpression on cell cycle regulation. Biochem. Biophys. Res. Com.239, 377-385 (1997).