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Figure 1

Figure 1. Analog-sensitive Cdc5 is inhibited by CMK. From: A Coupled Chemical Genetic and Bioinformatic Approach to Polo-like Kinase Pathway Exploration.

(A) Sequence alignment of kinase domain regions spanning the gatekeeper residue and the reactive cysteine. The Cdc5 sequence is in bold, and the specificity filters critical for RSK2 inhibition by CMK [22] are highlighted in gray.
(B) Chemical structure of CMK. CMK (in gray), with features of the kinase active site depicted, including a cysteine to react with the electrophilic chloromethyl ketone and a gatekeeper residue that controls access to a hydrophobic binding pocket. A predicted steric clash between the Cdc5 leucine gatekeeper residue and CMK is illustrated.
(C) Cell viability halo assay of wild type (CDC5) and cdc5-as1 yeast. Inhibition of cell growth in the region surrounding a disc spotted with 1 nmol CMK or scaffold molecule is observed only upon CMK application to cdc5-as1 (center), indicating a requirement for both the gatekeeper mutation and the electrophilic reactivity of CMK.

Jennifer L. Paulson, et al. Chem Biol. ;14(11):1261-1272.
Figure 4

Figure 4. Spc72 binds to the Cdc5 Polo-box domain in a cell cycle and phosphospecific manner. From: A Coupled Chemical Genetic and Bioinformatic Approach to Polo-like Kinase Pathway Exploration.

(A) Cdc5 domain structure indicating the kinase domain and Polo-box domain (PBD), and sequence alignment showing three conserved PBD residues required for phosphopeptide binding by Plk1 (highlighted in gray). The PBD* mutant contains mutations of the highlighted Cdc5 residues to the amino acids indicated for elimination of phospho-specific motif binding.
(B) Spc72 is bound by the Cdc5 PBD, and PBD* has reduced Spc72 binding. Anti-TAP (Spc72) western blot indicates Spc72 present in the input mitotic cell extract (I) or pulled down (P) with GST-PBD (PBD) or GST-PBD* (PBD*). Total protein staining indicates the amount of GST-fusion protein in pulldown lanes (P). I = 5% input, P = pulldown.
(C) Cdc5 preferentially binds mitotic Spc72. Wild type PBD pulldowns from mitotic cell extracts (as in (B)) or G1 phase cell extracts were probed for Spc72.
(D) Domain structure of Spc72 including coiled-coils predicted by COILS [59] and sites matching Cdc5 phosphorylation, (D/E/N)X(S/T) [58], and PBD binding, S(S/T) [27], minimal motifs. The best scoring PBD binding motif in Spc72 is indicated (*).
(E) Mutation of consensus Cdc5 binding residues in Spc72 reduces binding to the PBD. PBD binding to Spc72 or Spc72(S231A,S232A) as in (B).
(F) SPB proteins efficiently bound to the Cdc5 PBD. Anti-TAP western blot indicates tagged SPB proteins present in the input mitotic cell extract (5%, I) or pulled down (P) with GST-PBD. Selected SPB proteins efficiently detected in the pulldown are shown, along with functional information and protein abundance [7].

Jennifer L. Paulson, et al. Chem Biol. ;14(11):1261-1272.
Figure 3

Figure 3. A candidate-based in vivo screen identifies Spc72 as a Cdc5 substrate. From: A Coupled Chemical Genetic and Bioinformatic Approach to Polo-like Kinase Pathway Exploration.

(A) Schematic representation of substrate recognition by Cdc5. The substrate is depicted to contain a Cdc5 phosphorylation motif, (D/E/N)X(S/T) [58], and a binding motif, S(pS/pT)(P/X), which binds the Cdc5 Polo-boxes (PB1 and PB2) [27]. X represents any amino acid, p represents phosphorylation.
(B) Approach to screening for substrates phosphorylated by Cdc5 in vivo.
(C) Bioinformatic mining of the yeast proteome for candidate Cdc5 substrates. The distribution of assigned Cdc5 substrate likelihood scores is shown for 192 Cdc5 candidate substrates compared with the proteome, with low scores reflecting likely candidates.
(D) Cdc5 candidate substrates are enriched in low abundance proteins. Normalized distribution of protein abundance [7] comparing candidate Cdc5 substrates to the entire proteome. The data set means were statistically different (P=0.0003) by unpaired t-test. Proteins without abundance values [7] were excluded from the analysis.
(E) Result of the screen. The gel mobility of five TAP-tagged candidate substrates is altered upon Cdc5 inhibition with 10 μM CMK (+) as compared with a DMSO control treatment (-).
(F) Inhibition of Cdc5 with CMK eliminates the mitotic Spc72 upshift observed in a synchronized cell cycle. Cell cycle progression of CDC5 or cdc5-as1 cells expressing Spc72-TAP released from G1 into 10 μM CMK is indicated by budding index and Clb2 western blot. Spc72 is visualized by anti-TAP western blot (Spc72).
(G) In vitro phosphorylation of Spc72 by Cdc5. Immunopurified TAP-tagged Spc72 was incubated with [γ-32P]ATP, with and without purified Cdc5 (left panels). Phosphorylation (32P) and Spc72 western blot (α-TAP) are observed. No Spc72 phosphorylation is seen when Cdc5 is added to a mock pulldown reaction (untagged Spc72, - Spc72-TAP, right panels).

Jennifer L. Paulson, et al. Chem Biol. ;14(11):1261-1272.
Figure 2

Figure 2. CMK inhibition of Cdc5(L158G) leads to a first cell cycle anaphase arrest and delay in anaphase spindle migration. From: A Coupled Chemical Genetic and Bioinformatic Approach to Polo-like Kinase Pathway Exploration.

(A) CMK treatment causes of a first cell cycle arrest of cdc5-as1, but not wild type, cells in a dose-dependent manner. Time course of cell cycle synchronized CDC5 and cdc5-as1 cultures released from G1 arrest (unbudded) into the indicated concentrations of CMK (n = 200 cells for each point) is plotted. The y-axis represents the percentage of cells in S/G2 and M phases, as judged by the presence of a medium- to large-sized bud. A second cell cycle was prevented by re-arrest in the subsequent G1.
(B) cdc5-as1 cells arrest with an extracellular 50% inhibitory CMK concentration of 1.1 μM (dotted line, 95% confidence interval from 0.9 to 1.2 μM). The percent cdc5-as1 cells in S/G2 and M cell cycle phases at 180 min is shown for CMK-treated cultures prepared as in (A). Error bars represent standard errors of the mean for three experiments (n = 200 cells for each), and the data were fit to a sigmoidal dose response curve (R2 = 0.97).
(C) CMK-treated cdc5-as1 cells degrade Pds1 with wild type kinetics but maintain stabilized Clb2. Cell extracts from CDC5 and cdc5-as1 strains expressing Pds1-HA3 released from G1 into 5 μM CMK and re-arrested in the subsequent G1 were blotted for HA (Pds1) and Clb2.
(D) CMK-treated cdc5-as1 cells arrest as budded cells with segregated chromosomes. Budded cells (squares) and budded cells with DNA masses separated into mother cell and bud (circles) were quantified in CMK-treated CDC5 (closed symbols) and cdc5-as1 (open symbols) strains for the experiment shown in (C) (n = 100 cells per point).
(E) Mother cell localized anaphase spindles are transiently observed in CMK-treated cdc5-as1 cells. The percentage anaphase spindles with localization depicted (n = 100 cells) in CMK-treated cdc5-as1 and CDC5 cells at 105 min in the experiment shown in (C and D).
(F) Examples of CMK-treated cells with anaphase spindles (tubulin) observed at 90 min in the experiment shown in (C through E). The images show aberrant (cdc5-as1) and wild type (CDC5) anaphase spindle localization, but are not representative of all cells in the population. The scale bar represents 5 μm.
(G) CMK-treated cdc5-as1 cells have a delay in completing chromosome separation. CDC5 (black) and cdc5-as1 (gray) strains expressing tetR-GFP and containing a centromere-proximal tetO array, released from G1 into 5 μM CMK and re-arrested in the subsequent G1 were analyzed for budding (squares), separation of fluorescent GFP chromosomal dots (diamonds), and separation of DNA masses (circles) (n = 100 cells per point).
(H) CMK-treated cdc5-as1 cells have misaligned pre-anaphase spindles. Cells with an anaphase spindle (squares), with a pre-anaphase short spindle (diamonds), and with a misaligned pre-anaphase spindle (circles) in CMK-treated CDC5 (black) and cdc5-as1 (gray) strains were quantified for the experiment shown in (G) (n = 100 cells per point).
(I) Examples of cells with pre-anaphase spindles (tubulin) observed in the experiment shown in (G through H). The images show misaligned and normal pre-anaphase spindles, which are quantified in H.
(J) Detached cytoplasmic microtubules observed in a small number of CMK-treated cdc5-as1 cells. Examples of tubulin-stained cells observed in the experiment shown in (G through I) with detached microtubules indicated (arrows). The images represent only a small number of cells in the cdc5-as1 population. No detached microtubules were observed in CMK-treated CDC5 cells.

Jennifer L. Paulson, et al. Chem Biol. ;14(11):1261-1272.

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