Figure 1.

Strategy used by Snead et al. [1] to identify substrates of yeast Polo-like kinase (Cdc5). (a) A modified Cdc5 (Cdc5-as1) is generated to be selectively inhibited by a small-molecule inhibitor (CMK) that does not affect wild-type Cdc5. K, kinase domain; PBD, Polo-box domain. (b) Treatment of cdc5Δ cdc5-as1 cells with CMK reveals the cellular phenotypes associated with Cdc5 inhibition. (c) A sequence-scanning algorithm identifies proteins containing potential Plk phosphorylation motifs (blue), potential PBD-binding motifs (red) and satisfying functional criteria in database annotations (yellow). (d) Individual strains expressing candidate substrates (P1, P2, P3, and so on) from their normal loci in fusion with the TAP tag are screened for electrophoretic mobility shifts of the fusion protein between a G1 arrest (Cdc5 inactive) and an M-phase arrest (Cdc5 and other kinases active). (e) For fusion proteins showing a shift between G1 and M, strains combining the TAP-tagged gene with the cdc5Δ cdc5-as1 allele are generated. Those are then screened for a CMK-dependent (Cdc5-dependent) mobility shift in M-phase-arrested cells. Hits from that final step can be considered to be physiological Cdc5 substrates. The cellular phenotypes observed following Cdc5 inhibition in (b) may be hypothesized to result from a failure of phosphorylation of one or more of the Cdc5 substrates identified.