Properties of the kinome SDL interaction network. (A) Distribution of SDL (blue), negative (red), and positive (green) GIs across the kinome using the stringent cutoff. The number of interactions for SDL-positive queries is plotted. (*) Kinases that were not screened for GIs. (B) Conditional screening of hog1Δ and cmk1Δ mutants expands the SDL interaction network. SDL screening of a hog1Δ strain was performed on 0.2 M NaCl (osmotic stress) and on 200 mM CaCl₂ for a cmk1Δ strain. (C) Representative serial spot dilution assays illustrating the SDL phenotype caused by overexpression of RPB2 in a hog1Δ strain specifically in activating conditions. (D) Bar graph showing the prevalence of positive (green bar) and negative (red bar) GIs at the intermediate SGA cutoff among kinase–gene (all), kinase–substrate, kinase–kinase, and kinase–kinase pairs that share the same target according to the KID gold standard, as a fraction of gene pairs screened. Prevalence of SDL interactions for each subtype is shown in parallel (blue bar).

YMR074C is an inhibitor of the Slt2-MAP kinase cell wall integrity pathway. (A) Overrepresented triplet motif that identified YMR074C to be SDL with two kinases of the cell wall integrity pathway, Bck1, and its downstream target Slt2. (B) Model describing the role of Ymr074c as an inhibitor of cell wall integrity. (C) Overnight cultures of bck1Δ, slt2Δ, ymr074cΔ, single or double mutants were serially diluted and spotted onto YPD medium in the absence (left) and presence (right) of 10 mM caffeine. (*) Phenotypic rescue.

Overrepresented SDL motifs identified through combined analysis of SDL, SL/SSup GI data sets, and gold standard kinase–substrate pairs. Five types of sub-networks were enriched against the randomized data set: (A) counteracting; (B) balancing co-regulation; (C) converging regulation; (D) diverging regulation; and (E) SDL cascades. The number and fold-enrichment of triplet gene pairs identified are listed, as well as examples of known and novel motifs for each group. Possible mechanistic models describing each motif are highlighted. Some motifs fall under more than one subtype.

Kel1 protein is co-regulated by budneck kinases. (A) Overrepresented motif (gray triangle), supporting a functional connection between Kel1 and the two budneck kinases Cla4 and Hsl1 in a converging regulation model. (B) Endogenous Kel1 protein levels are reduced in hsl1Δ and cla4Δ but not in gin4Δ mutant strains. Kel1-TAP protein was immunoprecipitated from the indicated budneck kinase mutant strains and Western blots were probed with anti-PAP to detect total Kel1 protein. Electrophoretic mobility shift of Kel1 protein is shown (Kel1-P). Swi6 was used as a loading control. (C) Integrated Kel1-TAP protein was immunoprecipitated in the presence of Lte1-Flag and association of Lte1 with Kel1 was detected using α-flag antibody. The amounts of Lte1-Flag in the whole cell extract (WCE; top panel) and in the Kel1 immunoprecipitate (bottom panel) are shown. (Middle panel) The amount of Kel1-TAP in the immunoprecipitate (anti-PAP). Immunoprecipitation assays were performed in wild-type, hsl1Δ, gin4Δ background strains. cla4Δ strain was not tested, due to severe toxicity upon Kel1-TAP expression. (D) Model for regulation of Kel1 by budneck kinases. Lte1 is phosphorylated by Cla4, which triggers binding to its anchor, Kel1. Hsl1 regulates the stability of Kel1 protein, which allows the anchor to become available for binding to Lte1-P. Together, the complex inhibits mitotic exit possibly to delay budding to allow the cell to reach the correct size. Later, Cdc14 phosphatase releases Lte1 from its anchor in the cortex into the cytoplasm in order to activate mitotic exit in large budded cells.