Fig 2: Interaction of Elk-1 with mitotic kinases. (a) A schematic diagram of the domain structure and predicted mitotic kinase phosphorylation motifs of ternary complex factor Elk-1; predicted Plk phosphorylation motifs are shown in purple, Cdk motifs in orange, Aur-B motif in green, Aur-A motifs in red, and the known ERK/MAPK phosphorylation sites are shown in blue; ETS: E-twenty-six; DBD: DNA binding domain; B: SRF interaction domain; R: repression domain; D: docking domain; C: activation domain (AD). (b) Immunoprecipitation of endogenous Elk-1 in SH-SY5Y cell lines, followed by Western blot with antibodies specific for Aur-A, Aur-B, Cdk1, Plk1, and Elk-1 (IgG is used as IP control). (c) U87 cells were arrested with nocodazole treatment, released into mitosis at time 0 (indicated as 0′), and samples were taken at 30 min (30′), 60 min (60′), and 90 min (90′) after release. Immunoprecipitation was performed using Elk-1 antibody, and both input and IP samples were analyzed by Western blot with antibodies specific for Aurora-A, Aurora-B, Cdk1, Plk1, and Elk-1.

Fig 3: (a) Protein-protein interaction network resulted by integration of human whole PPI network with Elk-1 overexpression microarray in SH-SY5Y cells from KeyPathwayMiner algorithm. (b) Elk-1 and interaction partners in the PPI network. Color codes represent which phosphoproteome dataset of each protein is found in (filtered by mitotic phosphoproteome, Dephoure et al. [23]; only M and G1/M phase proteins are shown in figure for clarity); shape of the nodes represents the cell cycle phase each protein is associated with (filtered with Plk phosphoproteome, Grosstessner-Hain et al. [21], or Aur/Plk phosphoproteome, Kettenbach et al. [22]).

Fig 4: In vitro kinase assay for phosphorylation of various Elk-1 peptides by AurA, AurB, Plk1, and Cdk1 kinases. Unmodified peptides (for sequences see Suppl Table 4) were incubated with indicated kinases in the presence or absence of ATP, and phosphorylation was monitored by accumulation of ADP at 15, 30, 45, and 60 min time points. Reaction tube containing only reaction buffer was used as mock, and kinase activity was reported as relative fluorescence units: (a) phosphorylation of Ser106 peptide witth Plk1, (b) phosphorylation of Thr108 peptide with Plk1, (c) phosphorylation of Thr133 peptide with Cdk1, (d) phosphorylation of Ser198 peptide with AurA, (e) phosphorylation of Ser198 peptide with AurB, (f) phosphorylation of Thr199 peptide with AurA, (g) phosphorylation of Thr199 peptide with AurB, (h) phosphorylation of Ser200 peptide with AurA, (i) phosphorylation of Ser200 peptide with AurB, (j) phosphorylation of Ser202 peptide with Cdk1, (k) phosphorylation of Ser303 peptide with Cdk1, (l) phosphorylation of Ser304 peptide with Cdk1, (m) phosphorylation of Ser324 peptide with Cdk1, and (n) phosphorylation of Ser326 peptide with Cdk1. Reaction tube containing only reaction buffer was used as mock.

Fig 5: In vitro kinase assay for Elk-1 phosphorylation by (a) Aurora-A (AurA), (b) Aurora-B (AurB), (c) Cdk1, (d) Plk1, and (e) Hexokinase 2 kinases. Recombinant Elk-1 protein was incubated with indicated kinases in the presence or absence of ATP, and phosphorylation was monitored by accumulation of ADP at 15, 30, 45, and 60 min time points as determined by increase in fluorescence (i.e., kinase activity was reported as relative fluorescence units). Reaction tube containing only reaction buffer was used as mock.