Fig 1.
Cdc2 exists in three forms with distinct phosphorylation levels.
(A) Phosphorylation of S.pombe Cdc2 at threonine-14 (T14), tyrosine-15 (Y15) and threonine-167 (T167). Cdc2.1w is insensitive to inhibition by Wee1 kinase, whereas Cdc2.3w does not require activation by Cdc25 phosphatase. (B+C) Model of S.pombe Cdc2. The model was produced with the Swiss Model tool (template: PDB ID: 4EOM; identity: 66.78%; range: 1-296aa; coverage: 97%). The positions of T14, Y15, C67Y (cdc2.3w), G146D (cdc2.1w) and T167 are shown. The ATP ligand and the magnesium ion (yellow) are included. (D) Separation of total protein extracts of the indicated strains on a normal 10% SDS page and a 10% Phos-tag SDS page. The Cdc2 bands H, M and U are indicated. (E) IEF of total protein extracts of the indicated strains on a linear pH gradient (pH3-10).
Fig 2.
Wee1 kinase contributes to the aberrant modification of Cdc2.1w.
(A) IEFPT analysis: IEF strips were placed on top of a 10% Phos-tag SDS page. The IEF strip separates Cdc2 by its isoelectric values and the Phos-tag SDS page by its degree of phosphorylation. (B) IEFPT analysis of total extracts prepared from the indicated strains. The mobility of the Cdc2 bands H, M and U is indicated. The top panel shows an example of the normal IEF pattern of wild type cells. The two wild type and cdc2.1w experiments were conducted independently. The arrows indicate the acidic shift of forms A and B in the cdc2.1w mutant, its reversal in the cdc2.1w Δwee1 strain, and the additional, highly phosphorylated species in the H range in the cdc2.1w Δwee1 strain. (C-E) Phos-tag analysis of total protein extracts prepared from the indicated strains.
Fig 3.
Only Cdc2-He and Cdc2-Hf are phosphorylated at tyrosine 15.
(A) Serial dilutions (10-fold; starting with 107 cells/ml) of the listed strains were spotted onto YEA plates containing the indicated drugs. The plates were incubated at 30°C for 3 days. One YEA plate was incubated at 37°C. (B) IEFPT analysis of wild type and cdc2.1w cells grown in rich medium with 40μM camptothecin (CPT) or without the drug. The arrows indicate the increase in the abundance of the Cdc2 forms He and Hf. (C) Phos-tag analysis of total protein extracts prepared from wild type cells grown in rich medium with 12mM HU (hydroxyurea causes DNA replication arrest), with 40μM CPT (camptothecin causes DNA replication fork damage) or no drug. The total protein extracts were probed with the anti-Cdc2 antibody (left panel) or the anti-Cdc2-Y15 phospho-antibody (right panel). (D) Cdc2 was immunoprecipitated with an anti-Cdc2 antibody from CPT-treated wild type cells. The enriched kinase was either probed with the anti-Cdc2 antibody (top panel) or the anti-Cdc2-Y15 phospho-antibody (bottom panel). Only the H range is shown. (E) Cdc2 was immunoprecipitated with an anti-Cdc2 antibody from CPT-treated wild type cells and treated with Calf Intestinal Alkaline Phosphatase (CIP), which dephosphorylates tyrosine, with lambda protein phosphatase, which dephosphorylates tyrosine, threonine and serine, or left untreated. The protein was detected with the anti-Cdc2 antibody. (F) IEFPT analysis of wild type and cdc2.T14A cells grown in rich medium without a drug.
Fig 4.
Unphosphorylated Cdc2 associates with Chk1 kinase.
(A) Cyclin B (Cdc13) was immunoprecipitated from untreated and CPT-treated (40μM CPT, 4h) cdc13-HA cells and the precipitated protein was detected with the anti-Cdc2 antibody. Wild type (cdc13+) cells were included as a negative control. (B) The DNA damage checkpoint kinase Chk1 was immunoprecipitated from untreated and CPT-treated (40μM CPT, 4h) chk1-HA cells and the precipitated protein was detected with the anti-Cdc2 antibody and an anti-HA antibody (Chk1). The arrow indicates the DNA damage-induced phosphorylation of Chk1. Cdc2-U indicates the unphosphorylated Cdc2 species Ua and Ub. (C) Model. Since all four forms (C-F) in the H range bind to cyclin B (Cdc13) they are likely to be T167 phosphorylated. Cdc2-Hf may carry phosphate residues at T14, Y15 and T157, whereas Cdc2-He may be only phosphorylated at Y15 and T167. Cdc2-He and Cdc2-Hf are both involved in cell cycle regulation. Cdc2-Hc and Cdc2-Hd associate with cyclin B and may therefore be T167 modified. The unphosphorylated forms Cdc2-Ua and Cdc2-Ub associate with Chk1 and Cyclin B and may act in the response to DNA damage. Cdc2-M may be serine phosphorylated.