Two Distinct Cdc2 Pools Regulate Cell Cycle Progression and the DNA Damage Response in the Fission Yeast S.pombe
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.