CELL CYCLE AND GLUCOCORTICOID RECEPTOR PHOSPHORYLATION

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Glucocorticoids at high doses are used widely to treat disorders ranging from minor allergies to leukemias and lymphoma. They act on almost all cells. So much effort has gone into maximizing their therapeutic effectiveness and minimizing unwanted side effects. Sensitivity to glucocorticoids varies strikingly through the cell cycle, the hormones being effective only if present during late G1 and S (DNA synthesis) phases. Our long-term goal is to elucidate the basic mechanisms of this variation. In this proposal, the central objective is to test the hypothesis that phosphorylation of glucocorticoid receptors (GRs) changes through the cell cycle and accounts at least in part for the variation in sensitivity. This hypothesis, based on evidence for cell cycle-dependence of GR number, nuclear binding, and phosphorylation, has gained support -- and the means to test it -- from our finding of a rapid, hormone-dependent increase in GR phosphorylation, and our identification by phosphopeptide mapping and sequencing of seven phosphorylated sites in mouse GRs: four lie in consensus sequences for the p34cdc2 kinases that control the cell cycle. The proposal is designed to answer the following specific questions; 1. Do hormone dependent GR hyperphosphorylation and/or GR phosphorylated sites change through the cell cycle? (a) Synchronized WCL2 cells (CHO cells with overexpressed mouse GRs) will be labeled with 32P and treated with hormone to assay hormone-dependent phosphorylation of the GRs; (b) Phosphorylated sites in these GRs will be identified by phosphopeptide mapping, and sequencing of any new peptides. 2. How do glucocorticoid sensitivity and GR number change through the cell cycle in Cho cells? Synchronized CHO cells with overexpressed GRs will be used to: (a) measure hormone effects on activity of a stably transfected gene and thymidine kinase; (b) determine if the increased number of GR binding sites in G1/S is due to increased GR protein per cell. 3. Do GR phosphorylation mutants behave differently from wild-type GRs? CHO cells with overexpressed GRs mutated at normally phosphorylated sites will be studied as in (2a). 4. Are GR phosphorylation and glucocorticoid-induced apoptosis in lymphocytes cell cycle-dependent, and modifiable by chemotherapeutic agents? Synchronized WEHI-7 cells will be used to measure (a) GR phosphorylation as in la; (b) glucocorticoid-induced apoptosis; (c) effects in (a) and (b) of cell cycle-modifying agents. Our results may benefit therapy and enhance understanding of glucocorticoid resistance.