ABSTRACT
Cdc25B is a phosphatase that catalyzes the dephosphorylation and activation of the cyclin-dependent kinases, thus driving cell cycle progression. We have identified two residues, R488 and Y497, located >20 A from the active site, that mediate protein substrate recognition without affecting activity toward small-molecule substrates. Injection of Cdc25B wild-type but not the R488L or Y497A variants induces germinal vesicle breakdown and cyclin-dependent kinase activation in Xenopus oocytes. The conditional knockout of the cdc25 homolog (mih1) in Saccharomyces cerevisiae can be complemented by the wild type but not by the hot spot variants, indicating that protein substrate recognition by the Cdc25 phosphatases is an essential and evolutionarily conserved feature.
Subject(s)
cdc25 Phosphatases/chemistry , cdc25 Phosphatases/metabolism , Animals , Binding Sites/genetics , Female , Genes, Fungal , Genetic Complementation Test , In Vitro Techniques , Models, Molecular , Mutagenesis, Site-Directed , Oocytes/metabolism , Protein Conformation , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Substrate Specificity , Xenopus laevis , cdc25 Phosphatases/genetics , ras-GRF1ABSTRACT
The Cdc25 phosphatases function as key regulators of the cell cycle during normal eukaryotic cell division and as mediators of the checkpoint response in cells with DNA damage. The role of Cdc25s in cancer has become increasingly evident in recent years. More than 20 studies of patient samples from diverse cancers show significant overexpression of Cdc25 with frequent correlation to clinical outcome. Recent screening and design efforts have yielded novel classes of inhibitors that show specificity for the Cdc25s over other phosphatases and cause cell cycle arrest in vivo. Herein we provide a single source for those interested in the cellular functions of Cdc25 in cell cycle progression, its role in the progress of cancer and survival of cancer patients, and recent efforts in the design of specific inhibitors.
Subject(s)
Neoplasms/enzymology , cdc25 Phosphatases/metabolism , Animals , Cell Cycle , Cell Transformation, Neoplastic , Enzyme Inhibitors/therapeutic use , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , cdc25 Phosphatases/antagonists & inhibitors , cdc25 Phosphatases/chemistry , cdc25 Phosphatases/geneticsABSTRACT
Fas ligand (FasL) is expressed on some cancers and may play a role in the immune evasion of the tumour. We used immuno-histochemistry to study the expression of Fas and FasL in tissue samples from breast cancer patients, as well as normal breast tissue. Our results show that Fas and FasL are co-expressed both in normal tissue and in breast tumours. Fas and FasL mRNA were expressed in fresh normal and malignant breast tissue, as well as cultured breast epithelium and breast cancer cell lines. Flow cytometry analysis of live cells failed to detect FasL on the surface of normal or malignant breast cells; however, both stained positive for FasL after permeabilization. Fas was detected on the surface of normal breast cells and T47D and MCF-10A cell lines but only intracellularly in other breast cell lines tested. Neither normal breast epithelium nor breast cell lines induced Fas-dependent apoptosis in Jurkat cells. Finally, 20 tumour samples were stained for apoptosis. Few apoptotic cells were detected and there was no increase in apoptotic cells on the borders between tumour cells and lymphocytes. We conclude that FasL is expressed intracellularly in both normal and malignant breast epithelium and unlikely to be important for the immune evasion of breast tumours.
