Brian S Robinson | PubMed Health

PubMed2005

Cyclin E both regulates and is regulated by calpain 2, a protease associated with metastatic breast cancer phenotype.

Overexpression of cyclin E in breast tumors is associated with a poor response to tamoxifen therapy, greater genomic instability, more aggressive behavior, and a poor clinical prognosis. These tumors also express low molecular weight isoforms of cyclin E that are associated with higher kinase activity and increased metastatic potential. In the current study, we show that cyclin E overexpression in MCF7 cells transactivates the expression of calpain 2, leading to proteolysis of cyclin E as well as several known calpain substrates including focal adhesion kinase (FAK), calpastatin, pp60src, and p53. In vivo inhibition of calpain activity in MCF7-cyclin E cells impedes cyclin E proteolysis, whereas in vivo induction of calpain activity promotes cyclin E proteolysis. An analysis of human breast tumors shows that high levels of cyclin E are coincident with the expression of the low molecular weight isoforms, high levels of calpain 2 protein, and proteolysis of FAK. Lastly, studies using a mouse model of metastasis reveal that highly metastatic tumors express proteolyzed cyclin E and FAK when compared to tumors with a low metastatic potential. Our results suggest that cyclin E-dependent deregulation of calpain may be pivotal in modifying multiple cellular processes that are instrumental in the etiology and progression of breast cancer.

Animals↗

PubMed2004

Substrate capacity considerations in developing kinase assays.

In developing a screening assay for a serine/threonine kinase, we evaluated various formats of an in-plate enzyme-linked immunosorbent assay (ELISA), as well as solution-phase kinase assays using either ELISA or AlphaScreen detection. Substrate was available both as a biotinylated 15-residue peptide and as a 25-residue peptide containing the same sequence expressed as a glutathione S-transferase fusion protein. When increasing concentrations of either of these substrates were coated directly onto ELISA plates, the rates of the kinase reactions progressively increased. In contrast, when the biotin-peptide was captured onto NeutrAvidin-coated plates, the finite peptide binding capacity of the plates limited the amount of substrate that could be incorporated into the assay system and thereby limited the rate of the reaction at a given kinase concentration. Solution-phase kinase reactions can tolerate high substrate concentrations; however, analysis of kinase reaction samples containing biotin-peptide concentrations higher than the binding capacity of NeutrAvidin-coated plates resulted in an inability to detect differences between reactions run at different substrate concentrations. For AlphaScreen detection following solution-phase kinase reactions, limitations in the binding capacity of the donor and acceptor beads caused loss of signal for substrate concentrations above the maximum binding capacity. Overall, the solution-phase assays required significantly more kinase than the in-plate assays (1-4 microg/ml versus <100 ng/ml, respectively). These studies demonstrate that the amount of substrate that can be incorporated into an assay system substantially affects the rate of the kinase reaction and therefore the amount of kinase required for the assay.

Enzyme-Linked Immunosorbent Assay↗