The Tumor-specific Hyperactive Forms of Cyclin E Are Resistant to Inhibition by p21 and p27

Hannah Wingate,Ning Zhang,Mollianne J Mcgarhen,Isabelle Bedrosian,J Wade Harper,Khandan Keyomarsi

doi:10.1074/jbc.m409789200

Abstract

The low molecular weight (LMW) isoforms of cyclin E are unique to cancer cells. In breast cancer, such alteration of cyclin E is a very strong predictor of poor patient outcome. Here we show that alteration in binding properties of these LMW isoforms to CDK2 and the CDK inhibitors (CKIs), p21 and p27, results in their functional hyperactivity. The LMW forms of cyclin E are severalfold more effective at binding to CDK2. Additionally, compared with the full-length cyclin E-CDK2 complexes, the LMW cyclin E-CDK2 complexes are significantly more resistant to inhibition by p21 and p27, despite equal binding of the CKIs to the LMW complexes. When both the full-length and the LMW cyclin E are co-expressed, p27 preferentially binds to the LMW forms yet is unable to inhibit the CDK2 activity. Thus, the LMW forms of cyclin E may contribute to tumorigenesis through their resistance to the inhibitory activities of p21 and p27 while sequestering these CKIs from the full-length cyclin E.

Highlights

The low molecular weight (LMW) isoforms of cyclin E are unique to cancer cells
Compared with the full-length cyclin E-CDK2 complexes, the LMW cyclin E-CDK2 complexes are significantly more resistant to inhibition by p21 and p27, despite equal binding of the cyclin-dependent kinases (CDKs) inhibitors (CKIs) to the LMW complexes. When both the full-length and the LMW cyclin E are co-expressed, p27 preferentially binds to the LMW forms yet is unable to inhibit the CDK2 activity
Our results show that the LMW cyclin E-CDK2 complexes are significantly more resistant to inhibition by p21 and p27 both in vitro using purified CKIs and in vivo when the CKIs were co-infected with cyclin E and CDK2

Summary

EXPERIMENTAL PROCEDURES

Culture Conditions for Sf9 Cells—Insect Sf9 cells were cultured in Grace’s TNM-FH medium supplemented with 10% fetal calf serum in a 27 °C incubator in spinner cultures. For in vitro binding assays, purified HA-p27 or HA-p21 [26] was added, in a series of nine different concentrations from 0 up to 500 ␮M, to 300 ␮g of total cell lysate and incubated at 4 °C for 30 min followed by IP with anti-CDK2 antibody and coupling to protein A-Sepharose beads. 300 ␮g of protein extracts were first immunoprecipitated with polyclonal anti-p27 antibody (sc-528; Santa Cruz Biotechnology) and incubated with Sepharose-protein A beads and subjected to Western blot analysis with anti-FLAG or anti-p27 monoclonal antibodies, diluted at 1 ␮g/ml in Blotto

Hyperactive Cyclin E Isoforms Are Resistant to Inhibition

RESULTS

DISCUSSION