Abstract
Cyclin E, a positive regulator of the cell cycle, controls the transition of cells from G(1) to S phase. Deregulation of the G(1)-S checkpoint contributes to uncontrolled cell division, a hallmark of cancer. We have reported previously that cyclin E is overexpressed in breast cancer and such overexpression is usually accompanied by the appearance of low molecular weight isoforms of cyclin E protein, which are not present in normal cells. Furthermore, we have shown that the expression of cyclin E low molecular weight isoforms can be used as a reliable prognostic marker for breast cancer to predict patient outcome. In this study we examined the role of cyclin E in directly activating cyclin-dependent kinase (CDK) 2. For this purpose, a series of N-terminal deleted forms of cyclin E corresponding to the low molecular weight forms detected only in cancer cells were translated in vitro and mixed with cell extracts. These tumor-specific N-terminal deleted forms of cyclin E are able to activate CDK2. Addition of cyclin E into both normal and tumor cell extracts was shown to increase the levels of CDK2 activity, along with an increase in the amount of phosphorylated CDK2. The increase in CDK2 activity was because of cyclin E binding to endogenous CDK2 in complex with endogenous cyclin E, cyclin A, or unbound CDK2. The increase in CDK2 phosphorylation was through a pathway involving cyclin-activating kinase, but addition of cyclin E to an extract containing unphosphorylated CDK2 can still lead to increase in CDK2 activity. Our data suggest that the ability of high levels of full-length and low molecular weight forms of cyclin E to activate CDK2 may be one mechanism that leads to the constitutive activation of cyclin E.CDK2 complexes leading to G(1)/S deregulation and tumor progression.
Highlights
Cyclin E has an important role in the regulation of the restriction point transition in the cell cycle
The results revealed that all five cyclin E forms were able to co-immunoprecipitate with CDK2, suggesting each was able to bind to CDK2 (Fig. 1B)
Tumor cells overexpress cyclin E, which results in the constitutive expression and associated kinase activity of cyclin E throughout the tumor cell cycle [23]
Summary
Cyclin E has an important role in the regulation of the restriction point transition in the cell cycle. The phosphorylation state of CDK2 at three separate amino acids under the control of CAK (Thr-160 activating phosphorylation), KAP (Thr-160 inhibitory dephosphorylation), Wee (Tyr-15, Thr-14 inhibitory phosphorylation), and CDC25 (Tyr-15, Thr-14 activating dephosphorylation), give the cell additional levels of control over CDK2 activity and cell cycle progression, beyond cyclin binding. The role of cyclin E as a CDK2 activator in control of restriction point transition in the cell cycle [21] makes cyclin E an excellent candidate as a protein involved in tumor development. In addition to the full-length protein at 50-kDa, we have identified five LMW forms that are generated mainly by proteolysis at two domains in cyclin E, which results in two pairs of closely migrating doublets (EL-2/EL-3 and EL-5/EL-6) These isoforms, when in complex with CDK2, are biochemically hyperactive, as evidenced by their enhanced ability to phosphorylate histone H1 and GST-Rb [29] compared with full-length cyclin E1⁄7CDK2 complex. We have shown that the presence of the LMW forms in breast cancer patients is associated with increasing grade and stage [27], as well as a significantly worse prognosis [31, 32]
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