Abstract
CARP-1/CCAR1, a perinuclear phosphoprotein, is a regulator of cell growth and apoptosis signaling. Although CARP-1 is a regulator of chemotherapy-dependent apoptosis, it is also a part of the NF-κB proteome and a co-activator of steroid/thyroid nuclear receptors as well as β-catenin signaling. Our yeast two-hybrid screen revealed CARP-1 binding with the anaphase-promoting complex/cyclosome E3 ubiquitin ligase component APC-2 protein. CARP-1 also binds with anaphase-promoting complex/cyclosome co-activators Cdc20 and Cdh1. Following mapping of the minimal epitopes involved in CARP-1 binding with APC-2, a fluorescence polarization assay was established that indicated a dissociation constant (K(d)) of 480 nm for CARP-1/APC-2 binding. Fluorescence polarization assay-based high throughput screening of a chemical library yielded several small molecule antagonists of CARP-1/APC-2 binding, termed CARP-1 functional mimetics. CFM-4 (1(2-chlorobenzyl)-5'-phenyl-3'H-spiro[indoline-3,2'-[1,3,4]thiadiazol]-2-one), a lead compound, binds with and stimulates CARP-1 expression. CFM-4 prevents CARP-1 binding with APC-2, causes G(2)M cell cycle arrest, and induces apoptosis with an IC(50) range of 10-15 μm. Apoptosis signaling by CFM-4 involves activation of caspase-8 and -9 and caspase-mediated ubiquitin-proteasome pathway-independent loss of cyclin B1 and Cdc20 proteins. Depletion of CARP-1, however, interferes with CFM-4-dependent cell growth inhibition, activation of caspases, and apoptosis. Because CFM-4 also suppresses growth of drug-resistant human breast cancer cells without affecting the growth of human breast epithelial MCF-10A cells, elevating CARP-1 by CFM-4 and consequent apoptosis could in principle be exploited to further elucidate, and perhaps effectively target, often deregulated cell cycle pathways in pathological conditions, including cancer.
Highlights
CARP-1/CCAR1, a perinuclear phosphoprotein, is a regulator of cell growth and apoptosis signaling
CARP-1 Binds with APC/C Subunits APC-2, Cdc20, and Cdh1—Our previous studies have demonstrated that CARP-1 is phosphorylated by diverse signaling pathways [6, 19, 30] and that CARP-1 inhibited cell growth in part by its interactions with 14-3-3/Stratifin [5] and the PDZ-domain TAZ proteins [30]
Findings from other laboratories indicate that CARP-1 regulates ADR-dependent signaling by functioning as a co-activator of tumor suppressor p53 [7], whereas several proteomicbased studies indicate CARP-1 is a target of phosphorylation by the ataxia telangiectasia mutated kinase as well as EGF signaling [31, 32]
Summary
CARP-1/CCAR1, a perinuclear phosphoprotein, is a regulator of cell growth and apoptosis signaling. Our yeast twohybrid screen revealed CARP-1 binding with the anaphase-promoting complex/cyclosome E3 ubiquitin ligase component APC-2 protein. Apoptosis signaling by CFM-4 involves activation of caspase-8 and -9 and caspase-mediated ubiquitin-proteasome pathway-independent loss of cyclin B1 and Cdc proteins. A range of intracellular as well as extracellular signals are known to target the cell division cycle and apoptosis pathways and function to maintain homeostasis in normal tissues. A host of biochemical components that function to regulate apoptosis as well as cell division cycle pathways are known, it is likely that many crucial regulators that link these pathways have yet to be discovered In this context, identification of additional perhaps novel regulators of cell division and/or apoptosis signaling, and knowledge of their mechanism(s) of action, would be instrumental in further defining pathways for control of cell growth in normal and pathological conditions. Alterations in APC/C complex proteins have been noted in breast and colon cancer cells as well as primary colon cancers [12], whereas endogenous as well as synthetic inhibitors targeting APC/C-activating oncogenes or the APC/C complex have been recently described [13,14,15]
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