ABSTRACT
In response to DNA damage and genotoxic stress, the p53 tumor suppressor triggers either cell cycle arrest or apoptosis. The G(2) arrest after damage is, in part, mediated by the p53 target, 14-3-3final sigma (final sigma). Colorectal tumor cells lacking final sigma are exquisitely sensitive to DNA damage. Here we analyzed the mechanism of this sensitivity in final sigma(-/-) as compared with final sigma(+/+) human colorectal tumor cells. Exposure to adriamycin resulted in rapid apoptosis only in final sigma(-/-) cells. This was further characterized by caspase-3 activation, p21(CIP1) cleavage, and CDK2 activation. Moreover, Bax was rapidly translocated out of the cytoplasm, and cytochrome c was released in final sigma(-/-) cells. Transient adenovirus-mediated reconstitution of final sigma in the final sigma(-/-) cells led to effective rescue of this phenotype and protected cells against apoptosis. The association of final sigma, Bax, and CDK1 in protein complexes may be the basis for this antiapoptotic mechanism. In conclusion, final sigma not only enforces the p53-dependent G(2) arrest but also delays the apoptotic signal transduction.
Subject(s)
Apoptosis , G2 Phase , Mitosis , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/metabolism , Tyrosine 3-Monooxygenase/chemistry , Tyrosine 3-Monooxygenase/physiology , 14-3-3 Proteins , Adenoviridae/genetics , Caspase 3 , Caspases/metabolism , Cell Cycle , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Cytochrome c Group/metabolism , Cytoplasm/metabolism , Cytosol/metabolism , Doxorubicin/pharmacology , Enzyme Activation , Humans , Microscopy, Fluorescence , Mitochondria/metabolism , Phenotype , Precipitin Tests , Protein Binding , Protein Transport , Signal Transduction , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X ProteinABSTRACT
Calmodulin (CaM) is a small acidic protein essential to calcium-mediated signal transduction. Conformational change driven by calcium binding controls its selective activation of myriad target proteins. In most well characterized cases, both homologous domains of CaM interact with a target protein. However, physiologically separable roles for the two domains were demonstrated by mutants of Paramecium tetraurelia [Kung, C. et al. (1992) Cell Calcium 13, 413], some of which have altered calcium affinities [Jaren, O. R. et al. (2000) Biochemistry 39, 6881]. To determine whether these mutants can associate with canonical targets in a calcium-dependent manner, their ability to bind melittin was assessed using analytical gel permeation chromatography, analytical ultracentrifugation, and fluorescence spectroscopy. The Stokes radius of wild-type PCaM and 11 of the mutants decreased dramatically upon binding melittin in the presence of calcium. Fluorescence spectra and sedimentation velocity studies showed that melittin bound to wild-type PCaM and mutants in a calcium-independent manner. However, there were domain-specific perturbations. Mutations in the N-domain of PCaM did not affect the spectrum of melittin (residue W19) under apo or calcium-saturated conditions, whereas most of the mutations in the C-domain did. These data are consistent with a calcium-dependent model of sequential target association whereby melittin (i) binds to the C-domain of PCaM in the absence of calcium, (ii) remains associated with the C-domain upon calcium binding to sites III and IV, and (iii) subsequently binds to the N-domain upon calcium binding to sites I and II of CaM, causing tertiary collapse.
Subject(s)
Calcium/physiology , Calmodulin/deficiency , Calmodulin/genetics , Ion Channels/deficiency , Ion Channels/genetics , Melitten/metabolism , Mutation , Paramecium/genetics , Amino Acid Sequence , Animals , Calcium/metabolism , Calmodulin/chemistry , Calmodulin/metabolism , Chromatography, Gel , Ion Channels/metabolism , Melitten/chemistry , Models, Molecular , Molecular Sequence Data , Paramecium/metabolism , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Spectrometry, Fluorescence , Tryptophan/chemistry , UltracentrifugationABSTRACT
The cyclin-dependent kinase inhibitor p21(cip1) regulates cell cycle progression, DNA replication, and DNA repair by binding to specific cellular proteins through distinct amino- and carboxyl-terminal protein binding motifs. We have identified a novel human gene, CARB (CIP-1-associated regulator of cyclin B), whose product interacts with the p21 carboxyl terminus. Immunocytochemical analysis demonstrates that the CARB protein is perinuclear and predominantly associated with the centrosome and mitotic spindle poles. In addition, CARB is also able to associate with cyclin B1, a key regulator of mitosis. However, cyclin B1-CARB complex formation occurs preferentially in the absence of p21. Unexpectedly, overexpression of CARB is associated with a growth-inhibitory and ultimately lethal phenotype in p21(-/-) cells but not in p21(+/+) cells. These data identify a novel mechanism that may underlie the effects of p21 in the G(2)/M phases of the cell cycle.