14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression

Abstract

More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering.

Depletion of keratin 8/18 modulates oncogenic potential by governing multiple signaling pathways

“…This might be an important link between K8 downregulation and increased sensitivity of A431 cells toward apoptotic stimuli. Aside from this, upregulation in 14-3-3c levels and decreased NF-jB activity, further supports this hypothesis [68,69].…”

14-3-3γ prevents centrosome duplication by inhibiting NPM1 function

“…Given these observations, we decided to test if D129 and E136 in 14-3-3γ are essential for forming a complex with protein ligands and affecting ligand function. Previous work has demonstrated that 14-3-3γ is found in centrosome fractions (50) and that, loss of 14-3-3γ leads to an increase in centrosome number in human cell lines (24). We wanted to test if mutants of these conserved residues, D129 and E136 in 14-3-3γ, would have any effect on centrosome number.…”

“…To test if the effect of the mutants is modulated by the presence of the endogenous protein, we wished to determine the effect of these mutants on centrosome number in the absence of 14-3-3γ as cells with a decrease in 14-3-3γ expression show centrosome amplification (24). We transfected the HCT116 derived vector control or 14-3-3γ knockdown cells with the constructs described above and determined centrosome number in mitotic cells.…”

“…NPM1 associates exclusively with unduplicated centrosomes and expression of a phospho-site mutant of NPM1, T199A, results in the retention of NPM1 at the centrosome and inhibits centrosome duplication (22,23). In contrast, the expression of a phospho-mimetic mutant, T199D, leads to centrosome amplification (24). During Sphase, Polo-Like Kinase 4 (Plk4) recruitment at the site of procentriole biogenesis initiates centriole duplication (25,26).…”

“…Binding of 14-3-3 proteins to their ligands affects their localization, conformation, stability and function (33,(45)(46)(47)(48)(49)). 14-3-3γ and 14-3-3ε co-purify with the centrosomal fraction, (50,51) and loss of 14-3-3γ and 14-3-3ε leads to centrosome amplification in multiple cell lines due to the premature activation of CDC25C during interphase, leading to premature CDK1 activation and the increased phosphorylation of NPM1 at T199, which results in centrosome amplification (24).…”