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

14-3-3 proteins bind to ligands via phospho-serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between 14-3-3 proteins and their ligands remain unclear. We identified two conserved acidic residues in the 14-3-3 peptide-binding pocket (D129 and E136) that potentially regulate complex formation and dissociation. Altering these residues to alanine led to opposing effects on centrosome duplication. D129A inhibited centrosome duplication, whereas E136A stimulated centrosome amplification. These results were due to the differing abilities of these mutant proteins to form a complex with NPM1. Inhibiting complex formation between NPM1 and 14-3-3γ led to an increase in centrosome duplication and over-rode the ability of D129A to inhibit centrosome duplication. We identify a novel role of 14-3-3γ in regulating centrosome licensing and a novel mechanism underlying the formation and dissociation of 14-3-3 ligand complexes dictated by conserved residues in the 14-3-3 family.

Centrosome Amplification and Tumorigenesis: Cause or Effect?

Centrosome amplification is a feature of multiple tumour types and has been postulated to contribute to both tumour initiation and tumour progression. This chapter focuses on the mechanisms by which an increase in centrosome number might lead to an increase or decrease in tumour progression and the role of proteins that regulate centrosome number in driving tumorigenesis.

14-3-3 proteins mediate the localization of Centrin2 to centrosome.

14-3-3ε and 14-3-3γ localize to the centrosome and regulate centrosome duplication, by inhibiting cdc25C function. As 14-3-3γ and 14-3-3ε form a complex with centrosomal proteins, we asked if this ability was required to regulate centrosome duplication. The results in this report demonstrate that 14-3-3ε and 14-3-3γ form a complex with Centrin2 and that the binding site is located in the N-terminal EF hand in Centrin2, EF1. A Centrin2 mutant that does not form a complex with 14-3-3 proteins displays a punctate cytoplasmic localization and does not localize to the centrosome. These results suggest that in addition to negatively regulating centrosome duplication as previously reported, 14-3-3 proteins might also be required for centriole biogenesis by regulating the localization of Centrin2 at the centrosome.

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

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.