Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function

Abstract

The role of the mitotic phosphorylation of the amino (NH 2 ) terminus of Centromere Protein A (CENP-A), the histone variant epigenetic centromeric marker, remains elusive. Here, we show that the NH 2 terminus of human CENP-A is essential for mitotic progression and that localization of CENP-C, another key centromeric protein, requires only phosphorylation of the CENP-A NH 2 terminus, and is independent of the CENP-A NH 2 terminus length and amino acid sequence. Mitotic CENP-A nucleosomal complexes contain CENP-C and phosphobinding 14-3-3 proteins. In contrast, mitotic nucleosomal complexes carrying nonphosphorylatable CENP-A–S7A contained only low levels of CENP-C and no detectable 14-3-3 proteins. Direct interactions between the phosphorylated form of CENP-A and 14-3-3 proteins as well as between 14-3-3 proteins and CENP-C were demonstrated. Taken together, our results reveal that 14-3-3 proteins could act as specific mitotic “bridges,” linking phosphorylated CENP-A and CENP-C, which are necessary for the platform function of CENP-A centromeric chromatin in the assembly and maintenance of active kinetochores.

Posttranslational mechanisms controlling centromere function and assembly

“…Mutation of this site leads to errors in chromosome segregation and cytokinesis [5,6]. Ser7 phosphorylation is mediated by the Aurora kinases, and has been shown to indirectly recruit CENP-C through phospho-binding protein 14-3-3 [4–7]; although, the primary direct binding site for CENP-C is the carboxy terminus of CENP-A [8,9]. …”

CENP-A K124 Ubiquitylation Is Required for CENP-A Deposition at the Centromere

“…The phosphorylation of CENP-A serine 7 (S7) in human cells by Aurora B in the final stages of cytokinesis has been described (Zeitlin et al, 2001), and the phosphorylation of CenH3 serine 50 (S50) in maize has been characterized during chromosome segregation (Zhang et al, 2005). Studies on the CENP-A S7A mutant show that the phosphorylated human CENP-A nucleosomes might be “bridged” to CENP-C via the phospho-binding 14-3-3 proteins in mitosis (Goutte-Gattat et al, 2013). Bailey et al reported 3 PTMs on human CENP-A (trimethylation of Gly1 and phosphor-ylation of Ser16 and Ser18), suggesting that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere (Bailey et al, 2013).…”

Posttranslational modifications of CENP-A: marks of distinction

“…In this way, CENP-A Ser-7 phosphorylation and Aurora-B appear to function bi-directionally, where Aurora-B maintains the Ser-7 phosphorylation initiated by Aurora-A and phospho-CENP-A in turn recruits Aurora-B to the inner centromere. Mutational analysis further substantiates the role of CENP-A Ser-7 phosphorylation in chromosome segregation and cytokinesis, as CENP-A phospho-defective mutants fail to rescue the mitotic defects caused by the loss of endogenous CENP-A (Goutte-Gattat et al 2013). …”

“…CENP-A phosphorylation, acetylation, and trimethylation appear to affect the recruitment of CCAN-associated proteins to the centromere. Previously, Ser-7 phosphorylation was found to be involved in loading of CENP-C onto centromeres through phospho-binding protein 14-3-3, which acts as an intermolecular bridge between phospho-CENP-A and CENP-C (Goutte-Gattat et al 2013) (Fig. 2).…”