Functional impact of Aurora A-mediated phosphorylation of HP1γ at serine 83 during cell cycle progression

Abstract

BackgroundPrevious elegant studies performed in the fission yeast Schizosaccharomyces pombe have identified a requirement for heterochromatin protein 1 (HP1) for spindle pole formation and appropriate cell division. In mammalian cells, HP1γ has been implicated in both somatic and germ cell proliferation. High levels of HP1γ protein associate with enhanced cell proliferation and oncogenesis, while its genetic inactivation results in meiotic and mitotic failure. However, the regulation of HP1γ by kinases, critical for supporting mitotic progression, remains to be fully characterized.ResultsWe report for the first time that during mitotic cell division, HP1γ colocalizes and is phosphorylated at serine 83 (Ser83) in G2/M phase by Aurora A. Since Aurora A regulates both cell proliferation and mitotic aberrations, we evaluated the role of HP1γ in the regulation of these phenomena using siRNA-mediated knockdown, as well as phosphomimetic and nonphosphorylatable site-directed mutants. We found that genetic downregulation of HP1γ, which decreases the levels of phosphorylation of HP1γ at Ser83 (P-Ser83-HP1γ), results in mitotic aberrations that can be rescued by reintroducing wild type HP1γ, but not the nonphosphorylatable S83A-HP1γ mutant. In addition, proliferation assays showed that the phosphomimetic S83D-HP1γ increases 5-ethynyl-2´-deoxyuridine (EdU) incorporation, whereas the nonphosphorylatable S83A-HP1γ mutant abrogates this effect. Genome-wide expression profiling revealed that the effects of these mutants on mitotic functions are congruently reflected in G2/M gene expression networks in a manner that mimics the on and off states for P-Ser83-HP1γ.ConclusionsThis is the first description of a mitotic Aurora A-HP1γ pathway, whose integrity is necessary for the execution of proper somatic cell division, providing insight into specific types of posttranslational modifications that associate to distinct functional outcomes of this important chromatin protein.

Combined AURKA and H3K9 Methyltransferase Targeting Inhibits Cell Growth By Inducing Mitotic Catastrophe

“…An enhanced effect of the combination to inhibit PDAC growth is also observed with 3D spheroid and organoid cultures, as well as xenografts in vivo (Figure 3). This is consistent with the fact that these targets are in the same biological pathway that aids to the proper completion of mitosis [12]. Upon cell cycle arrest caused by AurkA inhibition, the mitotic machinery is exposed longer to targeting by the H3K9me pathway, which during mitosis regulates centromere structure.…”

“…Western blots were performed as previously described [12]. Membranes were blocked in 3% BSA/TBST and incubated overnight at 4°C with primary antibody (Supplementary Table 1).…”

“…We have shown that a member of the methyl lysine 9 histone 3 (H3K9me) pathway, HP1γ (CBX3), is phosphorylated by AurkA to support normal mitotic progression [12]. The K9H3me histone mark to which HP1 binds is established by the H3K9 histone methyltransferases (HMTs) G9a, GLP, SUV39H1 and SUV39H2.…”

Shigella flexneri targets the HP1γ subcode through the phosphothreonine lyase OspF

“…Previous reports have detected high levels of HP1γ protein associated with enhanced cell proliferation and oncogenesis in colon, breast, and cervical cancers (Takanashi et al , ; Abe et al , ; Slezak et al , ). Also, HP1γ S83 was reported enriched at the mitotic spindle, suggesting an additional role in proper mitotic cell division (Grzenda et al , ). Our in vivo analysis indicated that terminally differentiated columnar absorptive enterocytes were devoid of HP1γ, while the expression was detectable in the basal level of the crypt that contained the proliferative cell compartment.…”

Atypical heterochromatin organization and replication are rapidly acquired by somatic cells following fusion-mediated reprogramming by mouse ESCs

“…It has been proposed that HP1α localizes to heterochromatin by binding to the tri-methylated tails of histone H3 at Lysine 9 (H3K9me3). [26][27][28] It has also been shown that Aurora B-mediated phosphorylation of the adjacent H3 serine 10 residues displaces HP1 binding from mitotic heterochromatin and forms part of a binary "methyl/phospho" switch that is critical for proper cell division in yeast 29 and mammalian cells. 30 In mammals, recruitment of the AuroB/AIM-1 kinase complex to HP1α sites in heterochromatin is reported to occur in G 2 and is important for the G 2 /M transition.…”