The NIMA-family kinases Nek9/Nercc1, Nek6 and Nek7 form a signalling module required for mitotic spindle assembly. Nek9, the upstream kinase, is activated during prophase at centrosomes although the details of this have remained elusive. We now identify Plk1 as Nek9 direct activator and propose a two-step activation mechanism that involves Nek9 sequential phosphorylation by CDK1 and Plk1. Furthermore, we show that Plk1 controls prophase centrosome separation through the activation of Nek9 and ultimately the phosphorylation of the mitotic kinesin Eg5 at Ser1033, a Nek6/7 site that together with the CDK1 site Thr926 we establish contributes to the accumulation of Eg5 at centrosomes and is necessary for subsequent centrosome separation and timely mitosis. Our results provide a basis to understand signalling downstream of Plk1 and shed light on the role of Eg5, Plk1 and the NIMA-family kinases in the control of centrosome separation and normal mitotic progression.

Summary Nek6 and Nercc1/Nek9 belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase/metaphase arrest, and depletion of XNercc from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5, a kinesin necessary for spindle bipolarity. Nek6 phosphorylates Eg5 at several sites in vitro, and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. While Cdk1 phosphorylates nearly all Eg5 during mitosis at Thr926, Nek6 phosphorylates ~3% of Eg5, primarily at the spindle poles. Eg5 depletion arrests cells with a monopolar spindle; this can be rescued by Eg5 wildtype but not by Eg5(Thr926Ala). Eg5(Ser1033Ala) rescues half as well as wildtype whereas an Eg5(Ser1033Asp) mutant is nearly as effective. Thus Nek6 phosphorylates a subset of Eg5 polypeptides during mitosis at a conserved site, whose phosphorylation is critical for the mitotic function of Eg5.

Genetic variants affecting pancreatic islet enhancers are central to T2D risk, but the gene targets of islet enhancer activity are largely unknown. We generate a high-resolution map of islet chromatin loops using Hi-C assays in three islet samples and use loops to annotate target genes of islet enhancers defined using ATAC-seq and published ChIP-seq data. We identify candidate target genes for thousands of islet enhancers, and find that enhancer looping is correlated with islet-specific gene expression. We fine-map T2D risk variants affecting islet enhancers, and find that candidate target genes of these variants defined using chromatin looping and eQTL mapping are enriched in protein transport and secretion pathways. At IGF2BP2 , a fine-mapped T2D variant reduces islet enhancer activity and IGF2BP2 expression, and conditional inactivation of IGF2BP2 in mouse islets impairs glucose-stimulated insulin secretion. Our findings provide a resource for studying islet enhancer function and identifying genes involved in T2D risk.

The accumulation of γ-tubulin at the centrosomes during maturation is a key mechanism that ensures the formation of two dense microtubule (MT) asters in cells entering mitosis, defining spindle pole positioning and ensuring the faithful outcome of cell division. Centrosomal γ-tubulin recruitment depends on the adaptor protein NEDD1/GCP-WD and is controlled by the kinase Plk1. Surprisingly, and although Plk1 binds and phosphorylates NEDD1 at multiple sites, the mechanism by which this kinase promotes the centrosomal recruitment of γ-tubulin has remained elusive. Using Xenopus egg extracts and mammalian cells, we now show that it involves Nek9, a NIMA-family kinase required for normal mitotic progression and spindle organization. Nek9 phosphorylates NEDD1 on Ser377 driving its recruitment and thereby that of γ-tubulin to the centrosome in mitotic cells. This role of Nek9 requires its activation by Plk1-dependent phosphorylation but is independent from the downstream related kinases Nek6 and Nek7. Our data contribute to understand the mechanism by which Plk1 promotes the recruitment of γ-tubulin to the centrosome in dividing cells and position Nek9 as a key regulator of centrosome maturation.

Background Type 2 diabetes (T2D) is a common metabolic disease. Variants in human IGF2 mRNA binding protein 2 ( IMP2/IGF2BP2) are associated with increased risk of T2D. IMP2 contributes to T2D susceptibility primarily through effects on insulin secretion. However, the underlying mechanism is not known. Methods To understand the role of IMP2 in insulin secretion and T2D pathophysiology, we generated Imp2 pancreatic β-cell specific knockout mice (βIMP2KO) by recombining the Imp2 flox allele with Cre recombinase driven by the rat insulin 2 promoter. We further characterized metabolic phenotypes of βIMP2KO mice and assessed their β-cell functions. Results The deletion of IMP2 in pancreatic β-cells leads to reduced compensatory β-cell proliferation and function. Mechanically, IMP2 directly binds to Pdx1 mRNA and stimulates its translation in an m6A dependent manner. Moreover, IMP2 orchestrates IGF2-AKT-GSK3β-PDX1 signaling to stable PDX1 polypeptides. In human EndoC-βH1 cells, the over-expression of IMP2 is capable to enhance cell proliferation, PDX1 protein level and insulin secretion. Conclusion Our work therefore reveals IMP2 as a critical regulator of pancreatic β-cell proliferation and function; highlights the importance of posttranscriptional gene expression in T2D pathology.