Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. The Ser-Thr VRK1 (vaccinia-related kinase 1) kinase, whose activity is also cell cycle regulated, interacts with and phosphorylates coilin regulating assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and of CBs formation, which are rescued with an active VRK1, but not by kinase-dead VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. We conclude that VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome and propose a model of CB dynamics.

Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH) is an infantile SMA variant with additional manifestations, particularly severe microcephaly. We previously identified a nonsense mutation in Vaccinia-related kinase 1 (VRK1), R358X, as a cause of SMA-PCH.VRK1-R358X is a rare founder mutation in Ashkenazi Jews, and additional mutations in patients of different origins have recently been identified. VRK1 is a nuclear serine/threonine protein kinase known to play multiple roles in cellular proliferation, cell cycle regulation, and carcinogenesis. However, VRK1 was not known to have neuronal functions before its identification as a gene mutated in SMA-PCH. Here we show thatVRK1-R358X homozygosity results in lack of VRK1 protein, and demonstrate a role for VRK1 in neuronal migration and neuronal stem cell proliferation. Using shRNAin uteroelectroporation in mice, we show thatVrk1knockdown significantly impairs cortical neuronal migration, and affects the cell cycle of neuronal progenitors. Expression of wild-type human VRK1 rescues both proliferation and migration phenotypes. However, kinase-dead human VRK1 rescues only the migration impairment, suggesting the role of VRK1 in neuronal migration is partly noncatalytic. Furthermore, we found that VRK1 deficiency in human and mouse leads to downregulation of amyloid-β precursor protein (APP), a known neuronal migration gene. APP overexpression rescues the phenotype caused byVrk1knockdown, suggesting that VRK1 affects neuronal migration through an APP-dependent mechanism.

NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (-/-) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (-/-) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (-/-) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR.

Very rare polymorphisms in the human VRK1 (vaccinia-related kinase 1) gene have been identified in complex neuromotor phenotypes associated to spinal muscular atrophy (SMA), pontocerebellar hypoplasia (PCH), microcephaly, amyotrophic lateral sclerosis (ALS) and distal motor neuron dysfunctions. The mechanisms by which these VRK1 variant proteins contribute to the pathogenesis of these neurological syndromes are unknown. The syndromes are manifested when both of these rare VRK1 polymorphic alleles are implicated, either in homozygosis or compound heterozygosis. In this report, to identify the common underlying pathogenic mechanism of VRK1 polymorphisms, we have studied all human VRK1 variants identified in these neurological phenotypes from a biochemical point of view by molecular modeling, protein stability and kinase activity assays. Molecular modelling predicted that VRK1 variant proteins are either unstable or have an altered kinase activity. The stability and kinase activity of VRK1 pathogenic variants detected two groups. One composed by variants with a reduced protein stability: R133C, R358X, L195V, G135R and R321C. The other group includes VRK1variants with a reduced kinase activity tested on several substrates: histones H3 and H2AX, p53, c-Jun, coilin and 53BP1, a DNA repair protein. VRK1 variants with reduced kinase activity are H119R, R133C, G135R, V236M, R321C and R358X. The common underlying effect of VRK1 pathogenic variants with reduced protein stability or kinase activity is a functional insufficiency of VRK1 in patients with neuromotor developmental syndromes. The G135 variant cause a defective formation of 53BP1 foci in response to DNA damage, and loss Cajal bodies assembled on coilin.

GDAP1 pathogenic variants cause Charcot-Marie-Tooth (CMT) disease, the most common hereditary motor and sensory neuropathy. CMT-GDAP1 can be axonal or demyelinating, with autosomal dominant or recessive inheritance, leading to phenotypic heterogeneity. Recessive GDAP1 variants cause a severe phenotype, whereas dominant variants are associated with a milder disease course. GDAP1 is an outer mitochondrial membrane protein involved in mitochondrial membrane contact sites (MCSs) with the plasmatic membrane, the endoplasmic reticulum (ER), and lysosomes. In GDAP1-deficient models, the pathophysiology includes morphological defects in mitochondrial network and ER, impaired Ca2+ homeostasis, oxidative stress, and mitochondrial MCSs defects. Nevertheless, the underlying pathophysiology of dominant variants is less understood. Here, we study the effect upon mitochondria–lysosome MCSs of two GDAP1 clinical variants located in the α-loop interaction domain of the protein. p.Thr157Pro dominant variant causes the increase in these MCSs that correlates with a hyper-fissioned mitochondrial network. In contrast, p.Arg161His recessive variant, which is predicted to significantly change the contact surface of GDAP1, causes decreased contacts with more elongated mitochondria. Given that mitochondria–lysosome MCSs regulate Ca2+ transfer from the lysosome to mitochondria, our results support that GDAP1 clinical variants have different consequences for Ca2+ handling and that could be primary insults determining differences in severity between dominant and recessive forms of the disease.