Abstract
Although extracellular signal-related kinase 1/2 (ERK 1/2) activity is generally associated with cell survival, prolonged ERK activation induced by oxidative stress also mediates neuronal cell death. Here we report that oxidative stress-induced cyclin-dependent kinase 5 (CDK5) activation stimulates neuroprotective signaling via phosphorylation of vaccinia-related kinase 3 (VRK3) at Ser 108. The binding of vaccinia H1-related (VHR) phosphatase to phosphorylated VRK3 increased its affinity for phospho-ERK and subsequently downregulated ERK activation. Overexpression of VRK3 protected human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced apoptosis. However the CDK5 was unable to phosphorylate mutant VRK3, and thus the mutant forms of VRK3 could not attenuate apoptotic process. Suppression of CDK5 activity results in increase of ERK activation and elevation of proapoptotic protein Bak expression in mouse cortical neurons. Results from VRK3-deficient neurons were further confirmed the role of VRK3 phosphorylation in H2O2-evoked ERK regulation. Importantly, we showed an association between phospho-VRK3 levels and the progression of human Alzheimer’s disease (AD) and Parkinson’s disease (PD). Together our work reveals endogenous protective mechanism against oxidative stress-induced neuronal cell death and suggest VRK3 as a potential therapeutic target in neurodegenerative diseases.
Highlights
Protein kinases are crucial components involved in the integration of signal transduction networks, and the signaling pathways of mitogen-activated protein kinases (MAPKs)/Ras-Raf-MEK-ERK, which controls various cell responses, such as proliferation, differentiation, and metabolism, is most well-known in mammalian cells[1,2]
The MAPK signaling pathways have been implicated in Alzheimer’s disease (AD) pathogenesis through divergent mechanisms including induction of neuronal apoptosis[18,19,20] and phosphorylation of amyloid precursor protein (APP) and tau, which influence the cleavage of APP to generate amyloid-β (Aβ) and the formation of neurofibrillary tangle (NFT)s, two hallmarks of AD19,21
To assess whether Vaccinia-related kinase 3 (VRK3) is phosphorylated by cyclin-dependent kinase 5 (CDK5), and to identify the region of CDK5-mediated phosphorylation of VRK3, we performed an in vitro kinase assay by incubating GST-tagged recombinant full-length VRK3 and its fragments (F1, amino acids 1 to 165; F2, amino acids 166 to 340; F3, amino acids 166 to 474) with purified CDK5 complexed with its activator p35 (Fig. 1c)
Summary
Protein kinases are crucial components involved in the integration of signal transduction networks, and the signaling pathways of mitogen-activated protein kinases (MAPKs)/Ras-Raf-MEK-ERK, which controls various cell responses, such as proliferation, differentiation, and metabolism, is most well-known in mammalian cells[1,2]. Sustained ERK activation causes its nuclear translocation and contributes to neuronal cell death via transcriptional regulation of pro-apoptotic proteins[3]. Vaccinia-related kinase 3 (VRK3), a member of the VRK family, is widely expressed in human tissues and increases VHR phosphatase activity through a direct binding[6]. The identity of CDK5 substrates that link between CDK5 and the MAPK signaling pathway remains largely unknown Reactive oxygen species, such as hydrogen peroxide (H2O2), are generated during normal metabolic processes[13], and play crucial roles in cellular signaling cascades[14]. We found that CDK5-mediated VRK3 phosphorylation at Ser 108 suppresses H2O2-induced prolonged ERK activation and subsequent cell death via enhancement of ERK phosphatase VHR activity. We can conclude that phosphorylated VRK3 plays a neuroprotective role through regulation of protein activities that mediate cell death and stress resistance
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