Site-specific phosphorylation of Fbxw7 by Cdk5/p25 and its resulting decreased stability are linked to glutamate-induced excitotoxicity

Yeon Uk Ko,Chiho Kim,Dana Kim,Juhyung Lee,Young J Oh,Nuri Yun,Yoonkyung Kim

doi:10.1038/s41419-019-1818-4

Abstract

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine protein kinase that regulates brain development and neurodegeneration. Cdk5 is activated by p25 that is generated from calpain-dependent cleavage of p35. The generation of p25 is responsible for the aberrant hyper-activation of Cdk5, which causes neurodegeneration. Using in vitro assays, we discovered that F-box/WD repeat-containing protein 7 (Fbxw7) is a new substrate of Cdk5. Additionally, Cdk5-dependent phosphorylation of Fbxw7 was detected in the presence of p25, and two amino acid residues (S349 and S372) were determined to be major phosphorylation sites. This phosphorylation was eventually linked to decreased stability of Fbxw7. Using a culture model of cortical neurons challenged with glutamate, we confirmed that decreased stability of Fbxw7 was indeed Cdk5-dependent. Furthermore, diminished levels of Fbxw7 led to increased levels of transcription factor AP-1 (c-Jun), a known substrate of Fbxw7. Given that previous reports demonstrate that c-Jun plays a role in accelerating neuronal apoptosis in these pathological models, our data support the concepts of a molecular cascade in which Cdk5-mediated phosphorylation of Fbxw7 negatively regulates Fbxw7 expression, thereby contributing to neuronal cell death following glutamate-mediated excitotoxicity.

Highlights

Cyclin-dependent kinases (CDKs) comprise a family of protein kinases that regulates the cell cycle[1,2]
Phosphorylation of Fbxw[7] by Cdk[5], induced by transient transfection of Fbxw[7] and Cdk[5] with p25 or p35 activator into human embryonic kidney 293 (HEK293) cells, was observed in the presence of Cyclin-dependent kinase 5 (Cdk5)/p25 (Fig. 1a) and to a marginal degree in the presence of Cdk5/p35 (Supplementary Fig. 1a, b). This result could be due to the cellular localization of Fbxw[7], which has been reported to be primarily in the nucleus and cellular vesicle[22], whereas p35 is mainly localized in the plasma membrane by myristolylation[26]
We showed that Cdk5/p25-dependent degradation of Fbxw[7] was blocked in the presence of MG132 (Fig. 4e, f)

Summary

Introduction

Cyclin-dependent kinases (CDKs) comprise a family of protein kinases that regulates the cell cycle[1,2]. One member of the CDK family, Cdk[5], is primarily activated in the brain and plays a role in brain development, neuronal maturation, and synaptic plasticity[3,4]. This member has a unique physiological characteristic compared to other CDKs5. Cdk[5] is activated by neuron-specific regulatory proteins p35 or p39 instead of cyclin[6,7]. Cdk5/p25 acts as a regulator of cell death by phosphorylating tau and p5310,11.

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