Phosphorylation of CHIP at Ser20 by Cdk5 promotes tAIF-mediated neuronal death

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase and its dysregulation is implicated in neurodegenerative diseases. Likewise, C-terminus of Hsc70-interacting protein (CHIP) is linked to neurological disorders, serving as an E3 ubiquitin ligase for targeting damaged or toxic proteins for proteasomal degradation. Here, we demonstrate that CHIP is a novel substrate for Cdk5. Cdk5 phosphorylates CHIP at Ser20 via direct binding to a highly charged domain of CHIP. Co-immunoprecipitation and ubiquitination assays reveal that Cdk5-mediated phosphorylation disrupts the interaction between CHIP and truncated apoptosis-inducing factor (tAIF) without affecting CHIP''s E3 ligase activity, resulting in the inhibition of CHIP-mediated degradation of tAIF. Lentiviral transduction assay shows that knockdown of Cdk5 or overexpression of CHIP^S20A, but not CHIP^WT, attenuates tAIF-mediated neuronal cell death induced by hydrogen peroxide. Thus, we conclude that Cdk5-mediated phosphorylation of CHIP negatively regulates its neuroprotective function, thereby contributing to neuronal cell death progression following neurotoxic stimuli.Cyclin-dependent kinase 5 (Cdk5) is a member of cyclin-dependent kinase that is crucial for the regulation of various post-mitotic cellular processes in the nervous system.^{1, 2, 3, 4} Although Cdk5 is activated upon direct interaction with a neural-specific activator p35 in normal condition,¹ it can be hyperactivated in numerous pathological conditions by the conversion of p35 to p25 via calpain.⁵ Therefore, dysregulation of Cdk5 activity has been linked to an array of neurodegenerative diseases.^{2, 5, 6} Consequently, Cdk5-mediated phosphorylation of several key intracellular substrates has received significant attention and has been identified to associate with the pathophysiology of neurodegeneration.^{6, 7}Recently, our laboratory made an attempt to identify the novel substrate of Cdk5 and we came across that Cdk5 interacts with C-terminus of Hsc70-interacting protein (CHIP). CHIP is composed of three major domains: (1) an amino-terminal three tetratricopeptide repeat (TPR) domain interacting with Hsc/Hsp70 and Hsp90, (2) a highly charged central domain with unknown functions and (3) a carboxyl-terminal U-box domain conferring E3 ubiquitin ligase activity.^{8, 9, 10} Based on these domains, it has been indicated that CHIP has a crucial role as a molecular co-chaperone in the maintenance of protein homeostasis during cellular stress and recovery.^{8, 11, 12, 13} Indeed, CHIP has been demonstrated to serve as a crucial catalyst for ubiquitination of Hsp70 client proteins targeting for proteasome-dependent degradation.¹¹ The function of CHIP with its molecular chaperones has been well documented to reduce cellular toxicity associated with several neurodegenerative diseases.^{11, 12, 14, 15, 16, 17, 18} However, the mechanisms underlying how function of CHIP is regulated and, if any, the pathophysiological cascades contributing to CHIP-mediated neurodegeneration are not fully elucidated yet.Here, we propose the molecular cascade in which Cdk5 phosphorylation of CHIP at Ser20 disrupts the interaction between CHIP and truncated apoptosis-inducing factor (tAIF), thereby contributes to escaping of tAIF from CHIP-mediated proteasome-dependent degradation, and eventually leads to tAIF-mediated neuronal cell death. We argue that this cascade explains one plausible mechanism as to how dysregulation of Cdk5 leads to neuronal death by negatively regulating CHIP-mediated degradation of toxic molecules.