CDK5-mediated phosphorylation and autophagy of RKIP regulate neuronal death in Parkinson's disease

Abstract

Raf kinase inhibitor protein (RKIP) is a major negative mediator of the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. The downregulation of RKIP is correlated with many cancers, but the mechanisms that underlie this downregulation and its roles in the nervous system remain unclear. Here, we demonstrate that RKIP is a substrate of cyclin-dependent kinase 5 (CDK5) in neurons and that the phosphorylation of RKIP at T42 causes the release of Raf-1. Moreover, T42 phosphorylation promotes the exposure and recognition of the target motif “KLYEQ” in the C-terminus of RKIP by chaperone Hsc70 and the subsequent degradation of RKIP via chaperone-mediated autophagy (CMA). Furthermore, in the brain sample of Parkinson's disease (PD) patients and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride–induced and transgenic PD models, we demonstrate that CDK5-mediated phosphorylation and autophagy of RKIP are involved in the overactivation of the ERK/MAPK cascade, leading to S-phase reentry and neuronal loss. These findings provide evidence for the role of the CDK5/RKIP/ERK pathway in PD pathogenesis and suggest that this pathway may be a suitable therapeutic target in PD.

Introduction

In unstimulated cells, Raf kinase inhibitor protein (RKIP) is bound to Raf, thus preventing the phosphorylation of MEK1 by Raf-1 and blocking the Ras-Raf-MEK-extracellular signal-related kinase (ERK) signal transduction cascade (Yeung et al., 1999). Until now, research into RKIP has primarily focused on oncology, exploring the roles of this protein in cancers that are dependent on or independent of the effects of the ERK pathway (Al-Mulla et al., 2011, Beach et al., 2008, Beshir et al., 2010, Escara-Wilke et al., 2012). The loss of RKIP expression in cancer has long been known, and this downregulation has proven to be closely related to many significant aspects of tumors, including proliferation, metastasis, and prognosis (Beshir et al., 2010, Escara-Wilke et al., 2012, Granovsky and Rosner, 2008, Keller, 2011, Keller et al., 2004). However, the mechanism that underlies this downregulation is unclear, and the understanding of RKIP in other contexts is quite limited, particularly in the nervous system.

There are few studies regarding RKIP in the nervous system. Those that do exist include reports that the highest RKIP expression levels are detected in the brains and testes of mice and that RKIP knockout mice develop learning and olfaction deficits within a year, reflecting the function of RKIP in behavior modulation (Theroux et al., 2007). The knockdown of RKIP in SH-SY5Y cells has also been demonstrated to affect their differentiation (Hellmann et al., 2010). Moreover, RKIP expression was observed to be reduced in Alzheimer's disease (Maki et al., 2002) and was correlated with amyloid-β plaque formation (George et al., 2006). However, the mechanisms that underlie these phenomena remain poorly understood.

Here, we aim to explore the mechanism of RKIP downregulation and to identify its roles in the nervous system, especially in neurodegenerative diseases such as Parkinson's disease (PD). Certain reports have demonstrated a modification of RKIP by protein kinase C. This phosphorylation of S153 mediates a shift of RKIP from Raf-1 to G-protein–coupled receptor kinase (GRK)2, an inhibitor of G-protein–coupled receptor signaling, leading to the downstream signal conduction of the mitogen-activated protein kinase (MAPK) cascade (Corbit et al., 2003, Lorenz et al., 2003). With this phosphorylation-based modification of RKIP, the activity of ERK/MAPK is delicately regulated. The role of the ERK/MAPK cascade in postmitotic neurons has not been thoroughly demonstrated, although it is generally believed to promote cell survival (Bonni et al., 1999). Here, we discuss the influence of the ERK cascade on neurodegenerative diseases in the context of models of PD. We also intend to explore how RKIP regulates ERK signaling and how RKIP is downregulated.

In the present study, we provide evidence for a new pattern of phosphorylation-based regulation of RKIP by cyclin-dependent kinase 5 (CDK5). CDK5 is a unique member of the CDK family that is primarily active in neurons and is assisted by the cofactors P35 or P39. CDK5 controls multiple cellular events in postmitotic neurons and participates in neuronal disorders, including neurodegenerative diseases (Cheung and Ip, 2012, Dhavan and Tsai, 2001, Jessberger et al., 2009, Tian et al., 2009, Wong et al., 2011), and the overactivation of CDK5 in many pathologic conditions is mostly mediated by cleavage of P35 to P25, a stronger cofactor of CDK5, because of the activation of calpain in a Ca²⁺-dependent manner (Cruz et al., 2003, Patrick et al., 1999). The discovery of a relationship between CDK5 and RKIP enhances our understanding of the roles of the ERK cascade in neurons and further hints at the mechanism of RKIP downregulation.