Regular articleCDK5-mediated phosphorylation and autophagy of RKIP regulate neuronal death in Parkinson's disease
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.
Section snippets
Materials and methods
For details of the reagents, antibodies, animal models used, neuronal primary cultures, transfection, cdk5 RNA interference lentivirus, immunoprecipitation, pull-down assays, CDK5/p35 in vitro kinase assay, lysosome-binding plus uptake assay, luciferase reporter assay, cell survival assay, cell cycle analysis, fluorescence microscopy, real-time polymerase chain reaction, and statistical analysis, please see Supplementary Data.
CDK5 phosphorylates RKIP at Thr42 in vitro and in vivo
To verify a kinase-substrate relationship, we analyzed the protein sequence of RKIP with 2 bioinformatic tools, GPS 2.1 (Xue et al., 2011) and Scansite (http://scansite.mit.edu/), and we identified a candidate site, the threonine residue at position 42 of RKIP (Thr42), which was highly conserved across sequences of all available species in the examined databases (Fig. 1A). This site was identified in mass spectrometry assays of mouse brain samples and human HeLa cells but was never linked to
Discussion
RKIP is the primary negative mediator of the ERK/MAPK pathway (Yeung et al., 1999), and the downregulation of its expression contributes to many pathologies, including cancers and neurodegenerative diseases (Escara-Wilke et al., 2012, George et al., 2006, Maki et al., 2002). The mechanism of the downregulation of RKIP and its functions in nervous system have not been well recognized; we, therefore, addressed these questions in this study. Here, we report that RKIP is a substrate of the kinase
Disclosure statement
The authors declare no competing interest.
Acknowledgements
We are thankful to Kam C. Yeung (The University of Toledo, USA) for the plasmids of full-length RKIP and myc-tagged Raf-1. This work was partially sponsored by projects 30900423 (BT) and 31071208 (BT) supported by National Nature Science Foundation of China, project 20100142110053 (BT) supported by Specialized Research Fund for the Doctoral Program of Higher Education (grant no. 20100142110053), and project supported by Program for New Century Excellent Talents in University (BT).
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