CDK5 Phosphorylates eNOS at Ser-113 and Regulates NO Production Chien-Hsing Lee, 1 Yin-Win Wei, 2 Yi-Ting Huang, 2 Yuh-Te Lin, 1,3 Yu-Cheng Lee, 4 Kuen-Haur Lee, 4 and Pei-Jung Lu 1 * 1 Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan 2 Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 3 Section of Neurology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 4 Institute of Basic Sciences, National Cheng Kung University, Tainan, Taiwan ABSTRACT Phosphorylation of endothelial nitric oxide synthase (eNOS) is key mechanism in response to various forms of cellular stimulation. Through protein nitration by peroxynitrite, eNOS is believed to be responsible for the major abnormalities in several important neurodegenerative diseases including Alzheimer’s (AD) and Parkinson’s diseases (PD). Recent studies provide important in vivo evidence that hyperactivation of Cdk5 by p25 plays an essential role in the cell death of neurons in experimental models of AD and PD. This study focuses on the functional regulation of eNOS by Cdk5/p35 complex in a phosphorylation dependent manner. Our results showed that Cdk5 can phosphorylate eNOS both in vitro and in vivo. In vitro kinase assay together with the bioinformatic analysis and site direct mutagenesis revealed that Ser-113 is the major phosphorylation site for Cdk5. Most interestingly, the nitrite production was significantly reduced in eNOS and Cdk5/p35 co- transfected SH-SY5Y cells when compared with co-transfection of Cdk5/p35 and S113A. Together, our data suggest that Cdk5 can phosphorylate eNOS at the Ser-113 site and down-regulate eNOS-derived NO levels. J. Cell. Biochem. 110: 112–117, 2010. ß 2010 Wiley-Liss, Inc. KEY WORDS: Cdk5; eNOS; NO; ALZHEIMER’S DISEASES; PARKINSON’S DISEASES N itric oxide (NO) regulates multiple physiological and pathophysiological processes in the nervous system such as learning and memory, neuronal survival and differentiation [De Palma et al., 2008]. NO is generated from NO synthases (NOSs) through the oxidation of a guanidino nitrogen of L-arginine. The NOS isoforms have been identified and divided into two functional classes: constitutive and inducible. The two constitutive isoforms, initially identified in neurons (nNOS) and endothelial cells (eNOS), are expressed in central nervous system neurons [Dinerman et al., 1994; Campese et al., 2007]. In the nervous system, high levels of NO can be either detrimental or beneficial to cell viability and function. The cytotoxic effects of NO may be due to peroxynitrite formation and nitration of tyrosine residues in proteins, or the generation of superoxides [de la Monte et al., 2000]. Moreover, the presence of nitrotyrosination has been described in several neurodegenerative diseases linked to oxidative stress, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) [Guix et al., 2005]. Another important kinase linked to neurodegeneration diseases is cyclin-dependent kinase 5 (Cdk5). Cdk5 is an atypical member of the cyclin-dependent kinase family that has unique functions in the central nervous system. A marked interest among cell biologists and neuroscientists has been generated by the findings that Cdk5 and the regulatory protein p35, also named neuronal Cdk5 activator, which are expressed almost exclusively in postmitotic neurons [Ohshima et al., 2002]. Recent studies provide important in vivo evidence that hyperactivation of Cdk5 by p25 plays an essential role in the phosphorylation of tau and the cell death of neurons in experimental models of AD and PD [Ko et al., 2001; Camins et al., 2006]. Meanwhile, in enhanced NO production in AD and other neurodegenerative diseases implicates also a potential role for eNOS [De Palma et al., 2008]. However, the Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 110:112–117 (2010) 112 Chien-Hsing Lee and Yin-Win Wei contributed equally to this work. Grant sponsor: National Cheng Kung University Hospital; Grant number: NCKUH-9701004; Grant sponsor: National Science Council; Grant numbers: NSC 97-2311-B-006-003-MY3, NSC-98-3112-B-006-012. *Correspondence to: Prof. Pei-Jung Lu, PhD, Institute of Clinical Medicine, National Cheng Kung University, No 138, Sheng-Li Road, Tainan 704, Taiwan. E-mail: pjlu2190@mail.ncku.edu.tw Received 6 October 2009; Accepted 5 January 2010  DOI 10.1002/jcb.22515  ß 2010 Wiley-Liss, Inc. Published online 8 March 2010 in Wiley InterScience (www.interscience.wiley.com).