Original Contribution Serine-Threonine Kinase 38 is regulated by Glycogen Synthase Kinase-3 and modulates oxidative stress-induced cell death Atsushi Enomoto a, ⁎, Naoki Kido a , Michihiko Ito b , Nobuhiko Takamatsu b , Kiyoshi Miyagawa a a Laboratory of Molecular Radiology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113–0033, Japan b Department of Biosciences, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 252–0373, Japan abstract article info Article history: Received 27 May 2011 Revised 20 October 2011 Accepted 5 November 2011 Available online 13 November 2011 Keywords: STK38 GSK-3 Phosphorylation Oxidative stress JNK Free radicals Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-like family. In the present study, we investigated the regulatory mechanism of STK38 and assessed its role in the cellular stress response. Among various environmental stresses, STK38 was specifically activated by H 2 O 2 , and the phospha- tidylinositol 3-kinase inhibitor wortmannin or AKT inhibitor IV suppressed this activation. STK38 was also ac- tivated by a constitutively active AKT1 or by GSK-3β inhibitor VII. The phosphorylation level of GSK-3β was correlated with the STK38 activity, in response to various stimuli and in different cell lines. Co- immunoprecipitation analysis revealed that GSK-3β physically interacted with STK38 in cells. GSK-3β over- expression inhibited the H 2 O 2 -stimulated STK38 activity. GSK-3β phosphorylated STK38 on residues S6 and T7 in vitro, depending largely on a PKA-mediated priming phosphorylation of STK38 on residues S10 and S11, respectively. STK38's H 2 O 2 -stimulated activity was enhanced by alanine substitution at its priming sites and/ or at S6 and T7, and it was partially reduced by a phosphomimetic mutation at S6 or T7. STK38 knockdown enhanced the H 2 O 2 -induced JNK phosphorylation and cell death. Our results indicate that that GSK-3β in- hibits STK38's full activation, and suggest that STK38 activation is required to prevent cell death in response to oxidative stress. © 2011 Elsevier Inc. All rights reserved. Introduction STK38 ( Serine- Threonine Kinase 38, also known as NDR1; Nuclear Dbf-2 Related 1, GenBank accession number NP009202) is a serine/ threonine protein kinase belonging to a subclass of the protein kinase A (PKA)/PKG/PKC-like (AGC) family [1-3], which includes cAMP- dependent kinase, protein kinase B, and protein kinase C. The members of the STK38 family comprise Drosophila melanogaster TRC, Schizosac- charomyces pombe Orb6, Saccharomyces cerevisiae Cbk1 and Dbf2, the mammalian STK38, STK38L, LATS1 (Large tumor suppressor-1), and LATS2 [1-3]. Of these, Cbk1 and Orb6 are involved in regulating cell morphology [4,5], and Dbf2 is a cell cycle-regulated kinase whose activ- ity is required for progression through anaphase [6]. In mammals, LATS1/2 function as tumor suppressors, whereas STK38 and STK38L may function as proto-oncogenes [3]. Recently, STK38 was implicated in centrosome duplication [7], and we previously reported that STK38 interacts with the JNK kinase kinases MEKK1/2, and negatively regu- lates their signaling pathways [8]. STK38 and its relatives have a conserved N-terminal regulatory domain (NTR), a catalytic kinase domain containing the activation segment phosphorylation site, and a C-terminal regulatory domain [3]. The N-terminal regulatory region of the STK38 family includes a number of conserved basic hydrophobic residues and is predicted to form an amphiphilic α-helix. The C-terminal regulatory domain of the STK38 family protein kinases contains a hydrophobic motif that houses phosphorylation sites. As with many AGC kinases, STK38 is phosphorylated on two conserved residues, one in the activation loop of the catalytic domain and one in the C-terminal hydrophobic domain [9]. Phosphorylation of both residues is essential for the full activation of STK38 [10,11]. One protein kinase, mammalian STE20- like kinase 3 (MST3), has been shown to specifically phosphorylate only the hydrophobic motif of STK38s, but not the activation segment [11]. Otherwise, how STK38 is regulated by upstream kinases is largely unknown. In this study, we show that STK38 is activated by H 2 O 2 and pro- vide genetic, enzymatic, and pharmacological evidence that STK38 is regulated by GSK-3β. Moreover, we describe the role of STK38 in H 2 O 2 -induced cell death. Free Radical Biology & Medicine 52 (2012) 507–515 Abbreviations: STK38, Serine Threonine Kinase 38; NDR, Nuclear Dbf-2 Related; GSK-3, Glycogen Synthase Kinase-3; PKA, Protein Kinase A; CA-AKT1, Constitutive active AKT1; NTR domain, N-terminal regulatory domain. ⁎ Corresponding author. Fax: + 81 3 5841 3013. E-mail address: aenomoto-tky@umin.ac.jp (A. Enomoto). 0891-5849/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2011.11.006 Contents lists available at SciVerse ScienceDirect Free Radical Biology & Medicine journal homepage: www.elsevier.com/locate/freeradbiomed