Serine–Threonine Kinase 38 regulates CDC25A stability and the DNA damage-induced G2/M checkpoint

Highlights

• STK38 phosphorylates CDC25A at Ser-76 in vivo and in vitro.

• Phosphorylation of Ser-76 regulates CDC25A stability.

• STK38 modulates DNA-damage-induced G2/M checkpoint activation.

Abstract

Cells respond to DNA damage by activating protein kinase-mediated signaling pathways that promote cell-cycle arrest, DNA repair, or apoptosis. A key regulator of cell-cycle arrest is the CDC25A (cell division cycle 25 homologue A) phosphatase. CDC25A normally plays a pivotal role in regulating the G1/S and G2/M transitions by dephosphorylating and activating cyclin/cyclin-dependent kinase (CDK) complexes; however, CDC25A is specifically degraded in response to DNA damage. Here, we demonstrate that the depletion of serine–threonine kinase 38 (STK38) prevents the DNA-damage-induced degradation of CDC25A and subsequent G2 arrest, and that STK38 directly phosphorylates CDC25A at Ser-76, resulting in CDC25A's degradation. Taken together, these results indicate that the STK38-mediated phosphorylation of CDC25A at Ser-76 and the subsequent degradation of CDC25A are required to promote DNA damage-induced G2/M checkpoint activation.

Introduction

DNA damage and replication blocks activate an elaborate signaling pathway in cells known as the DNA damage stress response pathway [1], [2], [3]. This pathway is initiated by the sensing of DNA lesions, followed by the activation of damage-specific signaling. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) are two members of the phosphoinositide-3-kinase-related protein kinase (PIKK) family that, in collaboration with a host of other factors, recognize DNA damage or stalled replication forks [4], [5]. Once activated, ATM and ATR phosphorylate a number of substrates, including the checkpoint kinases CHK1 and CHK2, resulting in activation of the ATR–CHK1 and ATM–CHK2 signaling modules, respectively [2], [6]. These two parallel, but partially overlapping, kinase cascades are responsible for transducing the damage-induced signals to downstream effectors, which regulate cell-cycle arrest, DNA repair, and apoptosis [1], [2], [6].

CDC25 phosphatases are essential cell-cycle regulators that function by removing inhibitory phosphates from the tyrosine and threonine residues of cyclin-dependent kinases (CDKs), resulting in CDK activation and cell-cycle progression. Mammals possess three homologues of the ancestral CDC25 gene, designated CDC25A, -B, and -C [7], [8]. All three genes encode phosphatases that can dephosphorylate phosphotyrosine and phosphothreonine residues in CDKs, resulting in their activation [9]. However, despite these common features, the different CDC25 phosphatases exhibit distinct roles in regulating cell-cycle transition. Whereas CDC25B and CDC25C regulate only the G2/M transition, CDC25A regulates both the early G1/S and late G2/M cell-cycle transitions [10], [11]. CDC25A is a short-lived protein whose degradation is catalyzed by two different ubiquitin ligases, APC/C and SCF (Skp1/Cullin/F-box proteins). APC/C regulates CDC25A as cells exit mitosis, whereas SCF regulates the CDC25A level throughout the cell cycle [10], [12]. The binding of CDC25A to β-TrCP (β-transducin repeat-containing protein), a component of the SCF ubiquitin ligase complex, requires the CDC25A DSG motif [12]. In response to DNA damage, CHK1 phosphorylates CDC25A at Ser-76 to prime the protein for the NEK11-mediated phosphorylation of Ser-82, which is located within the DSG motif [13], [14], [15]. The phosphorylated DSG motif serves as a docking site for β-TrCP binding, thereby mediating the poly-ubiquitination and proteolysis of CDC25A [16].

STK38 (serine/threonine kinase 38), also known as NDR1 (nuclear Dbf2-related 1; GenBank accession number NP009202.1), is a serine/threonine protein kinase belonging to a subclass of the protein kinase A (PKA)/PKG/PKC-like (AGC) family, which includes cAMP-dependent kinase, protein kinase B, and protein kinase C. The STK38 family includes Schizosaccharomyces pombe Orb6, Saccharomyces cerevisiae Cbk1 and Dbf2, and mammalian LATS1 (large tumor suppressor 1), LATS2, STK38, and STK38L/NDR2 [17], [18], [19]. Cbk1 and Orb6 regulate cell morphology [20], [21], whereas Dbf2 is a cell-cycle-regulated kinase required for cell-cycle progression through anaphase [22]. LATS1 and LATS2 control mitotic exit and genomic stability [23], [24]. STK38 and STK38L are expressed throughout the mouse brain [25], and STK38 is implicated in the regulation of centrosome duplication and mitotic chromosome alignment [26], [27]. We previously demonstrated that STK38 regulates stress-activated MAPK signaling pathways and cellular sensitivity to DNA damage [28], [29], [30]. STK38's activity is regulated by MST3 (mammalian sterile 20-like 3) [31], the cofactors MOB1 (Mps one binder 1) and MOB2 [32], [33], or GSK-3 [29]. Although the functions and regulators of STK38 have been defined, its substrates and downstream signaling pathways remain to be determined. Here, we demonstrate that STK38 directly regulates CDC25A's stability by phosphorylating Ser-76, and that the STK38/CDC25A signaling module is required to regulate the DNA-damage-induced G2/M checkpoint.