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NIMA-related kinases (Neks) play divergent roles in mammalian cells. While several Neks regulate mitosis, Nek1 was reported to regulate DNA damage response, centrosome duplication and primary cilium formation. Whether Nek1 participates in cell cycle regulation is not known. Here we report that loss of Nek1 results in severe proliferation defect due to a delay in S-phase of the cell cycle. Nek1-deficient cells show replication stress and checkpoint activation under normal growth conditions. Nek1 accumulates on the chromatin during normal DNA replication. In response to replication stress, Nek1 is further activated for chromatin localization. Nek1 interacts with Ku80 and, in Nek1-deficient cells chromatin localization of Ku80 and several other DNA replication factors is significantly reduced. Thus, Nek1 may facilitate S-phase progression by interacting with Ku80 and regulating chromatin loading of replication factors.

Loss of function in either VHL or Nek1 leads to cyst formation in tissues, especially in kidneys. Whether there is a connection between pVHL and Nek1 regulation is unknown. Here, we report that the VHL protein (pVHL) may be a substrate of Nek1. While Nek1 can phosphorylate pVHL at multiple sites, the phosphorylation at serine-168 results in pVHL degradation. Nek1-mediated phosphorylation of pVHL does not significantly affect hypoxia-inducible factors (HIF), a known target of pVHL. However, non-phosphorylable pVHL reconstituted in VHL-deficient cells induces more stable cilia than wild-type VHL during serum stimulation and Nocodazole treatment. The results suggest a possible regulation of pVHL by Nek1 that may contribute to ciliary homeostasis and cystogenesis.

The usefulness and efficacy of cisplatin, a chemotherapeutic drug, are limited by its toxicity to normal tissues and organs, including the kidneys. The uptake of cisplatin in renal tubular cells is high, leading to cisplatin accumulation and tubular cell injury and death, culminating in acute renal failure. While extensive investigations have been focused on the signaling pathways of cisplatin nephrotoxicity, much less is known about the mechanism of cisplatin uptake by renal cells and tissues. In this regard, evidence has been shown for the involvement of organic cation transporters (OCT), specifically OCT2. The copper transporter Ctr1 is highly expressed in the renal tubular cells; however, its role in cisplatin nephrotoxicity is not known. In this study, we demonstrate that Ctr1 is mainly expressed in both proximal and distal tubular cells in mouse kidneys. We further show that Ctr1 is mainly localized on the basolateral side of these cells, a proposed site for cisplatin uptake. Importantly, downregulation of Ctr1 by small interfering RNA or copper pretreatment results in decreased cisplatin uptake. Consistently, downregulation of Ctr1 suppresses cisplatin toxicity, including cell death by both apoptosis and necrosis. Cimetidine, a pharmacological inhibitor of OCT2, can also partially attenuate cisplatin uptake. Notably, cimetidine can further reduce cisplatin uptake and cisplatin toxicity in Ctr1-downregulated cells. The results have demonstrated the first evidence for a role of Ctr1 in cisplatin uptake and nephrotoxicity.

Bcl-2 family proteins are central regulators of apoptosis. As the prototypic member, Bcl-2 protects various types of cells against apoptotic insults. In mammalian cells, Bcl-2 has a dual subcellular localization, in mitochondria and endoplasmic reticulum (ER). The respective roles played by mitochondrial and ER-localized Bcl-2 in apoptotic inhibition are unclear. Using Bcl-2 constructs for targeted subcellular expression, we have now determined the contributions of mitochondrial and ER-localized Bcl-2 to the antiapoptotic effects of Bcl-2 in renal tubular cells. Wild-type Bcl-2, when expressed in renal proximal tubular cells, showed partial colocalizations with both cytochrome cand disulfide isomerase, indicating dual localizations of Bcl-2 in mitochondria and ER. In contrast, Bcl-2 constructs with mitochondria-targeting or ER-targeting sequences led to relatively restricted Bcl-2 expression in mitochondria and ER, respectively. Expression of wild-type and mitochondrial Bcl-2 showed significant inhibitory effects on tubular cell apoptosis that was induced by cisplatin or ATP depletion; however, ER-Bcl-2 was much less effective. During ATP depletion, cytochrome cwas released from mitochondria into the cytosol. This release was suppressed by wild-type and mitochondrial Bcl-2, but not by ER-Bcl-2. Consistently, wild-type and mitochondrial Bcl-2, but not ER-Bcl-2, blocked Bax activation during ATP depletion, a critical event for mitochondrial outer membrane permeabilization and cytochrome crelease. In contrast, ER-Bcl-2 protected against apoptosis during tunicamycin-induced ER stress. Collectively, the results suggest that the cytoprotective effects of Bcl-2 in different renal injury models are largely determined by its subcellular localizations.

Cisplatin induces renal cell injury and death, resulting in nephrotoxicity that limits its use in cancer therapy. Using cell culture models, recent work has suggested the involvement of p53 in renal cell apoptosis during cisplatin treatment. However, the signals upstream of p53 remain elusive. ATM and ATR are critical regulators of p53 under various conditions of DNA damage. Here, we show that ATM, and not ATR, was proteolytically cleaved into specific fragments of ∼210 and 150 kDa during cisplatin-induced tubular cell apoptosis. ATM cleavage was paralleled by the development of apoptosis. VAD, a broad-spectrum inhibitor of caspases, attenuated the cleavage of ATM, whereas the inhibitors of specific caspases were less effective. In caspase-3-deficient MCF-7 cells, ATM was cleaved, releasing the 210- but not the 150-kDa fragment. Recombinant caspase-3 was much more effective than caspase-7 in cleaving ATM that was immunoprecipitated from cell lysates. During cisplatin incubation, VAD protected ATM and enhanced p53 phosphorylation. In vitro assay of protein kinase activity further showed that ATM immunoprecipitated from cisplatin-treated cells had significantly lower kinase activity toward p53 than that from control cells. Importantly, the protein kinase activity was restored in ATM that was protected by VAD during cisplatin incubation. ATM deficiency sensitized the cells to cisplatin-induced apoptosis, suggesting a cytoprotective role of ATM in this experimental model. Thus proteolysis of ATM by caspases may inactivate this regulatory molecule to facilitate the progression of apoptosis.

Comment on: Pabla N, et al. Proc Natl Acad Sci USA 2012; 109:197-202.