Oxidative stress can induce apoptosis through activation of MstI, subsequent phosphorylation of FOXO and nuclear translocation. MstI is a common component of apoptosis initiated by various stresses. MstI kinase activation requires autophosphorylation and proteolytic degradation by caspases. FOXO2 (forkhead box O; forkhead members of the O class) are transcription factors. FOXO factors mediate cell death by regulating numerous apoptotic genes transcription (1-3). FOXO factors are insulin-sensitive transcription factors with a variety of downstream targets and interacting partners. Insulinmediated inhibition of FOXO factors is predominantly regulated through a shuttling mechanism that distributes FOXO localization to the cytoplasm, thereby terminating its transcriptional function (4). FOXO factors contain three highly conserved putative Akt recognition motifs (RXRXX(S/T), where X denotes any residue), two at the N and C termini, respectively, and one located in the forkhead domain. All FOXO proteins require Akt phosphorylation in the N terminus and in the forkhead domain to translocate from the nucleus to the cytoplasm (5). The two phosphorylated residues are essential components for translocation, as they influence the NLS (nuclear localization sequence) function and the association with 14-3-3 proteins (2). Recently, Bonni and co-workers demonstrated that MstI mediates oxidative stress-induced neuronal apoptosis through FOXO factors by phosphorylating FOXO3 on Ser 207 . This phosphorylation triggers its nuclear translocation and disrupting the association between 14-3-3 and FOXO in the cytoplasm (6).MstI belongs to class II GC (protein Ser/Thr) kinase (7), which contains 487 residues and predominantly resides in the cytoplasm. MstI consists of an N-terminal catalytic domain in the Ste20 class, followed by a non-catalytic tail comprising of an autoinhibitory domain and a coiled-coil domain that mediates dimerization (8). It has been suggested that, physiologically, MstI exists as an autoinhibitory homodimer that is activated after post-translational modification such as phosphorylation and/or cleavage. Although caspase-mediated cleavage removes the C-terminal regulatory domain, which is associated with an increase in MstI activity, there is evidence that caspase-mediated cleavage alone cannot activate MstI and both phosphorylation and proteolysis are necessary to activate fully this enzyme (9 -11). Indeed, Lee et al. (12) (9,11). Of these, Thr 183 and Thr 187 appear to be essential for kinase activity (11). It has been proposed before that phosphorylation of MstI on Thr 183 and possibly Thr 187 is induced by an existing active MstI (11). Overexpression of MstI alone is sufficient to initiate apoptosis in various cells, which involves activation of SAPK (stress-activated protein kinase)/JNK (13), p53 (14), FOXO (6). MstI cycles rapidly and continuously through the nucleus (6, 15), and associates with DAP-4 (death-associated protein-4) in the nucleus (14), where the catalytic fragment generated during apoptosi...

Serine/arginine (SR) protein-specific kinase (SRPK), a family of cell cycle-regulated protein kinases, phosphorylate SR domain-containing proteins in nuclear speckles and mediate the pre-mRNA splicing. However, the physiologic roles of this event in cell cycle are incompletely understood. Here, we show that SRPK2 binds and phosphorylates acinus, an SR protein essential for RNA splicing, and redistributes it from the nuclear speckles to the nucleoplasm, resulting in cyclin A1 but not A2 up-regulation. Acinus S422D, an SRPK2 phosphorylation mimetic, enhances cyclin A1 transcription, whereas acinus S422A, an unphosphorylatable mutant, blocks the stimulatory effect of SRPK2. Ablation of acinus or SRPK2 abrogates cyclin A1 expression in leukemia cells and arrest cells at G 1 phase. Overexpression of acinus or SRPK2 increases leukemia cell proliferation. Furthermore, both SRPK2 and acinus are overexpressed in some human acute myelogenous leukemia patients and correlate with elevated cyclin A1 expression levels, fitting with the oncogenic activity of cyclin A1 in leukemia. Thus, our findings establish a molecular mechanism by which SR splicing machinery regulates cell cycle and contributes to leukemia tumorigenesis. [Cancer Res 2008;68(12):4559-70]

Molecular biological studies con¢rmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and speci¢c for neural and testicular tissues. A selective de¢-ciency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identi¢ed an amino acid residue involved in the di¡erent thermal stability of human GDH isozymes. At 45 ‡C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life = 45 min) than for hGDH1 (half-life = 310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 ‡C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not a¡ected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having di¡erences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the di¡erent thermal stability of human GDH isozymes.

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When the influence of ADP-ribosylation on the activities of the purified human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was measured in the presence of 100 lM NAD + for 60 min, hGDH isozymes were inhibited by up to 75%. If incubations were performed for longer time periods up to 3 h, the inhibition of hGDH isozymes did not increased further. This phenomenon may be related to the reversibility of ADP-ribosylation in mitochondria. ADP-ribosylated hDGH isozymes were reactivated by Mg 2+ -dependent mitochondrial ADP-ribosylcysteine hydrolase. The stoichiometry between incorporated ADP-ribose and GDH subunits shows a modification of one subunit per catalytically active homohexamer. Since ADP and GTP had no effects on the extent of modification, it would appear that the ADP-ribosylation is unlikely to occur in allosteric sites. It has been proposed that Cys residue may be involved in the ADP-ribosylation of GDH, although identification of the reactive Cys residue has not been reported. To identify the reactive Cys residue involved in the ADP-ribosylation, we performed cassette mutagenesis at three different positions (Cys59, Cys119, and Cys274) using synthetic genes of hGDH isozymes. Among the Cys residues tested, only Cys119 mutants showed a significant reduction in the ADP-ribosylation. These results suggest a possibility that the Cys119 residue has an important role in the regulation of hGDH isozymes by ADP-ribosylation.

Microglial cells are known as the main immune cells in the central nervous system, both regulating its immune response and maintaining its homeostasis. Furthermore, the antioxidant α-lipoic acid (LA) is a recognized therapeutic drug for diabetes because it can easily invade the blood–brain barrier. This study investigated the effect of α-LA on the inflammatory response in lipopolysaccharide (LPS)-treated BV-2 microglial cells. Our results revealed that α-LA significantly attenuated several inflammatory responses in BV-2 microglial cells, including pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-6, and other cytotoxic molecules, such as nitric oxide and reactive oxygen species. In addition, α-LA inhibited the LPS-induced phosphorylation of ERK and p38 and its pharmacological properties were facilitated via the inhibition of the nuclear factor kappa B signaling pathway. Moreover, α-LA suppressed the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasomes, multiprotein complexes consisting of NLRP3 and caspase-1, which are involved in the innate immune response. Finally, α-LA decreased the genes accountable for the M1 phenotype, IL-1β and ICAM1, whereas it increased the genes responsible for the M2 phenotype, MRC1 and ARG1. These findings suggest that α-LA alleviates the neuroinflammatory response by regulating microglial polarization.

S100A8 and S100A9 function as essential factors in inflammation and also exert antitumor or tumorigenic activity depending on the type of cancer. Chronic eosinophilic leukemia (CEL) is a rare hematological malignancy having elevated levels of eosinophils and characterized by the presence of the FIP1L1-PDGFRA fusion gene. In this study, we examined the pro-apoptotic mechanisms of S100A8 and S100A9 in FIP1L1-PDGFRα+ eosinophilic cells and hypereosinophilic patient cells. S100A8 and S100A9 induce apoptosis of the FIP1L1-PDGFRα+ EoL-1 cells via TLR4. The surface TLR4 expression increased after exposure to S100A8 and S100A9 although total TLR4 expression decreased. S100A8 and S100A9 suppressed the FIP1L1-PDGFRα-mediated signaling pathway by downregulating FIP1L1-PDGFRα mRNA and protein expression and triggered cell apoptosis by regulating caspase 9/3 pathway and Bcl family proteins. S100A8 and S100A9 also induced apoptosis of imatinib-resistant EoL-1 cells (EoL-1-IR). S100A8 and S100A9 blocked tumor progression of xenografted EoL-1 and EoL-1-IR cells in NOD-SCID mice and evoked apoptosis of eosinophils derived from hypereosinophilic syndrome as well as chronic eosinophilic leukemia. These findings may contribute to a progressive understanding of S100A8 and S100A9 in the pathogenic and therapeutic mechanism of hematological malignancy.

The molecular mechanism of hypoxia-induced apoptosis has not been clearly elucidated. In this study, we investigated the involvement of extracellular signal-regulated protein kinase (ERK 1/2) in hypoxia-induced apoptosis using cobalt chloride in HeLa human cervical cancer cells. The cobalt chloride was used for the induction of hypoxia, and its IC(50) was 471.4 microM. We demonstrated the DNA fragmentation after incubation with concentrations more than 50 microM cobalt chloride for 24 h, and also evidenced the morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signaling pathway of cobalt chloride-induced apoptosis in HeLa cells. ERK1/2 activation occurred 6 and 9 h after treatment with 600 microM cobalt chloride. Meanwhile, the pretreatment of the MEK 1 inhibitor (PD98059) completely blocked the cobalt chloride-induced ERK 1/2 activation. At the same time, the activated ERK 1/2 translocated into the nucleus and phosphorylated its transcriptional factor, c-Jun. In addition, the pretreatment of PD98059 inhibited the cobalt chloride-induced DNA fragmentation and apoptotic cell death. These results suggest that cobalt chloride is able to induce apoptotic activity in HeLa cells, and its apoptotic mechanism may be associated with signal transduction via ERK 1/2.