Faculty Bibliography

RAS proteins are GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, differentiation, and autophagy. Mutations or deregulated activities of RAS are frequently the driving force for oncogenic transformation and tumorigenesis. Given the important roles of the small ubiquitin-related modifier (SUMO) pathway in controlling the stability, activity, or subcellular localization of key cellular regulators, we investigated here whether RAS proteins are posttranslationally modified (i.e. SUMOylated) by the SUMO pathway. We observed that all three RAS protein isoforms (HRAS, KRAS, and NRAS) were modified by the SUMO3 protein. SUMOylation of KRAS protein, either endogenous or ectopically expressed, was observed in multiple cell lines. The SUMO3 modification of KRAS proteins could be removed by SUMO1/sentrin-specific peptidase 1 (SENP1) and SENP2, but not by SENP6, indicating that RAS SUMOylation is a reversible process. A conserved residue in RAS, Lys-42, was a site that mediates SUMOylation. Results from biochemical and molecular studies indicated that the SUMO-E3 ligase PIASγ specifically interacts with RAS and promotes its SUMOylation. Moreover, SUMOylation of RAS appeared to be associated with its activation. In summary, our study reveals a new posttranslational modification for RAS proteins. Since we found that HRAS, KRAS, and NRAS can all be SUMOylated, we propose that SUMOylation might represent a mechanism by which RAS activities are controlled.

Quantum dots (QDs) have shown great potential for biomedical use in a broad range including diagnostic agents. However, the regulatory mechanism of dermal toxicity is poorly understood. In this study, we investigated how QDs-induced apoptosis is regulated in human keratinocytes. We also examined the effect of carboxylic acid-coated QDs (QD 565 and QD 655) on reactive oxygen species (ROS) production and apoptosis-related cellular signalling. The viability of keratinocyte was inhibited by two types of QDs in a concentration-dependent manner. QDs induce ROS production and blockade of AKT phosphorylation. Moreover, the cleavage of AKT-dependent pro-apoptotic proteins such as poly (ADP-ribose) polymerase, caspases-3 and caspases-9 was significantly increased. We also found that a decrease in cellular ROS level by ROS scavenger, N-acetylcysteine (NAC), resulting in the abolishment of QDs-induced AKT de-phosphorylation and cellular apoptosis. Interestingly, QD 655 had a more cytotoxic effect including oxidative stress and AKT-dependent apoptosis than QD 565. In addition, QD 655 had the cytotoxic potential in the human skin equivalent model (HSEM). These data show that QD-induced intracellular ROS levels may be an important parameter in QD-induced apoptosis. These findings from this study indicate that intracellular ROS levels might determine the apoptotic potential of keratinocyte by QD via blockade of AKT phosphorylation.

Nuclear PTEN plays an important role during mitosis. To understand the molecular basis by which PTEN mediates mitotic progression, we examined whether PTEN regulated the formation of mitotic checkpoint complex (MCC). We observed that arsenic trioxide, a mitotic inducer, stimulated nuclear translocation of PTEN in a time-dependent manner. PTEN physically interacted with Cdc20 and Mad2, two important components of MCC. Arsenic treatment diminished the physical association of PTEN with BubR1 and Bub3 but not with Cdc20 and Mad2. Our further studies revealed that downregulation of PTEN via RNAi enhanced formation of MCC during the cell cycle. Moreover, PTEN silencing induced chromosomal instability. Given the crucial role of PTEN in suppressing tumor development, our study strongly suggests that PTEN also functions to maintain chromosomal stability, partly through suppressing unscheduled formation of MCC.

WWP2 is a ubiquitin E3 ligase belonging to the Nedd4-like family. Given that WWP2 target proteins including PTEN that are crucial for regulating cell proliferation or suppressing tumorigenesis, we have asked whether WWP2 plays a role in controlling cell cycle progression. Here we report that WWP2 is necessary for normal cell cycle progression as its silencing significantly reduces the cell proliferation rate. We have identified that an isoform of WWP2 (WWP2-V4) is highly expressed in the M phase of the cell cycle. Silencing of WWP2 accelerates the turnover of cyclin E, which is accompanied by increased levels of phospho-histone H3 (p-H3) and cyclin B. Moreover, silencing of WWP2 results in compromised phosphorylation of AktS473, a residue whose phosphorylation is tightly associated with the activation of the kinase. Combined, these results strongly suggest that WWP2 is an important component in regulating the Akt signaling cascade, as well as cell cycle progression.

A pool of PTEN localizes to the nucleus. However, the exact mechanism by which nuclear PTEN is regulated remains unclear. We have recently reported that Plk1 specifically phosphorylates PTEN on S380 during mitosis. Here we report that PTEN also localized to chromatin and that chromatin PTEN was removed by a proteasome-dependent process during mitotic exit. Pulldown analysis revealed that Cdh1, but not Cdc20, was significantly associated with PTEN. Cdh1 interacted with PTEN via two separate domains and their interaction was enhanced by MG132, a proteasome inhibitor. Cdh1 negatively controlled the stability of chromatin PTEN by polyubiquitination. Phosphorylation of PTEN on S380 impaired its interaction with Cdh1, thus positively regulating PTEN stability on chromatin. Significantly, The interaction of PTEN with Cdh1 was phosphatase-independent and Cdh1 knockdown via RNAi led to significant accumulation of chromatin PTEN, delaying mitotic exit. Combined, our studies identify Cdh1 as an important regulator of nuclear/chromatin PTEN during mitosis.

PTEN is a well-known tumor suppressor through the negative regulation of the PI3K signaling pathway. Here we report that PTEN plays an important role in regulating mitotic timing, which is associated with increased PTEN phosphorylation in the C-terminal tail and its localization to chromatin. Pull-down analysis revealed that Plk1 physically interacted with PTEN. Biochemical studies showed that Plk1 phosphorylates PTEN in vitro in a concentration-dependent manner and that the phosphorylation was inhibited by Bi2635, a Plk1-specific inhibitor. Deletional and mutational analyses identified that Plk1 phosphorylated S380, T382 and T383, but not S385, a cluster of residues known to affect the PTEN stability. Interestingly, a combination of molecular and genetic analyses revealed that only S380 was significantly phosphorylated in vivo and that Plk1 regulated the phosphorylation, which was associated with accumulation of PTEN on chromatin. Moreover, expression of phospho-deficient mutant, but not wild-type PTEN, caused enhanced mitotic exit. Taken together, our studies identify Plk1 as an important regulator of PTEN during the cell cycle.

A pool of PTEN localizes to the nucleus. However, the exact mechanism of action of nuclear PTEN remains poorly understood. We have investigated PTEN's role during DNA damage response. Here we report that PTEN undergoes chromatin translocation after DNA damage, and that its translocation is closely associated with its phosphorylation on S366/T370 but not on S380. Deletional analysis reveals that the C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3beta are involved in the phosphorylation of the S366/T370 epitope, as well as PTEN's association with chromatin after DNA damage. Significantly, PTEN specifically interacts with Rad52 and colocalizes with Rad52, as well as gammaH2AX, after genotoxic stress. Moreover, PTEN is involved in regulating Rad52 sumoylation. Combined, our studies strongly suggest that nuclear/chromatin PTEN mediates DNA damage repair through interacting with and modulating the activity of Rad52.

The human skin equivalent model (HSEM) is well known as an attractive alternative model for evaluation of dermal toxicity. However, only limited data are available on the usefulness of a HSEM for nanotoxicity testing. This study was designed to investigate cutaneous toxicity of polystyrene and TiO2 nanoparticles using cultured keratinocytes, a HSEM, and an animal model. In addition, we also evaluated the skin sensitization potential of nanoparticles using a local lymph node assay with incorporation of BrdU. Findings from the present study indicate that polystyrene and TiO2 nanoparticles do not induce phototoxicity, acute cutaneous irritation, or skin sensitization. Results from evaluation of the HSEMs correspond well with those from animal models. Our findings suggest that the HSEM might be a useful alternative model for evaluation of dermal nanotoxicity.