Jia-Ling Yang

Cooperation of ERK and SCFSkp2 for MKP-1 destruction provides a positive feedback regulation of proliferating signaling

The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feedback mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339–342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the Ser296/Ser323 domain, which was not affected by substituting Ala for Ser at Ser359/Ser364. The DEF and Ser296/Ser323 sites were essential for ubiquitin-mediated MKP-1 proteolysis stimulated by MKK1-ERK signaling in H293 cells, whereas the N-terminal domain and Ser359/Ser364 sites were dispensable. ERK activation by serum increased the endogenous level of ubiquitinated phospho-Ser296 MKP-1 and the degradation of MKP-1. Intriguingly, active ERK-promoted phospho-Ser296 MKP-1 bound to SCFSkp2 ubiquitin ligase in vivo and in vitro. Forced expression of Skp2 enhanced MKP-1 polyubiquitination and proteolysis upon ERK activation, whereas depletion of endogenous Skp2 suppressed such events. The kinetics of ERK signaling stimulated by serum correlated with the endogenous MKP-1 degradation rate in a Skp2-dependent manner. Thus, MKP-1 proteolysis can be achieved via ERK and SCFSkp2 cooperation, thereby sustaining ERK activation.

Formation of reactive oxygen species and DNA strand breakage during interaction of chromium(III) and hydrogen peroxide in vitro: Evidence for a chromium(III)-mediated Fenton-like reaction

The role of reactive oxygen species in causing DNA damage through interaction of chromium (III) and hydrogen peroxide was examined using plasmid relaxation assay and EPR spectroscopy. Marked DNA strand breakage was induced by CrCl3 plus H2O2 in a phosphate buffer at pH 6-8.9; whereas, only slight DNA strand breakage was observed during similar treatment at pH less than 4. DNA breakage also increased as the reaction temperature and Cr(III)/H2O2 concentrations increased. Control experiments with Cr(III) or H2O2 alone did not cause DNA breakage. Sodium azide, D-mannitol, Tris-HCl, or catalase completely inhibited Cr(III)/H2O2-induced DNA breakage, but superoxide dismutase did not. The D2O enhancing effect on DNA breaks was not observed. Cr(III) pre-incubated with a 30-fold molar excess of EDTA did not cause any significant DNA breakage in the presence of H2O2. In a phosphate buffer containing Cr(III) and H2O2, singlet oxygen and hydroxyl radicals were detected using EPR spectrometry with the spin traps 2,2,6,6-tetramethyl-4-piperidone and 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), respectively. DMPO/.OH adducts and DNA breakage induced by Cr(III)/H2O2 were markedly higher than those induced by Cr(VI)/H2O2. Furthermore, ascorbate decreased Cr(III)/H2O2-induced DNA breakage. EPR studies revealed that ascorbate (mole ratio to Cr(III) = 0.5:1) attenuated the DMPO/.OH signal generated by Cr(III)/H2O2/DMPO, but a Cr(V) signal and ascorbate radicals were detected. NADPH, GSH, and GSSG also decreased DMPO/.OH generated by Cr(III)/H2O2/DMPO; however, they were less efficient than ascorbate and no Cr(V) signals were detected. This study shows that Cr(III)/H2O2 generates oxidative damage to DNA through a Fenton-like reaction: Cr(III) + H2O2-->Cr(IV) + .OH + OH.

Lead acetate induces EGFR activation upstream of SFK and PKCα linkage to the Ras/Raf-1/ERK signaling

Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC --> ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1(S338) and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Gö6976 or depleting PKCalpha using specific small interfering RNA blocked Pb induction of Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKCalpha, Ras-GTP, phospho-Raf-1(S338) and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKCalpha activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKCalpha activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKCalpha and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade.

Cdc20 Proteolysis Requires p38 MAPK Signaling and Cdh1-Independent APC/C Ubiquitination During Spindle Assembly Checkpoint Activation by Cadmium

Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, initiates the destruction of key mitotic regulators to facilitate mitosis, while it is negatively regulated by the spindle assembly checkpoint (SAC) to prevent premature anaphase entry. Activation of the p38 mitogen‐activated protein kinase could contribute to mitotic arrest, but the underlying mechanism is unknown. Here we report a novel pathway in which the p38 signaling triggers Cdc20 destruction under SAC elicited by cadmium, a human carcinogen. We found that the cadmium‐induced prometaphase arrest was linked to decreased Cdc20 and accumulated cyclin A protein levels in human cells, whereas the activity of cyclin B1–Cdk1 was unaffected. The Cdc20 half‐life was markedly shortened along with its ubiquitination and degradation via 26S proteasome in cadmium‐treated asynchronous or G2‐enriched cells. Depletion of APC3 markedly suppressed the cadmium‐induced Cdc20 ubiquitination and proteolysis, while depletion of Cdh1, another activator of APC/C, did not. Intriguingly, blockage of p38 activity restored the Cdc20 levels for continuing mitosis under cadmium, while inhibition of JNK activity had no effect. The cadmium‐induced Cdc20 proteolysis was also suppressed during transient depletion of p38α or stable expression a dominant negative form of p38. Inhibition of p38 abolished the induction of Mad2–Cdc20–APC3 complex by cadmium. Moreover, forced expression of MKK6–p38 signaling could promote Cdc20 degradation in a Cdh1‐independent APC/C pathway. In summary, accelerated ubiquitination and proteolysis of Cdc20 is essential for prometaphase arrest that is mediated via the p38 signaling during SAC activation. J. Cell. Physiol. 223: 327–334, 2010.

APE1/Ref-1 prevents oxidative inactivation of ERK for G1-to-S progression following lead acetate exposure.

Apurinic/apyrimidinic endonuclease 1 (APE1)/redox effector factor-1 is a multifunctional enzyme involved in DNA base excision repair and protein redox regulation. Previously, we have showed that lead acetate (Pb) elicits EGFR activation to initiate the SFK/PKCα/Ras/Raf-1/MKK1/2/ERK signaling cascade functioning against genotoxicity. Here, we explore whether APE1 and reactive oxygen species (ROS) affect ERK signaling and cell cycle progression following Pb exposure. We found that Pb induced APE1 expression and ROS generation in CL3 human lung cancer cells. The Pb-elicited ROS levels and cytotoxicity were further enhanced by introducing small interfering RNA specific for APE1 (siAPE1). E3330, an inhibitor of APE1 redox activity, also augmented the ROS levels and cytotoxicity in Pb-treated cells. Intriguingly, the capability of Pb to activate ERK was abolished under siAPE1 or E3330 co-treatments; conversely, forced expression of APE1 up-regulated the ERK activation by Pb or serum in both Cys65-redox activity dependent and independent manners. Moreover, APE1 formed complex with ERK2, and its redox activity could rescue ERK oxidative inactivation. APE1 redox activity also facilitated the Cyclin D1 expression and G1-to-S progression following Pb exposure. In summary, the results indicate that APE1 is a direct redox regulator of ERK for maintaining the kinase activity to promote cell proliferation.

Cyclin B1 proteolysis via p38 MAPK signaling participates in G2 checkpoint elicited by arsenite.

Timely induction of cyclin B1 controls mitotic entry, whereas its proteolysis is essential for mitotic exit. By contrast, cyclin B1 transcription is repressed during G2 arrest induced by DNA damage. The p38 mitogen‐activated protein kinase is involved in the G2 checkpoint; yet, its impact on cyclin B1 protein levels remains unclear. Here we show that untimely proteolysis of cyclin B1 following p38 activation contributes to G2 checkpoint. Exposing early G2 cells to arsenite impeded cyclin B1 protein accumulation, Cdk1 activation, and G2‐to‐M progression. Conversely, cyclin B1 was non‐degradable in late G2 and mitotic cells after arsenite. Cyclin B1 proteolysis was enhanced by arsenite in early G2 and asynchronous cells. This rapid destruction of cyclin B1 was mediated via the ubiquitin–proteasome pathway probably in a Cdc20 and Cdh1 independent mechanism. Under arsenite, inhibition of p38 activation or depletion of p38α suppressed cyclin B1 ubiquitination and proteolysis, while forced expression of MKK6‐p38 accelerated these events. Inactivation of p38 in arsenite‐treated early G2 cells allowed G2‐to‐M progression, blocked apoptosis, increased cell viability, and decreased micronucleus formation. Thus, p38 signaling pathway triggering cyclin B1 proteolysis after arsenite may play an important role in connecting G2 arrest with apoptosis or genome instability. J. Cell. Physiol. 212: 481–488, 2007.

Activation of protein kinase Cα signaling prevents cytotoxicity and mutagenicity following lead acetate in CL3 human lung cancer cells

Protein kinase C (PKC) family of serine/threonine protein kinases is sensitive signaling transducers in response to lead acetate (Pb) that could transmit phosphorylation cascade for proliferation and de-differentiation of neural cells. However, little is known as to the impact of PKC on Pb genotoxicity. Here we investigate whether Pb activates the conventional/classical subfamily of PKC (cPKC) signaling to affect cytotoxicity and mutagenicity in CL3 human non-small-cell lung adenocarcinoma cells. Pb specifically promoted membrane localization of the alpha isoform of PKC in CL3 cells. Pb also elicited Raf-1 activation as measured by the induction of phospho-Raf-1S338 and the dissociation from the Raf-1 kinase inhibitor protein. Inhibition of cPKC activity using Gö6976 or depletion of PKCalpha by introducing specific small interfering RNA blocked the induction of phospho-Raf-1S338, phospho-MKK1/2 and phospho-ERK1/2 in cells exposed to Pb. Intriguingly, declining PKCalpha enhanced the Pb cytotoxicity and revealed the Pb mutagenicity at the hprt gene. The results suggest that PKCalpha is obligatory for activation of the Raf-1-MKK1/2-ERK1/2 signaling module and plays a defensive role against cytotoxicity and mutagenicity following Pb exposure. Results obtained in this study also support our previous report showing that ERK1/2 activity is involved in preventing Pb genotoxicity.

Role of extracellular signal-regulated kinase (ERK) signaling in nucleotide excision repair and genotoxicity in response to As(III) and Pb(II)

Arsenic and lead can induce genetic injuries and epigenetic signaling pathways in cultured mammalian cells. To test whether signaling pathways affect the extent of genetic injuries, we explored the impacts of extracellular signal-regulated kinase 1 and 2 (ERK) on nucleotide excision repair (NER), cytotoxicity, and genotoxicity following sodium arsenite [As(III)] and lead acetate [Pb(II)]. Sustained ERK activation was observed in human cells exposed to As(III) and Pb(II). As(III) inhibited the cellular NER synthesis capability; conversely, Pb(II) stimulated it. ERK activation contributed to the As(III)-induced NER inhibition and micronucleus formation. In contrast, this signal was required for inducing cellular NER activity and preventing mutagenesis following Pb(II). ERK activation by Pb(II) was dependent on protein kinase C (PKCα) that also exhibited anti-mutagenicity. Enforced expression of ERK signaling markedly elevated the cellular NER activity, which was suppressed by As(III). Nonetheless, ERK activation could counteract the cytotoxicity caused by these two metals. Together, the results indicate that pro-survival ERK signaling exhibits dual and opposing impacts on NER process following As(III) and Pb(II) exposures. The findings also suggest that ERK is an important epigenetic signaling in the determination of metal genotoxicity.