Amplification or overexpression of HER-2/neu in cancer cells confers resistance to apoptosis and promotes cell growth. The cellular localization of p21Cip1/WAF1 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Here we show that HER-2/neu-mediated cell growth requires the activation of Akt, which associates with p21Cip1/WAF1 and phosphorylates it at threonine 145, resulting in cytoplasmic localization of p21Cip1/WAF1. Furthermore, blocking the Akt pathway with a dominant-negative Akt mutant restores the nuclear localization and cell-growth-inhibiting activity of p21Cip1/WAF1. Our results indicate that HER-2/neu induces cytoplasmic localization of p21Cip1/WAF1 through activation of Akt to promote cell growth, which may have implications for the oncogenic activity of HER-2/neu and Akt.

HER-2/neu amplification or overexpression can make cancer cells resistant to apoptosis and promotes their growth. p53 is crucial in regulating cell growth and apoptosis, and is often mutated or deleted in many types of tumour. Moreover, many tumours with a wild-type gene for p53 do not have normal p53 function, suggesting that some oncogenic signals suppress the function of p53. In this study, we show that HER-2/neu-mediated resistance to DNA-damaging agents requires the activation of Akt, which enhances MDM2-mediated ubiquitination and degradation of p53. Akt physically associates with MDM2 and phosphorylates it at Ser166 and Ser186. Phosphorylation of MDM2 enhances its nuclear localization and its interaction with p300, and inhibits its interaction with p19ARF, thus increasing p53 degradation. Our study indicates that blocking the Akt pathway mediated by HER-2/neu would increase the cytotoxic effect of DNA-damaging drugs in tumour cells with wild-type p53.

Damaged mitochondria can be eliminated by autophagy, i.e. mitophagy, which is important for cellular homeostasis and cell survival. Despite the fact that a number of factors have been found to be important for mitophagy in mammalian cells, their individual roles in the process had not been clearly defined. Parkin is a ubiquitin-protein isopeptide ligase able to translocate to the mitochondria that are to be removed. We showed here in a chemical hypoxia model of mitophagy induced by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP) that Parkin translocation resulted in mitochondrial ubiquitination and p62 recruitment to the mitochondria. Small inhibitory RNAmediated knockdown of p62 significantly diminished mitochondrial recognition by the autophagy machinery and the subsequent elimination. Thus Parkin, ubiquitin, and p62 function in preparing mitochondria for mitophagy, here referred to as mitochondrial priming. However, these molecules were not required for the induction of autophagy machinery. Neither Parkin nor p62 seemed to affect autophagy induction by CCCP. Instead, we found that Nix was required for the autophagy induction. Nix promoted CCCP-induced mitochondrial depolarization and reactive oxygen species generation, which inhibited mTOR signaling and activated autophagy. Nix also contributed to mitochondrial priming by controlling the mitochondrial translocation of Parkin, although reactive oxygen species generation was not involved in this step. Deletion of the C-terminal membrane targeting sequence but not mutations in the BH3 domain disabled Nix for these functions. Our work thus distinguished the molecular events responsible for the different phases of mitophagy and placed Nix upstream of the events.

From soybean plant, 131 bZIP genes were identified and named as GmbZIPs. The GmbZIPs can be classified into ten groups and more than one third of these GmbZIPs are responsive to at least one of the four treatments including ABA, salt, drought and cold stresses. Previous studies have shown that group A bZIP proteins are involved in ABA and stress signaling. We now chose four non-group A genes to study their features. The four proteins GmbZIP44, GmbZIP46, GmbZIP62 and GmbZIP78 belong to the group S, I, C and G, respectively, and can bind to GLM (GTGAGTCAT), ABRE (CCACGTGG) and PB-like (TGAAAA) elements with differential affinity in both the yeast one-hybrid assay and in vitro gel-shift analysis. GmbZIP46 can form homodimer or heterodimer with GmbZIP62 or GmMYB76. Transgenic Arabidopsis plants overexpressing the GmbZIP44, GmbZIP62 or GmbZIP78 showed reduced ABA sensitivity. However, all the transgenic plants were more tolerant to salt and freezing stresses when compared with the Col plants. The GmbZIP44, GmbZIP62 and GmbZIP78 may function in ABA signaling through upregulation of ABI1 and ABI2 and play roles in stress tolerance through regulation of various stress-responsive genes. These results indicate that GmbZIP44, GmbZIP62 and GmbZIP78 are negative regulators of ABA signaling and function in salt and freezing tolerance.

SummarySoybean is one of the most important leguminous seed crops among the oil crops. Although the pathways for lipid biosynthesis have been identified, the factors that regulate the biosynthetic pathways at the transcriptional level are largely unknown. Here, we report our findings on the involvement of soybean Doftype transcription factor genes in the regulation of the lipid content in soybean seeds. We identified 28 Doftype transcription factor genes in soybean plants, and these genes displayed diverse patterns of expression in various organs. Seven flower/pod-specific genes and one constitutively expressed gene were further investigated. The proteins encoded by these seven genes were localized in the nucleus, and exhibited different abilities for transcriptional activation and DNA binding. Two genes, GmDof4 and GmDof11, were found to increase the content of total fatty acids and lipids in GmDof4 and GmDof11 transgenic Arabidopsis seeds. We also found that the 1000-seed weight was increased in the GmDof4 and GmDof11 transgenic plants. Using microarray and DNA binding analysis, we found that the two Dof-like proteins, GmDof4 and GmDof11, activated the acetyl CoA carboxylase gene and long-chain-acyl CoA synthetase gene, respectively, by direct binding to the cis-DNA elements in their promoter regions. In addition, both proteins downregulated the storage protein gene, CRA1, through direct binding. These results suggest that the two GmDof genes may augment the lipid content of soybean seeds by upregulating genes that are associated with the biosynthesis of fatty acids.

Myeloid cell leukemia-1 (Mcl-1), a Bcl-2-like antiapoptotic protein, plays a role in cell immortalization and chemoresistance in a number of human malignancies. A peptidylprolyl cis/trans isomerase, Pin1 is involved in many cellular events, such as cell cycle progression, cell proliferation, and differentiation through isomerizing prophosphorylated substrates. It has been reported that down-regulation of Pin1 induces apoptosis, and that Erk phosphorylates and upregulates Mcl-1; however, the underlying mechanisms for the two phenomena are not clear yet. Here, we showed that Pin 1 stabilizes Mcl-1, which is required for Mcl-1 posphorylation by Erk. First, we found expression of Mcl-1 and Pin1 were positively correlated and associated with poor survival in human breast cancer. We then showed that Erk could phosphorylate Mcl-1 at two consensus residues, Thr 92 and 163, which is required for the association of Mcl-1 and Pin1, resulting in stabilization of Mcl-1. Moreover, Pin1 is also required for the up-regulation of Mcl-1 by Erk activation. Based on this newly identified mechanism of Mcl-1 stabilization, two strategies were used to overcome Mcl-1-mediated chemoresistance: inhibiting Erk by Sorafenib, an approved clinical anticancer drug, or knocking down Pin1 by using a SiRNA technique. In conclusion, the current report not only unravels a novel mechanism to link Erk/Pin1 pathway and Mcl-1-mediated chemoresistance but also provides a plausible combination therapy, Taxol (Paclitaxel) plus Sorafenib, which was shown to be effective in killing breast cancer cells. [Cancer Res 2008;68(15):6109-17]

Background Adipokines are reported to participate in many common pathologic processes of glucose dysregulation, such as insulin resistance, β-cell dysfunction, and chronic inflammation. Objective To detect the concentrations of plasma asprosin in subjects with impaired glucose regulation (IGR) and newly diagnosed type 2 diabetes (nT2DM) and its relationship to parameters of glucose and lipid metabolism, insulin resistance, and pancreatic β-cell function. Methods 143 eligible participants were included and were divided into three groups including normal glucose regulation (NGR, n = 52), IGR (n = 40), and nT2DM group (n = 51). The intravenous glucose tolerance test (IVGTT) and clinical and biochemical parameters were measured in all participants. Results Plasma asprosin levels were higher in IGR (82.40 ± 91.06 ng/mL, P < 0.001) and nT2DM (73.25 ± 91.69 ng/mL, P < 0.001) groups compared with those in the NGR (16.22 ± 9.27 ng/mL) group, especially in IGR subjects. Correlation analysis showed that plasma asprosin levels were positively correlated with waist circumference (Wc), fasting plasma glucose (FPG), postchallenge plasma glucose (2hPG), HbA1c, triglyceride (TG), and homeostasis model assessment for insulin resistance (HOMA-IR) and negatively correlated with homeostasis model assessment for β-cell function (HOMA-β), area under the curve of the first-phase (0–10 min) insulin secretion (AUC), acute insulin response (AIR), and glucose disposition index (GDI) (all P < 0.05). Multiple logistical regression analyses revealed that plasma asprosin concentrations were significantly correlated with IGR and nT2DM after controlling for age, sex, BMI, and WHR. Conclusions Circulating asprosin might be a predictor of early diagnosis in DM and might be a potential therapeutic target for prediabetes and T2DM.

MYB-type transcription factors contain the conserved MYB DNA-binding domain of approximately 50 amino acids and are involved in the regulation of many aspects of plant growth, development, metabolism and stress responses. From soybean plants, we identified 156 GmMYB genes using our previously obtained 206 MYB unigenes, and 48 were found to have full-length open-reading frames. Expressions of all these identified genes were examined, and we found that expressions of 43 genes were changed upon treatment with ABA, salt, drought and/or cold stress. Three GmMYB genes, GmMYB76, GmMYB92 and GmMYB177, were chosen for further analysis. Using the yeast assay system, GmMYB76 and GmMYB92 were found to have transactivation activity and can form homodimers. GmMYB177 did not appear to have transactivation activity but can form heterodimers with GmMYB76. Yeast one-hybrid assay revealed that all the three GmMYBs could bind to cis-elements TAT AAC GGT TTT TT and CCG GAA AAA AGG AT, but with different affinity, and GmMYB92 could also bind to TCT CAC CTA CC. The transgenic Arabidopsis plants overexpressing GmMYB76 or GmMYB177 showed better performance than the GmMYB92-transgenic plants in salt and freezing tolerance. However, these transgenic plants exhibited reduced sensitivity to ABA treatment at germination stage in comparison with the wild-type plants. The three GmMYB genes differentially affected a subset of stress-responsive genes in addition to their regulation of a common subset of stress-responsive genes. These results indicate that the three GmMYB genes may play differential roles in stress tolerance, possibly through regulation of stress-responsive genes.