Identification of mitogen-activated protein kinases substrates in Arabidopsis using kinase client assay.

Mitogen-activated protein kinase (MPK) cascades are essential signal transduction components that control a variety of cellular responses in all eukaryotes. MPKs convert extracellular stimuli into cellular responses by the phosphorylation of downstream substrates. Although MPK cascades are predicted to be very complex, only limited numbers of MPK substrates have been identified in plants. Here, we used the kinase client (KiC) assay to identify novel substrates of MPK3 and MPK6. Recombinant MPK3 or MPK6 were tested against a large synthetic peptide library representing in vivo phosphorylation sites, and phosphorylated peptides were identified by high-resolution tandem mass spectrometry. From this screen, we identified 23 and 21 putative client peptides of MPK3 and MPK6, respectively. To verify the phosphorylation of putative client peptides, we performed in vitro kinase assay with recombinant fusion proteins of isolated client peptides. We found that 13 and 9 recombinant proteins were phosphorylated by MPK3 and MPK6. Among them, 11 proteins were proven to be the novel substrates of two MPKs. This study suggests that the KiC assay is a useful method to identify new substrates of MPKs.

Cross-Correlations between Scientific Physical Fitness, Body Mass Index Distribution, and Overweight/Obesity Risks among Adults in Taiwan.

Background and Objectives: Health-related physical fitness reduces the risk of chronic disease, promotes quality of life, and has enormous economic benefits considering the global health care costs resulting from obesity. However, relatively limited information is available regarding the dose-response relationship between scientific physical fitness and obesity risk. This study aimed to determine the associations of scientific physical fitness with body mass index (BMI) distribution and overweight/obesity risk among adults aged 23-64 years in Taiwan. Materials and Methods: We conducted a cross-sectional study and reviewed data derived from the Scientific Physical Fitness Testing Program, Sports Administration, Ministry of Education, Taiwan. Responses from 16,939 participants from the database (7761 men and 9178 women, aged 23-64 years) were collected in this study. Each participant completed a series of scientific physical fitness measurements, including cardiorespiratory fitness (3 min progressive knee-up and step [3MPKS] test), muscular fitness (hand grip strength), and flexibility (sit-and-reach test). Anthropometric measurements included body height, weight, and BMI. The quartiles of scientific physical fitness results were identified as the dependent variable in the multiple linear and multiple logistic regression analysis to determine the associations of the scientific physical fitness measurements with BMI distribution and overweight/obesity risk, as well as the dose-response relationship. Results: The 3MPKS test was significantly associated with BMI (quartile 1 (Q1): ß = 1.900; quartile 2 (Q2): ß = 1.594; quartile 3 (Q3): ß = 1.079 for men, and Q1: ß = 1.454; Q2: ß = 0.882; Q3: ß = 0.555 for women), overweight (Q1: odds ratio (OR) = 2.117; Q2: OR = 2.056; Q3: OR = 2.063 for men, and Q1: OR = 3.036; Q2: OR = 2.542; Q3: OR = 1.959 for women), and obesity (Q1: OR = 6.530; Q2: OR = 5.747; Q3: OR = 3.557 for men, and Q1: OR = 3.238; Q2: OR = 1.431 for women) risk compared with quartile 4 (Q4) as the reference group with a dose-response relationship. In addition, relative hand grip strength was significantly associated with BMI (Q2: ß = -0.922; Q3: ß = -1.865; Q4: ß = -3.108 for men, and Q2: ß = -1.309; Q3: ß = -2.161; Q4: ß = -2.759 for women), overweight (Q2: OR = 0.806; Q3: OR = 0.697; Q4: OR = 0.278 for men, and Q2: OR = 0.667; Q3: OR = 0.398; Q4: OR = 0.228 for women), and obesity (Q1: OR = 0.528; Q2: OR = 0.206; Q3: OR = 0.049 for men, and Q1: OR = 0.351; Q2: OR = 0.129; Q3: OR = 0.051 for women) risk compared with Q1 as the reference group with a dose-response relationship. Conclusions: Higher levels of performance of the 3MPKS and relative grip strength tests were associated with lower BMI and overweight/obesity risk in both sexes. However, the sit-and-reach test was only partially related to BMI and overweight/obesity risk in both sexes. Cardiorespiratory fitness and muscular fitness were effective predictors of BMI distribution and overweight/obesity risk in Taiwanese adults.

MxMPK4-1 phosphorylates NADPH oxidase to trigger the MxMPK6-2-MxbHLH104 pathway mediated Fe deficiency responses in apple.

Iron (Fe) deficiency is a nutritional stress in plants that commonly occurs in alkaline and calcareous soils. Mitogen-activated protein kinases (MPKs), the terminal player of MAPK cascade, are involved in distinct physiological processes. Once plants suffer from Fe deficiency stress, the mechanism of MPK function remains unclear owing to limited study on the MPK networks including substrate proteins and downstream pathways. Here, the MAP kinase MPK4-1 was induced in roots of Fe efficient apple rootstock Malus xiaojinensis but not in Fe inefficient rootstock Malus baccata under Fe deficiency conditions. Overexpression of MxMPK4-1 in apple calli and apple roots enhanced the responses to Fe deficiency. We found that MxMPK4-1 interacted with NADPH oxidases (NOX)-respiratory burst oxidase homologs MxRBOHD1 and MxRBOHD2, which positively regulated responses to Fe deficiency. Moreover, MxMPK4-1 phosphorylated the C terminus of MxRBOHD2 at Ser797 and Ser906 and positively and negatively regulated NOX activity through these phospho-sites, respectively. When compared with apple calli that overexpressed MxRBOHD2, the coexpression of MxMPK4-1 and MxRBOHD2 prominently enhanced the Fe deficiency responses. We also demonstrated that hydrogen peroxide derived from MxMPK4-1-MxRBOHD2 regulated the MxMPK6-2-MxbHLH104 pathway, illuminating a systematic network that involves different MPK proteins in M. xiaojinensis under Fe deficiency stress.

Sophoridine Inhibits the Tumour Growth of Non-Small Lung Cancer by Inducing Macrophages M1 Polarisation via MAPK-Mediated Inflammatory Pathway.

Lung cancer is one of the most common and lethal neoplasms for which very few efficacious treatments are currently available. M1-like polarised tumour-associated macrophages (TAMs) are key mediators to modulate the tumour microenvironment, which play a key role in inhibiting cancer cell growth. Sophoridine, a naturally occurring alkaloid, exerts multiple pharmacological activities including anti-tumour and anti-inflammatory activities, but it has not been characterised as a regulator of tumour microenvironment towards NSCLC. Herein, the regulatory effects of sophoridine on the polarisation of THP-1 cells into TAMs and the anti-tumour effects of sophoridine-stimulated M1 polarised macrophages towards lung cancer cells were carefully investigated both in vitro and in vivo. The results showed that sophoridine could significantly promote M1 polarisation of RAW264.7 and THP-1-derived macrophages, leading to increased expression of pro-inflammatory cytokines and the M1 surface markers CD86 via activating MAPKs signaling pathway. Further investigations showed that sophoridine-stimulated RAW264.7 and THP-1-derived M1 macrophages effectively induced cell apoptosis as well as inhibited the cell colony formation and cell proliferation in both H460 and Lewis lung cancer cells. In Lewis-bearing mice model, sophoridine (15 or 25 mg/kg) significantly inhibited the tumour growth and up-regulated the expression of CD86/F4/80 in tumour tissues. Collectively, the findings clearly demonstrate that sophoridine promoted M1-like polarisation in vitro and in vivo, suggesting that sophoridine held a great therapeutic potential for treating lung cancer.