Methylglyoxal downregulates Raf-1 protein through a ubiquitination-mediated mechanism.
Abnormal accumulation of methylglyoxal, a physiological glucose metabolite, is considered a potential link between hyperglycemia and diabetes complications. Evidence has shown that methylglyoxal modifies cellular proteins by glycation and oxidation, resulting in dysfunction or loss of cellular proteins. Raf-1 protein-serine/threonine kinase serves as a central switch board in the transmission of many growth and developmental signals. It was reported that Raf-1 levels appear to decrease in some diabetic subjects. But the potential mechanisms have not yet been clarified. Here, we tested the hypothesis that methylglyoxal-mediated proteolysis might contribute to the downregulation of Raf-1 levels. We observed that a rapid and detectable decrease in Raf-1 protein levels was induced by methylglyoxal, which was accelerated by treating with a Raf-1 activator, phorbol-12-myristate-13-acetate, and by expressing active forms of Raf-1 and Ras. Moreover, immunoprecipitation and immunoblotting assays showed that co-treatment of cells with methylglyoxal and phorbol-12-myristate-13-acetate caused dramatic ubiquitination in both total intracellular proteins and Raf-1. Blocking phosphorylation with the protein kinase C inhibitor bisindolylmaleimide, or inhibiting intracellular oxidation by addition of the antioxidant N-acetyl-l-cysteine could reverse the ubiquitination and downregulation of Raf-1 induced by methylglyoxal and phorbol-12-myristate-13-acetate. These results suggest that methylglyoxal-mediated intracellular oxidation and ubiquitin/proteasome-dependent proteolysis are involved in the downregulation of Raf-1, which may be closely related to the development complications in diabetes.
Du
Jun
J
Center of Microbiology, Biochemistry and Pharmacology, School of Pharmaceutical Sciences, Sun Yat-Sen University. Guangzhou 510080, China.
Zeng
Jun
J
Ou
Xueling
X
Ren
Xianda
X
Cai
Shaohui
S
D018929:Cell Culture Techniques; D048909:Diabetes Complications / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D010084:Oxidation-Reduction; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000187:drug effects / Q000378:metabolism*; D011765:Pyruvaldehyde / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism*
D011765:Pyruvaldehyde; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D046988:Proteasome Endopeptidase Complex; D044764:Ubiquitin-Activating Enzymes; D013755:Tetradecanoylphorbol Acetate