Although it is well appreciated that arachidonic acid, a second messenger molecule that is released by ligand-stimulated phospholipase A₂, stimulates a wide range of cell types, the mechanisms that mediate the actions of arachidonic acid are still poorly understood. We now report that arachidonic acid stimulated the appearance of dual-phosphorylated (active) p38 mitogen-activated protein kinase as detected by Western blotting in HeLa cells, HL60 cells, human neutrophils, and human umbilical vein endothelial cells but not Jurkat cells. An increase in p38 kinase activity caused by arachidonic acid was also observed. Further studies with neutrophils show that the stimulation of p38 dual phosphorylation by arachidonic acid was transient, peaking at 5 min, and was concentration-dependent. The effect of arachidonic acid was not affected by either nordihydroguaiaretic acid, an inhibitor of the 5-, 12-, and 15-lipoxygenases or by indomethacin, an inhibitor of cyclooxygenase. Arachidonic acid also stimulated the phosphorylation and/or activity of the extracellular signal-regulated protein kinase and of c-jun N-terminal kinase in a cell-type-specific manner. An examination of the mechanisms through which arachidonic acid stimulated the phosphorylation/activity of p38 and extracellular signal-regulated protein kinase in neutrophils revealed an involvement of protein kinase C. Thus, arachidonic acid stimulated the translocation of protein kinase C α, βI, and βII to a particulate fraction, and the effects of arachidonic acid on mitogen-activated protein kinase phosphorylation/activity were partially inhibited by GF109203X, an inhibitor of protein kinase C. This study is the first to demonstrate that a polyunsaturated fatty acid causes the dual phosphorylation and activation of p38.

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the ...

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R–mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Methylglyoxal (MGO) is a highly reactive cellular metabolite that glycates lysine and arginine residues to form post-translational modifications known as advanced glycation end products. Because of their low abundance and low stoichiometry, few studies have reported their occurrence and site-specific locations in proteins. Proteomic analysis of WIL2-NS B lymphoblastoid cells in the absence and presence of exogenous MGO was conducted to investigate the extent of MGO modifications. We found over 500 MGO modified proteins, revealing an over-representation of these modifications on many glycolytic enzymes, as well as ribosomal and spliceosome proteins. Moreover, MGO modifications were observed on the active site residues of glycolytic enzymes that could alter their activity. We similarly observed modification of glycolytic enzymes across several epithelial cell lines and peripheral blood lymphocytes, with modification of fructose bisphosphate aldolase being observed in all samples. These results indicate that glycolytic proteins could be particularly prone to the formation of MGO adducts.