Essential Role of the MEKK3-ERK5 Module in Endothelial Cell Death

Jorge Pincay ’20

Figure 1. The MEKK3-ERK5 module in blood serum can help mitigate apoptosis in endothelial cells and therefore, help prevent atherosclerosis. 

Atherosclerosis is a chronic inflammatory disease characterized by the build-up of plaque in artery walls, which can lead to coronary artery disease, heart failure, and stroke. Since atherosclerosis results from endothelial cell injury and death, researchers are making greater efforts to acquire a better understanding of the cellular mechanisms that may lead to these unfavorable changes in endothelial cells. In particular, the MEKK3-MEK5-ERK5 kinase module, an essential tertiary kinase signaling pathway known to be involved in mediating endothelial cell survival, is a current key area of study; the mechanisms by which the module regulates endothelial cell survival remain unclear. Scientists from the University of South Australia and the University of Adelaide have recently conducted studies on human umbilical cord vein endothelial cells (HUVEC) to investigate the MEKK3-MEK5-ERK5 module signaling pathways and how certain kinase interactions may lead to endothelial cell survival or death.

HUVECs were first isolated from human umbilical cords by treating the cords with collagenase, an enzyme that breaks the peptide bonds in collagen. In order to investigate potential proteins that may interact with the ternary kinase complex within cells, HUVECs were incubated in the presence or absence of blood serum, respectively, and then lysed. The ERK5 protein of the ternary kinase complex within cells incubated in serum was then immuno-precipitated using antibodies and incubated in a kinase assay to measure the activity of ERK5. The product of this assay was then fractionated by SDS-page, and the gel was used to produce a radiogram. A band on the SDS-page with a Mr. (relative molecular weight) of about 113,000 was found to generate the highest peak on the radiogram, which is consistent with known value for ERK5. Another band on the gel with an Mr. of about 36,000 generated a more faint peak that was believed to be the substrate of the ERK5 kinase. Proteins from 36,000 band were excised and subjected to HPLC (high pressure liquid chromatography). These proteins were identified to be lactate dehydrogenase and glyceraldehyde dehydrogenase (GAPDH), an enzyme that has been found to induce apoptosis in certain cell types. Additionally, immuno-precipitation followed by western blotting was able to show co-precipitation of ERK5 and GAPDH. These results as a whole  provided evidence of notable interaction between the tertiary kinase complex and GAPDH. In order to assess the effects of the MEKK3-MEK5-ERK5 module on GAPDH, levels of GAPDH in the nucleus of HUVECs were measured in the presence and absence of blood serum. It was found in the absence of blood serum, cytotoxic levels of GAPDH accumulated in HUVEC nuclei, causing cellular damage and apoptosis. After replenishing HUVECs with blood serum, MEKK3-GAPDH interactions were observed and nuclear GAPDH concentrations decreased significantly. Furthermore, a pull-down assay revealed that GAPDH binds specifically to MEKK3. In addition to  providing strong evidence for the interaction between GAPDH and MEKK3, these results implicate the accumulation of nuclear GADPH as a cause of endothelial cell death and suggest that MEKK3 may be a possible target for regulating GADPH function and thus promoting endothelial cell survival. 

The results of this study have furthered the understanding of an essential kinase interaction in the signaling pathways of endothelial cells and may potentially lay the groundwork for future studies on the development of novel atherosclerosis treatments.




  • Li et al., Regulation of endothelial cell survival and death by the MAP kinase/ERK kinase kinase3-glyceraldehyde-3-phosphate dehydrogenase signaling axis, Cellular Signaling 58, 20-23 (2019),




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