Regulation of glucose uptake by the stress-activated p38 MAPK pathway in skeletal muscle cells: implications for obesity and diabetes.

Dematin is a cytoskeletal associated protein that was first identified in erythrocytes, where it binds directly to the glucose transporter GLUT1 and provides mechanical support to the cell. The expression of dematin has since been identified in many tissues of the body, but the function and regulation of dematin in non-erythroid cells is poorly understood.

This research project aimed to determine whether the stability of dematin is regulated by its PEST motif, and to investigate the potential role of p38 phosphorylation in regulating this process. The presence of a PEST motif within a protein has been linked to an increased tendency for degradation, however not all proteins that contain these motifs are unstable. Our experiments have clearly confirmed that dematin does have a short half-life, which we have calculated to be approximately 51⁄2 hours. We have further shown that the enhanced degradation of dematin is attributable to the presence of the PEST motif. Previous research in our lab has characterised dematin as a novel substrate of the stress-activated p38 MAPK.

The PEST motif contains two potential p38 phosphorylation sites and is flanked by a further two motifs, leading to the hypothesis that p38 is a regulator of dematin’s stability. However, our experiments indicate that the PEST-mediated degradation of dematin is not regulated by p38 phosphorylation. This also suggests that p38 is not a likely regulator of dematin’s ability to regulate glucose uptake. Indeed, other experiments performed in parallel have confirmed that the role of dematin in glucose uptake is not direct and that other regulators may be involved.

Our findings form the first available data on the regulation of dematin in skeletal muscle cells. The half-life of dematin, which depends on the presence of the PEST domain, indicates that dematin expression can be controlled by the cell over a short time scale, suggesting that it is involved in processes which must be tightly regulated. This data therefore forms a solid basis for further research into the potential regulators of the dematin PEST motif.

The funding received allowed us to purchase the necessary consumables for the cloning and mutagenesis of the dematin constructs, and for conducting the degradation experiments - this included the purchase of cell culture and transfection reagents, antibodies, a p38 inhibitor, and

the specialist media and radioisotopes required. Without this grant it would not have been possible to conduct these experiments as no alternative funding was available. The results collected here will be combined with additional data on the regulation of dematin to produce a manuscript for publication. These results will also be extremely useful as preliminary data for future grant applications to further investigate the regulatory mechanisms involved in controlling the function of dematin. Finally, the data generated has been included in the doctoral thesis of Holly Baum, which was submitted in 2014.