Functional study of O-GlcNAcylation on Cdk4 during G1 S transition

Abstract

O-GlcNAcylation is a post-translational modification which occurs on proteins located in the cytosol and nucleus of cells. This modification regulates the expression and functions of proteins, thereby participating in various cellular signalings and cell mechanisms. UDP-GlcNAc, the O-GlcNAc donor of the O-GlcNAcylation is synthesized from glucose via hexosamine biosynthetic pathway. Production of UDP-GlcNAc is greatly reliant on the cellular glucose condition. It is also known that O-GlcNAcylation is involved in numerous diseases such as cancer, diabetes, and Alzheimer's disease.

Recent scientific reports showed that cancer cells are in an elevated state of O-GlcNAcylation when compared with normal cells and furthermore, it was also shown that O-GlcNAcylation is involved in cell cycle regulation. From this we hypothesized that higher O-GlcNAcylation in cancer cells promotes cell cycle transition. In order to verify this hypothesis, we generated an OGT knock-down stable cell line using lung cancer cell line A549. Through FACS analysis, we observed a relatively delayed G1/S cell cycle transition in these cells. Using Western blot, we determined that hypophosphorylation of pRB was the cause of the delayed G1/S cycle transition.

To gain a better understanding of the relationship between the decrease in O-GlcNAcylation and pRB hypophosphorylation, we examined whether cyclin-dependent kinase 4 (Cdk4), a protein crucially involved in phosphorylation of pRB, is O-GlcNAcylated. By Western blot, we detected the presence of O-GlcNAcylation on overexpressed Cdk4 and observed an increase in detected O-GlcNAcylation when OGT was also overexpressed. Furthermore, using protein O-GlcNAc moiety binding sWGA precipitation, we observed O-GlcNAcylation on Cdk4 regulation during G1/S cell cycle transition.

To determine the functions of O-GlcNAcylation on Cdk4, we focused on Cdk4 and cyclin D binding affinity which is critical to the G1/S transition. By coimmunoprecipitation of overexpressed Cdk4 and cyclin D, we observed increased binding between Cdk4 and cyclin D1, D2 under an OGT overexpressed condition.

In this research we aim to identify that G1/S cell cycle transition is regulated by the O-GlcNAcylation on Cdk4, which increases the binding between Cdk4 and cyclin D. Such relationship serve to show that increased global O-GlcNAcylation of cancer cells contribute to cancer cell proliferation.