Submitted to: Protein Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/21/2007
Publication Date: 3/31/2008
Citation: Cao, H., Rui, L. 2008. Phosphorylation of recombinant tristetraprolin in vitro. Protein Journal. 27(3):163-169. Interpretive Summary: Diet plays an important role in the prevention of inflammation-related diseases, such as arthritis, obesity, and type 2 diabetes. The diets consumed in the United States and other developed countries appear to increase the risks of developing these diseases. Drug treatment for these diseases is not feasible for most of the world population and alternative treatments need to be explored. Previous studies suggest that cinnamon and green tea extracts exhibit insulin-like activity in cells and intact animals to induce gene expression of tristetraprolin (TTP), a regulatory protein with anti-inflammatory function. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity, and myeloid hyperplasia. TTP expression is reduced in fats of obese people with metabolic syndrome and brains of suicide victims. These previous studies suggest that a modest increase of TTP activity may probably have a positive effect on improving these disease conditions. One of the ways to regulate TTP activity is to add phosphate to the protein, a process called protein phosphorylation. In living cells, this process is catalyzed by a class of proteins called protein kinases. In this paper, TTP phosphorylation was explored. To this end, TTP proteins of human and mouse origins were produced in bacterial cells as recombinant forms since pure forms of these proteins were not available from native cells. These proteins were then used for the study of phosphorylation in test tubes. Our results demonstrate that TTP is an in vitro substrate for a number of protein kinases. These findings should be of interest to scientists for further study of the regulation of TTP activity by phosphorylation.
Technical Abstract: Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some AU-rich-containing mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of proinflammatory cytokines. TTP gene expression is induced by various factors including insulin, cinnamon, and green tea extracts. Previous studies have shown that TTP is highly phosphorylated in vivo; and multiple phosphorylation sites are identified in human TTP. This study evaluated the potential protein kinases that could phosphorylate recombinant TTP in vitro. Motif scanning suggested that TTP was a potential substrate for various kinases. SDS-PAGE showed that in vitro phosphorylation of TTP with p42 and p38 MAP kinases resulted in visible electrophoretic mobility shift of TTP to higher molecular masses. Autoradiography showed that TTP was phosphorylated in vitro by GSK3b, PKA, PKB, PKC, but not Cdc2, in addition to p42, p38, and JNK kinases. These results demonstrate that TTP is a substrate for a number of protein kinases in vitro.