Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

Abstract

Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods, including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry (MS), including MALDI/MS, MALDI/MS/MS, liquid chromatography/MS/MS, immobilized metal ion affinity chromatography (IMAC)/MALDI/MS/MS and multidimensional protein identification technology has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP’s regulatory functions in controlling proinflammatory cytokines.

Keywords::

• cytokine
• in vivo radiolabeling
• inflammation
• mass spectrometry
• mRNA instability
• phosphorylation site
• protein
• sequencing
• site-directed
• mutagenesis
• tristetraprolin
• zinc finger protein

Acknowledgement

We greatly appreciate Joseph F Urban Jr (US Department of Agriculture – Agricultural Research Service) and Kenneth B Tomer (NIH-National Institute of Environmental Health Sciences) for their strong support. We also thank John D Venable in John R Yates III’s Laboratory (Scripps Research Institute) for the collaboration on the MudPIT MS analyses, and Kiran S Panickar (USDA-ARS) and Joseph F Urban Jr for their helpful comments on the manuscript.

Financial & competing interests disclosure

This work was supported in part by USDA-ARS Human Nutrition Research Program and the Intramural Research Program of the NIH, NIEHS. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.