Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin.
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.
Cao
Heping
H
US Department of Agriculture, Agricultural Research Service, Beltsville Human Nutrition Research Center, Diet, Genomics, and Immunology Laboratory, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. heping.cao@ars.usda.gov
Deterding
Leesa J
LJ
Blackshear
Perry J
PJ
D000595:Amino Acid Sequence; D000893:Anti-Inflammatory Agents / Q000737:chemistry*; D001665:Binding Sites; D016207:Cytokines / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010802:Phylogeny; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051816:Tristetraprolin / Q000737:chemistry* / Q000502:physiology*
D000893:Anti-Inflammatory Agents; D016207:Cytokines; D010455:Peptides; D012333:RNA, Messenger; D051816:Tristetraprolin