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 including MALDI/MS, MALDI/MS/MS, LC/MS/MS, IMAC/MALDI/MS/MS and multidimensional protein identification technology (MudPIT) 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 pro-inflammatory cytokines.

Phosphorylation of Tristetraprolin by MK2 Impairs AU-Rich Element mRNA Decay by Preventing Deadenylase Recruitment

“…TTP has the potential to be phosphorylated at a number of residues by various kinases (6,7,13), and inhibition of PP2A results in dramatic hyperphosphorylation of TTP (Fig. 6) (57).…”

dTIS11 Protein-dependent Polysomal Deadenylation Is the Key Step in AU-rich Element-mediated mRNA Decay in Drosophila Cells

“…Previous studies have demonstrated that TTP, a human protein of the TIS11 family, induces the degradation of ARE-containing mRNAs through a large variety of mechanisms including deadenylation, decapping, and P-body targeting. Sequence alignment of TIS11 family members reveals a high degree of conservation in the CCCH zing finger RNA-binding region (34), whereas other regions of the protein are poorly conserved between invertebrates and mammals, suggesting that the molecular mechanism controlling the activity of TIS11 proteins may have evolved in the course of eukaryotic evolution.…”

Direct Binding of Specific AUF1 Isoforms to Tandem Zinc Finger Domains of Tristetraprolin (TTP) Family Proteins

“…4, 3, lanes 1 and 5). These results demonstrate that AUF1p45 can bind to all known mammalian TTP family members, presumably reflecting the high degree of amino acid conservation in their TZF domains (38,39).…”