Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 5, 2007
Publication Date: June 4, 2007
Citation: Cao, H., Anderson, R.A., Blackshear, P.J. Protein Kinase Targets[CD-ROM]. Version ?, where? Cambridge Healthtech Institute. Technical Abstract: Transgenic mice without tristetraprolin (TTP) protein develop a profound inflammatory syndrome with erosive arthritis, autoimmunity, and myeloid hyperplasia. TTP is a hyperphosphorylated zinc finger protein that binds to AU-rich elements within certain mRNAs such as tumor necrosis factor mRNA and causes destabilization of those mRNAs. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. In this study, we investigated the phosphorylation sites in human TTP by mass spectrometry and site-directed mutagenesis. A number of phosphorylation sites including S66, S88, T92, S169, S186, S197, S218, S228, S276, and S296 were identified by MS analyses using MALDI/MS, MALDI/MS/MS, LC/MS/MS, and multidimensional protein identification technology. Alanine mutations at S197, S218, and S228 of the human protein significantly increased TTP’s gel mobility (likely to be stoichiometric). Dephosphorylation and in vivo labeling studies showed that mutant proteins containing multiple mutations were still phosphorylated. S197, S218, and S228 are predicted by motif scanning to be potential sites for protein kinase A, extracellular-signal-regulated kinase 1 and glycogen synthase kinase-3, respectively. We further determined that TTP was phosphorylated in vitro by a number of protein kinases including p42 MAP kinase, p38 MAP kinase, JNK, GSK3, MK2, PKA, PKB, and PKC. This study demonstrates that the anti-inflammatory protein TTP is phosphorylated at multiple sites by multiple protein kinases and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines.