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ARS Home » Northeast Area » Beltsville, Maryland (BHNRC) » Beltsville Human Nutrition Research Center » Diet, Genomics and Immunology Laboratory » Research » Publications at this Location » Publication #208955
Title: Post-translational modifications of the mRNA destabilizing protein tristetraprolin

Author
item Cao, Heping
item Anderson, Richard
item BLACKSHEAR, PERRY - DHHS-NIH-NIEHS-LN, NC

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/17/2007
Publication Date: 6/6/2007
Citation: Cao, H., Anderson, R.A., Blackshear, P.J. 2007. Post-translational modifications of the mRNA destabilizing protein tristetraprolin. [CD-ROM].Version ---. . Cambridge Health Tech Institute Conference. [CD-ROM].

Interpretive Summary:

Technical Abstract: Tristetraprolin(TTP)is a hyperphosphorylated zinc finger protein that binds to AU-rich elements within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity, and myeloid hyperplasia. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected 293 cells 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), while mutations at the other sites had little effect on its gel mobility. Dephosphorylation and in vivo labeling studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AU-rich elements. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. S197, S218, and S228 are predicted by motif scanning to be potential sites for protein kinase A, extracellular-signal-regulated kinase 1 (both S218 and S228) and glycogen synthase kinase-3, respectively. This study has identified multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells, and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines.