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United States Department of Agriculture

Agricultural Research Service

Research Project: Value-Added Products from Cottonseed

Location: Commodity Utilization Research

Title: Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry

Authors
item Cao, Heping
item Deterding, Lessa -
item Blackshear, Perry -

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 2, 2014
Publication Date: July 10, 2014
Citation: Cao, H., Deterding, L.J., Blackshear, P.J. 2014. Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry. PLoS One. 9(7):1-13.

Interpretive Summary: Alzheimer’s disease, arthritis, cardiovascular diseases (CVD) and diabetes affect millions of people today. For example, CVD accounted for 38.5% of all deaths in 2001. CVD and stroke accounted for 15% of the total health expenditures in 2007 and was estimated to cost the United States $444 billion in 2010. Cytokines including tumor necrosis factor alpha (TNF) are important in the development and progression of these diseases. However, anti-TNF therapies targeting TNF and its receptors failed in clinical trials. An alternative strategy to reduce pro-inflammatory cytokines in the relevant tissues is to promote degradation of multiple cytokine mRNAs because these mRNAs possess AU-rich elements (AREs) and that their stabilities are largely controlled by ARE binding proteins. Tristetraprolin (TTP), an anti-inflammatory protein, binds to the AREs of clinically important mRNAs such as TNF mRNA and promotes the destruction of those transcripts. TTP-deficient mice develop a profound inflammatory syndrome with cachexia, dermatitis, erosive arthritis, autoimmunity and myeloid hyperplasia, due to excessive production of TNF and other cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated in vivo and is a substrate for several protein kinases. Multiple phosphorylation sites are identified in human TTP, but it is difficult to assign major vs. minor phosphorylation sites. This study generated additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry.

Technical Abstract: Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated in vivo and is a substrate for several protein kinases. Multiple phosphorylation sites are identified in human TTP, but it is difficult to assign major vs. minor phosphorylation sites. This study aimed to generate additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry (MS). Wild-type and site-directed mutant TTP proteins were expressed in transfected human cells followed by in vivo radiolabeling with [32P]-orthophosphate. Histidine-tagged TTP proteins were purified with Ni-NTA affinity beads and digested with trypsin and lysyl endopeptidase. The digested peptides were separated by C18 column with high performance liquid chromatography. Wild-type and all mutant TTP proteins were localized in the cytosol, phosphorylated extensively in vivo and capable of binding to ARE-containing RNA probes. Mutant TTP with S90 and S93 mutations resulted in the disappearance of a major phosphopeptide peak. Mutant TTP with an S197 mutation resulted in another major phosphopeptide peak being eluted earlier than the wild-type. Additional mutations at S186, S296 and T271 exhibited little effect on phosphopeptide profiles. MS analysis identified the peptide that was missing in the S90 and S93 mutant protein as LGPELSPSPTSPTATSTTPSR (corresponding to amino acid residues 83-103 of human TTP). MS also identified a major phosphopeptide associated with the first zinc-finger region. These analyses suggest that the tryptic peptide containing S90 and S93 is a major phosphopeptide in human TTP.

Last Modified: 7/25/2014
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