NDLI: Identification of a Major Phosphopeptide in Human Tristetraprolin by Phosphopeptide Mapping and Mass Spectrometry

Content Provider	PubMed Central
Author	Cao, Heping Deterding, Leesa J. Blackshear, Perry J.
Editor	Wang, Tianyi
Copyright Year	2014
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.
Related Links	http://dx.doi.org/10.1371/journal.pone.0100977
Starting Page	100977
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	7
Volume Number	9
Language	English
Publisher	Public Library of Science
Publisher Date	2014-07-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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