Related Experiment Video

Updated: Aug 29, 2026

Assaying Protein Kinase Activity with Radiolabeled ATP
08:05

Assaying Protein Kinase Activity with Radiolabeled ATP

Published on: May 26, 2017

MAPKAP kinase 2 phosphorylates tristetraprolin on in vivo sites including Ser178, a site required for 14-3-3 binding

Carol A Chrestensen1, Melanie J Schroeder, Jeffrey Shabanowitz

  • 1Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.

The Journal of Biological Chemistry
|December 23, 2003
PubMed

Insights

MAPKAP kinase 2 (MK2) phosphorylates Tristetraprolin (TTP) at key sites, creating a 14-3-3 binding site. This phosphorylation may control mRNA stability and tumor necrosis factor synthesis.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Signaling

Background:

  • Tumor necrosis factor (TNF) synthesis is regulated by Tristetraprolin (TTP), which binds to TNF mRNA.
  • MAPKAP kinase 2 (MK2) is essential for TNF production.

Purpose of the Study:

  • To identify phosphorylation sites on TTP.
  • To investigate the role of MK2 in TTP phosphorylation.
  • To determine if TTP phosphorylation by MK2 affects its function.

Main Methods:

  • Nanoflow high-pressure liquid chromatography microelectrospray ionization tandem mass spectrometry.
  • Direct digestion of TTP bound to affinity matrices (GSH-beads or anti-Myc magnetic beads).
  • Immobilized metal affinity chromatography for phosphopeptide enrichment.

Main Results:

  • MK2 phosphorylates TTP in vitro at Ser(52) and Ser(178) as major sites.
  • MK2 phosphorylation creates a 14-3-3 binding site on TTP.
  • In cells, TTP is phosphorylated at Ser(52) and Ser(178) upon MK2 overexpression or anisomycin treatment, suggesting these are in vivo MK2 sites.

Conclusions:

  • Serine 52 and Serine 178 are identified as major in vitro and putative in vivo phosphorylation sites of TTP by MK2.
  • TTP phosphorylation by MK2 may act as a biologic switch regulating mRNA stability and translation.
  • Understanding TTP phosphorylation provides insights into TNF synthesis regulation.

More Related Videos

Identification of Kinase-substrate Pairs Using High Throughput Screening11:13

Identification of Kinase-substrate Pairs Using High Throughput Screening

Published on: August 29, 2015

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate11:31

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Published on: September 18, 2013

Related Concept Videos

PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...
MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
Receptor Tyrosine Kinases01:26

Receptor Tyrosine Kinases

Receptor tyrosine kinases or RTKs are membrane-bound receptors that phosphorylate specific tyrosine on protein substrates. RTKs regulate cellular growth, differentiation, survival, and migration. They contain an extracellular ligand binding domain, a transmembrane domain, and a cytosolic tail with intrinsic kinase activity. Several extracellular signaling molecules activate RTKs in one or more ways and relay the signal downstream. Ligands such as platelet-derived growth factor (PDGF) or...
The JAK-STAT Signaling Pathway01:20

The JAK-STAT Signaling Pathway

Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as  SH2...
Enzyme-linked Receptors01:13

Enzyme-linked Receptors

Enzyme-linked receptors are proteins that act as both receptor and enzyme, activating multiple intracellular signals. This is a large group of receptors that include the receptor tyrosine kinase (RTK) family. Many growth factors and hormones bind to and activate the RTKs.Neurotrophin (NT) receptors are a family of RTKs, including trkA, trkB, and trkC (tropomyosin-related kinase) receptors. TrkA is specific for nerve growth factor (NGF), neurotrophin-6, and neurotrophin-7. TrkB binds...
Microtubule Associated Proteins (MAPs)01:42

Microtubule Associated Proteins (MAPs)

Microtubule function and architecture are regulated by an array of specialized proteins called microtubule-associated proteins or MAPs. These proteins are widespread across different organisms and have conserved protein motifs, like the multi-TOG domain for tubulin binding found in the CLASP family of MAPs. Some MAPs are lineage-specific based on their conserved domains. Their functions depend upon the cytoskeletal architecture and cell type they are located within. In-plant cells, a specific...