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Identification of the Anti-proliferative protein Tob as a MAPK substrate
Momoko Maekawa1, Eisuke Nishida, Takuji Tanoue
1Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Abstract:
Mitogen-activated protein kinases (MAPKs) regulate a wide variety of cellular functions by phosphorylating their specific substrates. Here we have identified Tob as a novel substrate of MAPK. Tob, a member of the Tob and B-cell translocation gene anti-proliferative protein family, is shown to negatively regulate the proliferation of osteoblasts and T cells. In this study, our two-hybrid screening has identified Tob as an ERK2-interacting protein. Biochemical analyses have then shown that ERK MAPK (ERK2) and JNK/SAPK (JNK2) bind to and phosphorylate Tob in vitro. ERK catalyzes the phosphorylation more efficiently than JNK. When the ERK pathway is activated in cells, phosphorylation of Tob is induced. An ERK-binding or -docking site locates in the N-terminal portion of Tob, and phosphorylation sites reside in the C-terminal stretch region. The docking is crucial for efficient phosphorylation. Mutant forms of Tob, in which serines are replaced by glutamic acids to mimic phosphorylation, show a much reduced ability to inhibit the cell cycle progression to S phase from G(0)/G(1) phase, as compared with wild-type Tob, indicating that ERK phosphorylation negatively regulates the anti-proliferative function of Tob.
Insights
Mitogen-activated protein kinases (MAPKs) phosphorylate Tob, a protein that inhibits cell proliferation. This phosphorylation by ERK MAPK reduces Tob's anti-proliferative function, impacting cell cycle progression.
Area of Science:
- Cellular Biology
- Molecular Biology
- Signal Transduction
Background:
- Mitogen-activated protein kinases (MAPKs) are crucial regulators of cellular processes through substrate phosphorylation.
- Tob proteins are known inhibitors of cell proliferation in osteoblasts and T cells.
Purpose of the Study:
- To identify novel MAPK substrates.
- To investigate the interaction between Tob and MAPKs.
- To elucidate the functional consequences of Tob phosphorylation by MAPKs.
Main Methods:
- Yeast two-hybrid screening to identify interacting proteins.
- In vitro kinase assays using purified proteins.
- Cell-based assays to assess cell cycle progression and protein phosphorylation.
- Site-directed mutagenesis to create Tob phosphorylation mimics.
Main Results:
- Tob was identified as a novel substrate for ERK2 and JNK2 MAPKs.
- ERK MAPK demonstrated more efficient phosphorylation of Tob compared to JNK.
- Activation of the ERK pathway in cells led to Tob phosphorylation.
- Phosphorylation of Tob by ERK MAPK significantly reduced its ability to inhibit cell cycle progression from G(0)/G(1) to S phase.
Conclusions:
- Tob is a direct substrate of ERK and JNK MAPKs.
- ERK-mediated phosphorylation negatively regulates the anti-proliferative activity of Tob.
- This regulation involves specific N-terminal docking and C-terminal phosphorylation sites, impacting cell cycle control.