Related Experiment Videos
Akt-dependent phosphorylation specifically regulates Cot induction of NF-kappa B-dependent transcription
Lawrence P Kane1, Marianne N Mollenauer, Zheng Xu
1Department of Medicine. The Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, California 94143, USA.
Abstract:
The Akt (or protein kinase B) and Cot (or Tpl-2) serine/threonine kinases are associated with cellular transformation. These kinases have also been implicated in the induction of NF-kappa B-dependent transcription. As a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, Cot can also activate MAP kinase signaling pathways that target AP-1 and NFAT family transcription factors. Here we show that Akt and Cot physically associate and functionally cooperate. Akt appears to function upstream of Cot, as Akt can enhance Cot induction of NF-kappa B-dependent transcription, and dominant-negative Cot blocks the activation of this element by Akt. Furthermore, deletion analysis shows that binding to Akt is critical for Cot function. The regulation of NF-kappa B-dependent transcription by Cot requires Akt-dependent phosphorylation of serine 400 (S400), near the carboxy terminus of Cot. However, phosphorylation at this site is not required for Cot kinase activity or AP-1 induction, suggesting it specifically regulates Cot effector function at the level of the NF-kappa B pathway. Mutation of S400 in Cot does indeed abolish its ability to activate I kappa B-kinase (IKK) complexes, but paradoxically it allows for increased Cot association with the IKK complex. This mutated form of Cot also acts as a dominant negative for T-cell antigen receptor/CD28- or Akt/phorbol myristate acetate-induced NF-kappa B induction, while having relatively little effect on tumor necrosis factor induction of NF-kappa B. These findings suggest that the activation of different signaling pathways by MAP3Ks may be regulated separately and may provide evidence for how such discrimination by one member of this kinase family occurs.
Insights
The Akt and Cot kinases physically associate and cooperate to regulate NF-kappa B transcription. Akt-dependent phosphorylation of Cot at serine 400 is critical for this NF-kappa B pathway activation.
Area of Science:
- Cellular Biology
- Molecular Signaling
- Signal Transduction
Background:
- Akt (protein kinase B) and Cot (Tpl-2) are serine/threonine kinases linked to cellular transformation and NF-kappa B transcription.
- Cot, a MAP3K family member, also activates MAP kinase pathways impacting AP-1 and NFAT transcription factors.
Purpose of the Study:
- To investigate the physical and functional relationship between Akt and Cot kinases.
- To elucidate the mechanism by which Cot regulates NF-kappa B-dependent transcription.
Main Methods:
- Co-immunoprecipitation to assess physical association.
- Dominant-negative constructs and site-directed mutagenesis to analyze functional cooperation.
- Reporter gene assays to measure NF-kappa B and AP-1 transcriptional activity.
Main Results:
- Akt and Cot physically associate and functionally cooperate, with Akt acting upstream of Cot.
- Akt-dependent phosphorylation of Cot at serine 400 (S400) is essential for NF-kappa B induction but not for kinase activity or AP-1 induction.
- Mutation of S400 abolishes I kappa B-kinase (IKK) activation by Cot and impairs NF-kappa B induction via T-cell receptor/CD28 or Akt signaling.
Conclusions:
- Akt and Cot signaling pathways are integrated, with Akt regulating Cot's specific function in NF-kappa B transcription.
- Phosphorylation of Cot at S400 by Akt is a key regulatory event for NF-kappa B pathway activation.
- This study suggests distinct regulatory mechanisms for different signaling pathways activated by MAP3K family members.