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Phosphorylation of Ets1 regulates the complementation of a CSF-1 receptor impaired in mitogenesis
B Rabault1, M F Roussel, C T Quang
1Laboratoire d'Oncologie Virale et Cellulaire, CNRS UMR 146, Institut Curie, Centre Universitaire, Orsay, France.
Abstract:
Ets1, the founder member of the Ets transcription factor family, is involved in a variety of developmental and cellular processes. Previous studies have shown that serine phosphorylation of Ets1 inhibits its DNA binding activity, suggesting that phosphorylation is important in the regulation of Ets1 function. To further examine Ets1 phosphorylation, we ectopically expressed Ets1 in fibroblasts and stimulated these cells with serum. Using two-dimensional tryptic phosphopeptide analysis and site-directed mutagenesis, we found that Ets1 was phosphorylated on threonine 38, a residue conserved in several Ets proteins. Substitution of this residue with alanine enhanced CSF-1-dependent colony formation in semi-solid medium of NIH3T3 cells expressing a mitogenically defective CSF-1 receptor [Y809F]. Threonine 38 is part of a consensus amino-acid sequence frequently recognized and targeted by members of the MAP kinase family. Moreover, this residue is phosphorylated in vitro by recombinant ERK2, which suggests that the kinase which phosphorylates threonine 38 in vivo is a member of the MAP kinase family. In addition, phosphorylation on threonine 38 seems to negatively regulate Ets1 activity in response to growth-factor stimulation.
Insights
Ets1 transcription factor phosphorylation at threonine 38 by MAP kinase negatively regulates its activity. This finding is crucial for understanding cellular processes and growth factor signaling pathways.
Area of Science:
- Molecular Biology
- Cellular Biology
- Signal Transduction
Background:
- Ets1, a key transcription factor, regulates vital cellular and developmental processes.
- Serine phosphorylation of Ets1 is known to inhibit its DNA binding activity.
- Understanding Ets1 phosphorylation is critical for deciphering its regulatory mechanisms.
Purpose of the Study:
- To investigate the specific phosphorylation sites and regulatory roles of Ets1.
- To identify the kinases involved in Ets1 phosphorylation.
- To elucidate the functional consequences of Ets1 phosphorylation on cellular responses.
Main Methods:
- Ectopic expression of Ets1 in fibroblasts followed by serum stimulation.
- Two-dimensional tryptic phosphopeptide analysis to map phosphorylation sites.
- Site-directed mutagenesis to alter specific residues and assess functional impact.
- In vitro kinase assays using recombinant ERK2.
Main Results:
- Ets1 is phosphorylated on threonine 38, a conserved residue.
- Substitution of threonine 38 with alanine enhanced colony formation in NIH3T3 cells with a defective CSF-1 receptor.
- Threonine 38 is phosphorylated by ERK2, a MAP kinase family member.
- Phosphorylation at threonine 38 negatively regulates Ets1 activity in response to growth factors.
Conclusions:
- Threonine 38 is a critical phosphorylation site on Ets1, targeted by MAP kinases.
- Phosphorylation of Ets1 at threonine 38 plays a negative regulatory role in growth factor signaling.
- This phosphorylation event influences cellular proliferation and differentiation processes.