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Phosphorylation of the oncogenic transcription factor interferon regulatory factor 2 (IRF2) in vitro and in vivo
M J Birnbaum1, B van Zundert, P S Vaughan
1Department of Cell Biology, Cancer Center, University of Massachusetts Medical Center, Worcester 01655, USA.
Abstract:
IRF2 is a transcription factor, possessing oncogenic potential, responsible for both the repression of growth-inhibiting genes (interferon) and the activation of cell cycle-regulated genes (histone H4). Surprisingly little is known about the post-translational modification of this factor. In this study, we analyze the phosphorylation of IRF2 both in vivo and in vitro. Immunoprecipitation of HA-tagged IRF2 expressed in 32P-phosphate labelled COS-7 cells demonstrates that IRF2 is phosphorylated in vivo. Amino acid sequence analysis reveals that several potential phosphorylation sites exist for a variety of serine/threonine protein kinases, including those of the mitogen activated protein (MAP) kinase family. Using a battery of these protein kinases we show that recombinant IRF2 is a substrate for protein kinase A (PKA), protein kinase C (PKC), and casein kinase II (CK2) in vitro. However, other serine/threonine protein kinases, including the MAP kinases JNK1, p38, and ERK2, do not phosphorylate IRF2. Two-dimensional phosphopeptide mapping of the sites phosphorylated by PKA, PKC, and CKII in vitro demonstrates that these enzymes are capable of phosphorylating IRF2 at multiple distinct sites. Phosphoaminoacid analysis of HA-tagged IRF2 immunoprecipitated from an asynchronous population of proliferating, metabolically phosphate-labelled cells indicates that this protein is phosphorylated exclusively upon serine residues in vivo. These results suggest that the oncogenic protein IRF2 may be regulated via multiple pathways during cellular growth.
Insights
Interferon regulatory factor 2 (IRF2), an oncogenic transcription factor, undergoes phosphorylation in vivo and in vitro. This post-translational modification, occurring exclusively on serine residues, suggests IRF2
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- IRF2 is an oncogenic transcription factor involved in cell growth regulation.
- Its post-translational modifications, crucial for function, are largely uncharacterized.
- IRF2 represses interferon genes and activates cell cycle genes like histone H4.
Purpose of the Study:
- To investigate the in vivo and in vitro phosphorylation of the oncogenic transcription factor IRF2.
- To identify specific kinases that phosphorylate IRF2.
- To determine the sites and nature of IRF2 phosphorylation.
Main Methods:
- Immunoprecipitation of HA-tagged IRF2 from 32P-phosphate labeled COS-7 cells.
- In vitro kinase assays using recombinant IRF2 and various serine/threonine protein kinases.
- Two-dimensional phosphopeptide mapping and phosphoaminoacid analysis.
Main Results:
- IRF2 is phosphorylated in vivo in COS-7 cells.
- Recombinant IRF2 is phosphorylated in vitro by Protein Kinase A (PKA), Protein Kinase C (PKC), and Casein Kinase II (CK2).
- MAP kinases (JNK1, p38, ERK2) do not phosphorylate IRF2; phosphorylation occurs exclusively on serine residues in vivo.
Conclusions:
- IRF2 is a substrate for PKA, PKC, and CK2, indicating multiple phosphorylation sites.
- In vivo IRF2 phosphorylation occurs exclusively on serine residues.
- These findings suggest IRF2's oncogenic activity may be regulated by multiple signaling pathways during cellular growth.