Phosphorylation-dependent Down-regulation of c-Myb DNA Binding Is Abrogated by a Point Mutation in the v-mybOncogene

Kristin Brevik Andersson,Elisabeth Kowenz-Leutz,Elen Margrethe Brendeford,Ann-Helen Herwig Tygsett,Achim Leutz,Odd S Gabrielsen

doi:10.1074/jbc.m209404200

Kristin Brevik Andersson, Elisabeth Kowenz-Leutz + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m209404200

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Journal: Journal of Biological Chemistry	Publication Date: Feb 1, 2003
Citations: 21	License type: cc-by

Affiliation: University of Oslo

Abstract

The viral Myb (v-Myb) oncoprotein of the avian myeloblastosis virus (AMV) is an activated form of the cellular transcription factor c-Myb causing acute monoblastic leukemia in chicken. Oncogenic v-Myb alterations include N- and C-terminal deletions as well as point mutations. Whereas truncations in Myb cause loss of various protein modifications, none of the point mutations in v-Myb has been directly linked to protein modifications. Here we show that the DNA-binding domain of c-Myb can be phosphorylated on serine 116 by the catalytic subunit of protein kinase A. Phosphorylation of Ser(116) differentially destabilizes a subtype of c-Myb-DNA complexes. The V117D mutation of the AMV v-Myb oncoprotein abolishes phosphorylation of the adjacent Ser(116) residue. Modification of Ser(116) was also detected in live cells in c-Myb, but not in AMV v-Myb. Phosphorylation-mimicking mutants of c-Myb failed to activate the resident mim-1 gene. Our data imply that protein kinase A or a kinase with similar specificity negatively regulates c-Myb function, including collaboration with C/EBP, and that the leukemogenic AMV v-Myb version evades inactivation by a point mutation that abolishes a phosphoacceptor consensus site. This suggests a novel link between Myb, a signal transduction pathway, cooperativity with C/EBP, and a point mutation in the myb oncogene.

Highlights

Introduction of Charge inSer116. We developed a phospho-c-Myb (Ser116) Affects Resident Myb Target Gene Expression—In effector-reporter experiments, we failed to observe significant differences between wild type c-Myb, S116A, and phospho-mimicking S116D and S116E mutations
Pilot biochemical experiments with recombinant c-Myb minimal DNA-binding domain (mDBD) domain corresponding to the AMVmyb version showed that this domain was readily phosphorylated in vitro by protein kinase A (PKA) catalytic subunit
We show that c-Myb is phosphorylated by the catalytic subunit of PKA on Ser116 in the DNA-binding domain

Summary

Introduction

Introduction of Charge inSer116 Affects Resident Myb Target Gene Expression—In effector-reporter experiments, we failed to observe significant differences between wild type c-Myb, S116A, and phospho-mimicking S116D and S116E mutations (data not shown). A mitogen-activated protein kinase phosphorylation site (Ser528) in the C-terminal negative regulatory domain is deleted in AMV v-Myb. Substitution of this serine to alanine increased the transcriptional capacity of c-Myb on some promoters but not on others (29 –31).

Results

Conclusion