Differential Regulation of c-Myb-induced Transcription Activation by a Phosphorylation Site in the Negative Regulatory Domain

doi:10.1074/jbc.271.37.22697

Differential Regulation of c-Myb-induced Transcription Activation by a Phosphorylation Site in the Negative Regulatory Domain*

From the ^‡ Departments of Microbiology and
^¶ Pharmacology, University of Virginia, Charlottesville, Virginia 22908

^″ To whom correspondence should be addressed:
Dept. of Microbiology, University of Virginia, Box 441, 1300 Jefferson Park Ave., Charlottesville, VA 22908.
Tel.: 804-924-1246; Fax: 804-982-1071; E-mail: tpb3e{at}virginia.edu.

Abstract

The c-myb protooncogene encodes a highly conserved 75-89-kDa transcription factor that contains three functional domains, an amino-terminal DNA binding domain (DBD), a central acidic transactivation domain, and a carboxyl-terminal negative regulatory domain (NRD). Two acute transforming retroviruses, avian myeloblastosis virus and the E26 leukemia virus, transduced portions of c-myb and encode Myb proteins that are truncated in both the DBD and the NRD. Several conserved potential sites for phosphorylation by proline-directed serine/threonine protein kinases reside in or near the NRD, suggesting that phosphorylation might play a role in regulating c-Myb. We have previously demonstrated that serine 528, located in the NRD, is a target for p42^mapk in vitro. Serine 528 is phosphorylated in vivo in several cell lines, and substitution of serine 528 to alanine (S528A) resulted in an increased ability of Myb to transactivate a synthetic promoter containing five copies of the mim-1A Myb-responsive element and a minimal herpes tk promoter. We have tested the ability of S528A Myb to transactivate a series of cellular target promoters and report that the serine to alanine substitution increased the ability of Myb to activate transcription from the CD34 promoter but not the c-myc or mim-1 promoters. This suggests that phosphorylation of serine 528 may differentially regulate c-Myb activity at different promoters. The DNA binding and multimerization activities of c-Myb appear to be unaffected by the S528A substitution, suggesting that phosphorylation of serine 528 may mediate its effect on the transcription transactivating activity of c-Myb by regulating interactions with other proteins.

Footnotes

↵^§ Supported in part by Public Health Service Training Grant CA-09109 from the National Cancer Institute. Present address: Pfizer Central Research, Eastern Point Road, Groton, CT 06340.
^∥ Supported in part by Public Health Service Training Grant GM-07055 from the National Institutes of Health. Present address: DNAX Research Institute, 901 California Ave., Palo Alto, CA 94304
↵* This work was supported by Public Health Service Grant CA-40042 from the National Cancer Institute and American Cancer Society Faculty Research Award FRA-425 (to T. P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

AMV

avian myeloblastosis virus

DBD

DNA binding domain

MRE

Myb-responsive element

NRD

negative regulatory domain

NF-M

nuclear factor-myeloid

EMSA

electrophoretic mobility shift assay

bp

base pair

GST

glutathione S-transferase

mAb.

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline.
↵²A. F. Richardson and T. P. Bender, unpublished observations.
↵³M. R. Miglarese and T. P. Bender, unpublished observations.
↵⁴J. Lipsick, M. R. Miglarese and T. P. Bender, unpublished observations.
↵⁵N. Aziz, M. R. Miglarese and T. P. Bender, unpublished observation.
↵⁶N. Aziz and T. P. Bender, unpublished observation.
- Received April 19, 1996.