Sequential Phosphorylation by Mitogen-activated Protein Kinase and Glycogen Synthase Kinase 3 Represses Transcriptional Activation by Heat Shock Factor-1

doi:10.1074/jbc.271.48.30847

Sequential Phosphorylation by Mitogen-activated Protein Kinase and Glycogen Synthase Kinase 3 Represses Transcriptional Activation by Heat Shock Factor-1*

From the Dana Farber Cancer Institute and Joint Center for Radiation Therapy, Harvard Medical School, Boston, Massachusetts 02115

^‡ To whom correspondence should be addressed:
Dana Farber Cancer Institute and Joint Center for Radiation Therapy, Harvard Medical School, 44 Binney St., Boston, MA 02115.
Tel.: 617-632-3885; Fax: 617-632-4599; E-mail: stuart_calderwood{at}dfci.harvard.edu.

Abstract

Mammalian heat shock genes are regulated at the transcriptional level by heat shock factor-1 (HSF-1), a sequence-specific transcription factor. We have examined the role of serine phosphorylation of HSF-1 in the regulation of heat shock gene transcription. Our experiments show that mitogen-activated protein kinases (MAPKs) of the ERK-1 family phosphorylate HSF-1 on serine residues and repress the transcriptional activation of the heat shock protein 70B (HSP70B) promoter by HSF-1 in vivo. These effects of MAPK are transmitted through a specific serine residue (Ser-303) located in a proline-rich sequence within the transcriptional regulatory domain of human HSF-1. However, despite the importance of Ser-303 in transmitting the signal from the MAPK cascade to HSP70 transcription, there was no evidence that Ser-303 could be phosphorylated by MAPK in vitro, although an adjacent residue (Ser-307) was avidly phosphorylated by MAPK. Further studies revealed that Ser-303 is phosphorylated by glycogen synthase kinase 3 (GSK3) through a mechanism dependent on primary phosphorylation of Ser-307 by MAPK. Secondary phosphorylation of Ser-303 by GSK3 may thus repress the activity of HSF-1, and its requirement for priming by MAPK phosphorylation of Ser-307 provides a potential link between the MAPK cascade and HSF-1. Our experiments thus indicate that MAPK is a potent inhibitor of HSF-1 function and may be involved in repressing the heat shock response during normal growth and development and deactivating the heat shock response during recovery from stress.

Footnotes

↵* This work was supported by National Institutes of Health Grants CA47407, CA31303, and CA50642. 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:

HSP

heat shock protein

HSF

heat shock factor

HSE

heat shock element

GSK3

glycogen synthase kinase 3

MAP

mitogen-activated protein

MAPK

MAP kinase

MKK

MAP kinase kinase

ERK

extracellular regulated kinase

PAGE

polyacrylamide gel electrophoresis

EMSA

electrophoretic mobility shift assay

CAT

chloramphenicol acetyltransferase

MBP

myelin basic protein

wt

wild-type

MOPS

4-morpholinepropanesulfonic acid.
↵² F. Soncin, R. Prevelige, and S. K. Calderwood, manuscript in preparation.
↵³ J. Landry, F. Soncin, and S. K. Calderwood, unpublished results.
- Received August 13, 1996.
- Revision received September 19, 1996.