Phosphorylation of the Transactivation Domain of Pax6 by Extracellular Signal-regulated Kinase and p38 Mitogen-activated Protein Kinase

doi:10.1074/jbc.274.21.15115

Phosphorylation of the Transactivation Domain of Pax6 by Extracellular Signal-regulated Kinase and p38 Mitogen-activated Protein Kinase*

From the Department of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway

Abstract

The transcription factor Pax6 is required for normal development of the central nervous system, the eyes, nose, and pancreas. Here we show that the transactivation domain (TAD) of zebrafish Pax6 is phosphorylated in vitro by the mitogen-activated protein kinases (MAPKs) extracellular-signal regulated kinase (ERK) and p38 kinase but not by Jun N-terminal kinase (JNK). Three of four putative proline-dependent kinase phosphorylation sites are phosphorylated in vitro. Of these sites, the serine 413 (Ser⁴¹³) is evolutionary conserved from sea urchin to man. Ser⁴¹³ is also phosphorylatedin vivo upon activation of ERK or p38 kinase. Substitution of Ser⁴¹³ with alanine strongly decreased the transactivation potential of the Pax6 TAD whereas substitution with glutamate increased the transactivation. Reporter gene assays with wild-type and mutant Pax6 revealed that transactivation by the full-length Pax6 protein from paired domain-binding sites was strongly enhanced (16-fold) following co-transfection with activated p38 kinase. This enhancement was largely dependent on the Ser⁴¹³ site. ERK activation, however, produced a 3-fold increase in transactivation which was partly independent of the Ser⁴¹³ site. These findings provide a starting point for further studies aimed at elucidating a post-translational regulation of Pax6 following activation of MAPK signaling pathways.

Footnotes

↵* This work was supported by grants from the Norwegian Cancer Society, the Norwegian Research Council, the Aakre Foundation, and the Blix Foundation (to T. J.).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.
↵‡ Fellows of the Norwegian Research Council.
↵§ Fellow of the Norwegian Cancer Society.
↵¶ To whom correspondence should be addressed: Dept. of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway. Tel.: 47-776-44720; Fax: 47-776-45350; E-mail:terjej{at}fagmed.uit.no.
↵2 I. Mikkola, unpublished data.
Abbreviations:

Pax

paired box

GST

glutathione S-transferase

MAPK

mitogen-activated protein kinase

ERK

extracellular signal-regulated kinase

MEK

MAPK/ERK kinase

JNK

c-Jun N-terminal kinase

TPA

12-O-tetradecanoylphorbol-13-acetate

PAGE

polyacrylamide gel electrophoresis

CAT

chloramphenicol acetyltransferase

LUC

luciferase

TAD

transactivation domain

wt

wild-type

DBD

DNA-binding domain

PVDF

polyvinylidene difluoride

CMV

cytomegalovirus

PBS

phosphate-buffered saline

PST

proline, serine, and threonine residues
- Received January 14, 1999.
- Revision received February 24, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.