Phosphorylation-Elicited Quaternary Changes of GA Binding Protein in Transcriptional Activation

Abstract

Enrichment of nicotinic acetylcholine receptors (nAChR) on the tip of the subjunctional folds of the postsynaptic membrane is a central event in the development of the vertebrate neuromuscular junction. This is attained, in part, through a selective transcription in the subsynaptic nuclei, and it has recently been shown that the GA binding protein (GABP) plays an important role in this compartmentalized expression. The neural factor heregulin (HRG) activates nAChR transcription in cultured cells by stimulating a signaling cascade of protein kinases. Hence, it is speculated that GABP becomes activated by phosphorylation, but the mechanism has remained elusive. To fully understand the consequences of GABP phosphorylation, we examined the effect of heregulin-elicited GABP phosphorylation on cellular localization, DNA binding, transcription, and mobility. We demonstrate that HRG-elicited phosphorylation dramatically changes the transcriptional activity and mobility of GABP. While phosphorylation of GABPβ seems to be dispensable for these changes, phosphorylation of GABPα is crucial. Using fluorescence resonance energy transfer, we furthermore showed that phosphorylation of threonine 280 in GABPα triggers reorganizations of the quaternary structure of GABP. Taken together, these results support a model in which phosphorylation-elicited structural changes of GABP enable engagement in certain interactions leading to transcriptional activation.

ACKNOWLEDGMENTS

We thank H. H. Sitte for the tandem construct. We appreciate the technical support of S. Pons, L. Strochlic, and R. Grailhe. We thank L. Schaeffer and N. Mechawar for critically reading the manuscript.

M.S. was supported by the Carlsberg Foundation, The Danish Research Council, and The Lundbeck Foundation. M.O.C. was supported by the Deutsche Forschungsgemeinschaft (Bo 910/3-1 and Bo 910/4-1).