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 GABPbeta seems to be dispensable for these changes, phosphorylation of GABPalpha is crucial. Using fluorescence resonance energy transfer, we furthermore showed that phosphorylation of threonine 280 in GABPalpha 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.

FIG. 1.

Schematic presentation of the fluorescently tagged GABP subunits α and β. (A) GABPα contains a threonine phosphorylation site at amino acid 280, which is replaced with an alanine in GABPα(T280A). Introduction of a stop codon after position 399 deletes the dimerization domain in the GABPα(Δ399) protein. The fluorescent tag is positioned either N or C terminally. (B) By using site-directed mutagenesis, two putative phosphorylation sites of GABPβ are replaced with alanines [GABPβ(S170A-T180A)]. GABPβ(Δ330) contains a truncation mutation at amino acid 330 that deletes the homodimerization domain. The fluorescent tag is positioned either N or C terminally.

FIG. 2.

Localization of ECFP- or EYFP-tagged GABP subunits in living C2C12 myotubes. Epifluorescence microscope images were taken with the filter set given by the color in the panel. Images were taken 4 to 6 days after induction of differentiation. C2C12 myoblasts were transfected with GABPα-N1-ECFP (A) or GABPβ-C1-EYFP (B) or cotransfected with GABPα-N1-ECFP and GABPβ-C1-EYFP (ECFP filter set) (C), GABPα(T280A)-N1-EYFP and GABPβ-C1-ECFP (EYFP filter set) (D), GABPα-N1-EYFP and GABPβ(S170A-T180A)-C1-ECFP (ECFP filter set) (E), or GABPα(Δ399)-N1-EYFP and GABPβ-C1-ECFP (EYFP filter set) (F).

FIG. 3.

nAChRɛ promoter-driven luciferase reporter gene activation. C2C12 cells were cotransfected with the indicated alleles of GABPα and -β and a luciferase gene controlled by a 2,200-bp fragment of the mouse nAChRɛ promoter, which harbors the N-box response element. HRG was added when myoblasts were in the middle of differentiating into myotubes. PD was added 30 min prior to HRG. Measurements of luciferase activity were performed 48 h after addition of HRG. The results are presented as the average ratio of luciferase activities from HRG-treated and nontreated cells (corrected for transfection efficiency) for ≥3 independent experiments done in triplicate. The error bars correspond to the standard errors of the means.

FIG. 4.

DNA-binding properties of GABP in EMSA and protein fractionation. (A) EMSA comparison of the DNA-binding efficiencies of GABP wild-type and mutant proteins. C2C12 myotubes transfected with GABPαβ (lanes 1 and 4), GABPα(T280A)β (lanes 2 and 5), or GABPαβ(S170A-T180A) (lanes 3 and 6) were mock treated (−) or treated (+) with HRG 15 min prior to preparation of lysates. C2C12 lysates (30 μg/lane) were incubated with ³²P-labeled N-box-containing oligoduplex DNA for 30 min at room temperature. (B) C2C12 myotubes transfected with GABPβ-ECFP were either not treated (lane 1) or treated (lanes 2 and 3) with HRG for 15 min before preparation of the IPFs. Twenty-five micrograms of IPF was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4 to 15% polyacrylamide), blotted onto a polyvinylidene difluoride membrane, and reacted with anti-EGFP (JL-8).

FIG. 5.

FRAP analysis of the mobilities of GABP proteins upon HRG stimulation. (A) C2C12 myotubes expressing EGFP-GABPα and nontagged GABPβ were bleached at 2.2 s in the region indicated by the arrow. Images were taken before bleaching and after bleaching with an interval of 0.3-s. Only selected images from the recovery period are shown. (B) FRAP analysis of living cells expressing GABPβ, GABPαβ, and GABPα(T280A)β either mock treated or treated with HRG. (C) FRAP analysis of living cells expressing GABPαβ either mock treated, treated with HRG, or treated with HRG in the presence of PD. Quantitative data of fluorescence recovery kinetics are plotted. Fluorescence intensities in the bleached region were measured and expressed as the relative recovery over time after the bleach pulse. The relative intensity is shown as the average from ≥3 independent experiments with ≥6 individual cells per experiment. Standard deviations were in each case less than 5% of the average value (not shown).

FIG. 6.

FRET analysis of the GABPαβ heterodimer after acceptor photobleaching. C2C12 cells were transfected with equal amounts of vectors encoding EYFP- and ECFP-GABP fusion proteins. Images were taken before and after the bleach pulse, using both the CFP and FRET filter sets. Left panels, after the cells were differentiated into myotubes, representative images (before and after photobleaching) of the following GABP wild-type and mutant protein combinations were obtained with the ECFP filter set (λ = 480 nm): 1, EYFP-N1-GABPα plus ECFP-C1-GABPβ; 2, EYFP-N1-GABPα(T280A) plus ECFP-C1-GABPβ; 3, EYFP-N1-GABPα plus ECFP-C1-GABPβ(S170A-T180A). Right panel, dequenching calculated as the increase in fluorescent intensity of the donor fluorophore ECFP after acceptor bleaching. Bars, 1, EYFP-N1-GABPα plus ECFP-C1-GABPβ; 2, EYFP-N1-GABPα(T280A) plus ECFP-C1-GABPβ; 3, EYFP-N1-GABPα plus ECFP-C1-GABPβ(S170A-T180A). Data are represented as the averages from ≥3 independent experiments with ≥3 individual cells. Error bars correspond to the standard errors of the means.

FIG. 7.

FRET analysis after phosphorylation modifications. (A) Stimulation protocol. At time zero, PD was added and incubated for 4 h. The cells were then washed and reincubated in differentiation medium for 1 h. The medium was again changed, and stimulation with HRG was initiated. (B) In order to determine the impact of phosphorylation on FRET efficiencies, dequenching experiments similar to those presented in Fig. 6 were performed. Left panel, images obtained before and after acceptor bleaching from C2C12 cells cotransfected with EYFP-N1-GABPα plusECFP-C1-GABPβ before (− PD) or after (+ PD) PD treatment or after medium change and reincubation with HRG (+PD + HRG). Right panel, dequenching calculations as described in the legend to Fig. 6.

FIG. 8.

Speculative model of the mechanism of action of GABP in transcriptional activation. The subunits of GABP hardly exist as monomers in solution; heterodimers are quickly formed and transported into the nucleus (54). Once GABPα interacts with GABPβ, a high-affinity DNA-binding complex is formed. Binding to DNA promotes the formation of a GABPα₂β₂ heterotetrameric complex (8), which is able to scan DNA for N-box sites but not to initiate transcription. HRG-induced phosphorylation generates structural changes of the heterotetrameric GABP complex, which allow interaction with other cellular components of the preinitiation complex (PIC). This will ultimately lead to the assembly of the general transcription factors (GTFs) around the start site, and RNA polymerase II transcription of an N-box-containing gene can be initiated. Fast and slow refer to the mobility of GABP as determined from Fig. 5.