The phosphorylation and DNA binding of the DNA-binding domain of the orphan nuclear receptor NGFI-B.

Y Hirata,K Kiuchi,H C Chen,J Milbrandt,G Guroff

doi:10.1016/s0021-9258(19)74536-0

Y Hirata, K Kiuchi + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(19)74536-0

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Abstract

NGFI-B is an orphan member of the nuclear receptor superfamily encoded by an immediate-early gene. It is rapidly synthesized and phosphorylated in PC12 cells in response to nerve growth factor (NGF) and other agents and is differentially phosphorylated dependent upon the inducing stimulus. The DNA-binding domain (DBD) of NGFI-B has been expressed in bacteria and purified. The purified protein is phosphorylated by protein kinase A or by extracts from NGF-treated PC12 cells. The phosphorylated residues within the DBD have been identified as Ser-340 and Ser-350. The use of mutants in which either or both of these residues were replaced with alanines revealed that phosphorylation of Ser-350, located within the "A box," a motif necessary for DNA binding by NGFI-B, resulted in a decrease in binding to the NGFI-B response element, while phosphorylation of Ser-340 had little or no effect. These findings demonstrate that phosphorylation of a nuclear receptor DBD results in a change in DNA binding and provides another potential mechanism for regulating NGFI-B activity.

Highlights

NGFI-B is an orphan member of the nuclear receptor tyrosine kinase initiates a number of signaling cascades, the superfamily encoded by an immediate-early gene
Responseto nerve growth factor (NGF) and othaegrents Thetranscription of IEGsisactivated rapidly andtranand is differentiallyphosphorylateddependentupon siently within minuteosf stimulation and is independeonf t he the inducing stimulusT. he DNA-binding domain(DBD) synthesis of new protein [12, 13].Many of them are known to of NGFI-B has been expressed in bacteria and purified.encode transcription factors that are thought to control the
NGFI-B is strongly induced by NGF in PC12 cells, by which either or both of these residues were replaced with alanines revealed that phosphorylatoifonSer-350, located within the “A box,” a motif necessary for DNA binding by NGFI-B,resulted in a decrease in binding the NGFI-B response element, while phosphorylationf Ser-340 had little or no effect

Summary

NA Zn

Box,”and the arrowisndicate the positions of phosphorylation by protein kinase A. For protein kinase A the reaction mixture contained Tris-HC1 (40 m ~ ) p,H 7.4, magnesium acetate (20m d , ATP (0.2 mM), [y-32PlATP(1pCi), NGFI-B327 (0.5pg), and various concentrations of the catalytic subunit of protein kinase A in a final volume of 20 pl. For phosphorylation with protein kinase C, the reaction mixture contained HEPES (20 m ~ ) p, H 7.4 MgClz (10mM), EDTA (0.34 mM), EGTA (0.34 mM), CaClz (1.67 mM), DTT (1m ~ )p,hosphatidylserine (50 pg/ml), ATP (0.15 rm),[Y-~~PIAT(1PpCi), NGFI-B327 (0.5 pg), and various concentrations of protein kinase C in a total volume of 20 pl. The incubations were terminated by the addition of SDS-PAGE sample buffer andthe samples analyzed as described above. Phosphoamino Acid Analysis-Purified NGFI-B was phosphorylated with purified protein kinase A or C, and thesamples were resolved by SDS-PAGEand transferredelectrolytically to Immobilon-P membranes (Millipore). The suspension was incubated on ice for 15 min and thencentrifuged at 15,000 x g for 15 min a t 4 “C. The supernatant portion was used as the nuclear extract

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