Phosphorylation of the retinoic acid receptor-alpha by protein kinase A.

Abstract

The phosphorylation of retinoic acid receptor-α1 (RAR α1) by PKA was investigated both in vitro and in vivo. We show that bacterially expressed RAR α1 is phosphorylated in vitro by protein kinase A (PKA) at the unique serine residue 369 located in the C-terminal end of the E region. We also show that RAR α1 overexpressed in COS-1 cells is phosphorylated on multiple serine residues and that phosphorylation at serine 369 occurs only when COS-1 cells are cotransfected with PKA or treated with forskolin. RAR α1 mutants were constructed in which serine 369 was replaced by an alanine (S369A) or a glutamic acid (S369E) residue. Comparison of the tryptic phosphopeptide patterns of wild type and mutated RAR α1 overexpressed in COS-1 cells allowed us to confirm that serine 369 is the unique phosphorylation site for PKA in cultured cells. The DNA-binding efficiency of RAR α/retinoid X receptor-α (RXR α) heterodimers was enhanced in vitro by the S369E mutation. However, in transfected RAC65 cells, the same S369E mutation did not affect the ligand-dependent transcriptional activation by RAR α1 of reporter genes containing a retinoic acid (RA)-response element. In contrast, the S369A mutation slightly decreased both DNA binding and the efficiency of PKA to enhance RA-induced transactivation by RAR α1. Finally, we show that endogenous RAR α is also phosphorylated in vivo at serine 369 in forskolin-treated F9 cells, supporting the idea that phosphorylation of RARs at this site is involved in the modulation of the RA-induced differentiation of F9 cells by (Bu)2cAMP. © 1995 by The Endocrine Society.