Analysis of Retinoid Receptor Phosphorylation

Abstract

Protein kinases (PKs) and protein phosphatases (PPases) are activated in response to extracellular stimuli such as peptidic hormones and growth factors. The resulting alteration of the phosphorylation state of target proteins is a common post-translational modification used by cells to modulate rapidly the activity of enzymes and transcription factors (1, 2). Most of the investrgations have focused on the role of O-phosphorylation on serme, threonine, and tyrosine residues, and consequently, most of the techniques developed for these studies apply to these acid-stable residues. Although we will only consider methods to assess the functional importance of O-phosphorylation, it should be kept in mind that N-phosphorylation is very likely to be as important as O-phosphorylation (3). Indeed, the most common biochemical techniques, from gel fixing to peptide analysis and purification, rely on the use of low pH solutions in which N-phosphorylated residues are unstable. These technical aspecis have so far limited the study of N-phosphorylation processes on the function of nuclear receptors, but appropriate methods from the study of N-phosphoproteins have been developed and described in detail by Matthews and coworkers (3). In addition, mammalian N-PKs are yet to be fully characterized, and the cloning of these enzymes catalyzing the transfer of phosphate to lysine, histidine, and also arginine residues will undoubtedly pave the way for a better understanding of the physiological role of N-phosphorylation. Considering this, the initial choice of the experimental strategy is very important and should take into account both technical-feasibility parameters and access to biological reagents such as purified enzymes, antibodies, and possibly expression vectors coding for PKs and PPases.