Post-translational modifications have major importance for the structure and function of a multiplicity of proteins. Phosphorylation is a widespread phenomenon among eukaryotic proteins. Whereas O-phosphorylation on the side chains of serine, threonine, and tyrosine in proteins is well known and has been studied extensively, to our knowledge the endogenous phosphorylation of hydroxyproline has not previously been reported. In the present work, we provide evidence for the first time that O-phosphohydroxyproline (Hyp(P)) is a proteinogenic amino acid. To detect Hyp(P) in proteins we generated a Hyp(P)-specific polyclonal antibody. We could identify Hyp(P) in various proteins by Western blot analysis, and we characterized the sequence position of Hyp(P) in the protein ␣-crystallin A by electrospray ionization-tandem mass spectrometry. Our experiments clearly demonstrate hydroxylation and subsequent phosphorylation of a proline residue in ␣-crystallin A in the eye as well as in heart tissue of rat.A wide range of biological processes are controlled by reversible protein phosphorylation, and conservative estimates indicate reversible phosphorylation targets of up to one-third of cellular proteins (1). To understand protein regulation through phosphorylation it is essential to find and characterize all sites and types of phosphorylation on proteins and to identify the kinases involved.Besides the three genetically encoded hydroxyamino acids serine (Ser), threonine (Thr), and tyrosine (Tyr), there are two other hydroxyamino acids, hydroxyproline (Hyp) 2 and hydroxylysine (Hyl). They are not encoded in the genome, although they can be identified in mature proteins. They are generated by enzymatic hydroxylation of proline (Pro) and lysine (Lys) residues (2). All genetically encoded hydroxyamino acids in polypeptide chains, such as serine, threonine, and tyrosine have been shown that they can undergo posttranslational phosphorylation (3, 4). Although phosphorylation of Hyl has been described in collagens (5) phosphohydroxyproline (Hyp(P)) has not been identified in native proteins.In addition to the O-phosphorylation of the hydroxyl group, O-glycosylation is another important modification. There is a growing speculation that phosphorylation and glycosylation share a reciprocal relationship. Both Hyp and Hyl are known to undergo glycosylation in a few proteins. In collagens up to 70% of Hyl residues are modified with galactose and glycosyl-galactose (6). Hyl was also found to be phosphorylated in collagens (5). In extensins (hydroxyproline-rich glycoproteins) of the cell wall in plants, ϳ50% of protein mass is glycosylated at Hyp residues by mono-, di-, tri-(132)--linked arabinoses (7,8,9). Furthermore, cytoplasmic F box-binding protein SKP1 in Dictyostelium contains a pentasaccharide linked to Hyp (10). These findings suggest that subsequent modification of Hyp is widespread and that, in analogy to all other hydroxyamino acids, phosphorylation of Hyp should be possible in native proteins.Hydroxylation of Pro is the preco...
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