Abstract
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 α-crystallin A by electrospray ionization-tandem mass spectrometry. Our experiments clearly demonstrate hydroxylation and subsequent phosphorylation of a proline residue in α-crystallin A in the eye as well as in heart tissue of rat.
Highlights
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]
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
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
Summary
Hydroxyproline; Hyp(P), phosphohydroxyproline; Hyl, hydroxylysine; Hyl(P), phosphohydroxylysine; Fmoc, N-(9-fluorenyl)methoxycarbonyl; MS/MS, tandem mass spectrometry; sHsp, small heat shock protein. 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. It was interesting to know whether MS/MS fragmentation was able to characterize the configuration of Hyp(P) in post-translationally modified proteins and to find parameters that would allow for the identification of Hyp(P) in unknown proteins. This would permit to distinguish between Hyp(P) and other phosphoamino acids
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