Phosphorylation of hydroxylysine residues in collagen synthesized by cultured aortic smooth muscle cells.

Abstract

05-Phosphohydroxylysine was chemically synthesized and techniques were established for its identification by combined use of cation-exchange chromatography, thin-layer electrophoresis at pH 1.9 and 3.5, and thin-layer chromatography. Clean separation of phosphohydroxylysine from the other phospho amino acids, phosphoethanolamine, and phosphocholine was achieved. Conditions were also determined to permit hydrolysis of proteins in 2 M HCO without loss of the phosphono group of phosphohydroxylysine residues. Experiments were then performed showing that 32p was incorporated into the hydroxylysine residues of cell-associated collagens when cultured calf aorta medial smooth muscle cells were incubated with [32P]orthophosphate. In other experiments, the cells incorporated [3Hllysine into hydroxylysine residues of cell-associated collagen and then 32p into phosphohydroxylysine residues. The doubly labeled phosphohydroxylysine subsequently isolated showed nearly 1:1 stoichiometry with respect to incorporation of precursor lysine and phosphorus. Finally, in preliminary experiments done with a cell-free extract of the smooth muscle cells, 32p was transferred from [y-32P]ATP to hydroxylysine residues in several kinds of collagenous substrates. Thus, this work shows that smooth muscle cells have the capacity to phosphorylate hydroxylysine residues in their cell-associated collagens and provides preliminary evidence that a protein kinase is involved.With the discovery of proteins containing phosphotyrosine residues (1, 2), all of the coded hydroxy amino acids inserted into polypeptide chains have been shown to undergo posttranslational phosphorylation by action of protein kinases and ATP. Two other hydroxy amino acids are in themselves formed by post-translational hydroxylation reactions; these are hydroxyproline in collagens and elastins, formed from proline residues, and 5-hydroxylysine in collagens, formed from lysine residues. We have begun studies of the phosphorylation of hydroxylysine and hydroxyproline residues in proteins.In its free form, 05-phosphohydroxylysine was identified tentatively in the extracellular fluids of bovine embryos in the early screening applications of paper chromatography (3). More recently, a system has been found in liver for phosphorylation of free hydroxylysine by a hydroxylysine kinase that utilizes GTP as the phosphono donor (4, 5). That reaction is the first step in the complete degradative metabolism of hydroxylysine liberated by digestion of collagenous proteins as, for instance, shown by our laboratory, by the combined actions of trypsin and carboxypeptidase B (6).For many years, we have been concerned with possible functions for hydroxylysine residues. It is known that such residues, like those of lysine, can undergo oxidation at C-6 to form corresponding aldehyde groups, which can participate in crosslink formation (7). The hydroxyl groups of hydroxylysine residues can undergo glycosylation by action of a transferase and UDP-galactose, and the substituted galactose residue c...

Glycosylation Modulates Melanoma Cell α2β1 and α3β1 Integrin Interactions with Type IV Collagen

“…Platelets can supply sugar donor substrates for extracellular glycosylation (94). Collagen Hyl residues can also be phosphorylated (95), and phosphorylation of collagen can occur extracellularly (96).…”

Development of a convenient peptide‐based assay for lysyl hydroxylase

“…The stationary phases used were C 18 , C 8 O, or methanol. The pH values for mobile phase A were 2, 4, and 6.…”

“…4 Cross-links formed from a Hyl-derived aldehyde are more stable than those formed from a Lys-derived aldehyde. 4 Hyl hydroxy groups can also be phosphorylated, 8 but the significance of this modification has not been described.…”

Synthesis and Characterization of a Collagen Model δ-O-Phosphohydroxylysine-Containing Peptide

“…Although HylP residues have been identified in collagens isolated from cultured aortic smooth muscle cells (6) and placoid-scale dentines of sharks (7) as early as in 1985, there is no definitive proof of such modification at the amino acid sequence level. It has been suggested that, in these tissues, phosphorylation of Hyl may play a regulatory role similar to that which serine, threonine, and tyrosine phosphorylation plays in many signal transduction pathways.…”