Phosphorylation and nucleotide-dependent dephosphorylation of hepatic polypeptides related to the plasma cell differentiation antigen PC-1

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Abstract
A glycoprotein fraction was isolated from rat liver membranes by affinity chromatography on immobilized wheat-germ lectin. Incubation of this fraction with MgATP or MgGTP resulted in a sequential phosphorylation and dephosphorylation of a complex of three polypeptides (118, 128 and 197 kDa on SDS/PAGE) with N-linked sialyloligosaccharides. Each polypeptide was recognized by polyclonal antibodies against recombinant plasma cell differentiation antigen PC-1. The relationship of the 118 kDa and 128 kDa polypeptides with PC-1 was confirmed by observations that they are linked by disulphide bonds into a larger protein, and that they are exclusively phosphorylated on Thr residues. Phosphorylation of p118, p128 and p197 only occurred after a lag period (up to 90 min at 30 degrees C), which lasted until most of the ATP had been converted to adenosine and Pi, with ADP and AMP as intermediate products. The length of the latency period increased with the concentration of initially added ATP (5-1000 microM) and could be prolonged by a second addition of similar concentrations of ATP, ADP, AMP and various nucleotide analogues. Most potent were the alpha beta-methylene derivatives of ADP and ATP. Adenosine was poorly effective. AMP, ADP, and perhaps ATP, emerge as the direct determinants of the latency. After further purification of the lectin-purified membrane fraction on anion-exchange and molecular-sieve columns, the complex of p118, p128 and p197 was still capable of autophosphorylation and dephosphorylation. The dephosphorylation was not affected by classical inhibitors (NaF, okadaic acid, EDTA, EGTA, phenylalanine). It was stimulated about 20-fold by various adenine nucleotides and analogues, with the same order of efficiency as noted for the induction of the latency. A similar stimulation of dephosphorylation was caused by 0.5 mM Na3VO4, which also prevented the phosphorylation of the three polypeptides. The likely explanation for the latency that precedes the phosphorylation of the membrane proteins is that the action of a protein kinase is initially offset by nucleotide-stimulated dephosphorylation.