PHOSPHORYLATION AND NUCLEOTIDE-DEPENDENT DEPHOSPHORYLATION OF HEPATICPOLYPEPTIDES RELATED TO THE PLASMA-CELL DIFFERENTIATION ANTIGEN PC-1

A glycoprotein fraction was isolated from rat liver membranes by affin ity chromatography on immobilized wheat-germ lectin. Incubation of thi s fraction with MgATP or MgGTP resulted in a sequential phosphorylatio n and dephosphorylation of a complex of three polypeptides (118, 128 a nd 197 kDa on SDS/PAGE) with N-linked sialyloligosaccharides. Each pol ypeptide was recognized by polyclonal antibodies against recombinant p lasma cell differentiation antigen PC-1. The relationship of the 118 k Da and 128 kDa polypeptides with PC-1 was confirmed by observations th at 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 converte d to adenosine and P(i), with ADP and AMP as intermediate products. Th e length of the latency period increased with the concentration of ini tially added ATP (5-1000 muM) and could be prolonged by a second addit ion of similar concentrations of ATP, ADP, AMP and various nucleotide analogues. Most potent were the alphabeta-methylene derivatives of ADP and ATP. Adenosine was poorly effective. AMP, ADP, and perhaps ATP, e merge as the direct determinants of the latency. After further purific ation of the lectin-purified membrane fraction on anion-exchange and m olecular-sieve columns, the complex of p118, p128 and p197 was still c apable of autophosphorylation and dephosphorylation. The dephosphoryla tion was not affected by classical inhibitors (NaF, okadaic acid, EDTA , EGTA, phenylalanine). It was stimulated about 20-fold by various ade nine nucleotides and analogues, with the same order of efficiency as n oted for the induction of the latency. A similar stimulation of dephos phorylation was caused by 0.5 mM Na3VO4, which also prevented the phos phorylation 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.