Purified
RNA polymerase I was phosphorylated by the endogenous
protein kinase or dephosphorylated by
alkaline phosphatase and used as
antigen in a radioimmunoassay with sera from
systemic lupus erythematosus patients or serum from an immunized rabbit.
Enzyme incubated in the absence of
ATP or
phosphatase served as control. Three to seven times more of the
autoantibodies in the patients' sera reacted with phosphorylated
RNA polymerase I than with control
enzyme. The reactivity of the dephosphorylated
enzyme with lupus
autoantibodies was only 50-60% of that observed with control
enzyme. Neither phosphorylation nor dephosphorylation of the
enzyme had an effect on its reaction with the rabbit
antibodies. The effect of phosphorylation on the reaction of each
RNA polymerase I subunit (S1-S8; Mr = 190,000-17,000) with the patients'
antibodies was determined by an immunoblot procedure following resolution of the subunits on
polyacrylamide gels. Prior phosphorylation of the
enzyme resulted in a dramatic increase in binding of each patient's
antibodies to all polymerase subunits with the exception of S4. Anti-S4 antibody was not detected with either phosphorylated or control
enzyme. Strikingly,
antibodies in each patients' sera reacted with S6 only after its phosphorylation. Similarly, anti-S5
antibodies in the serum of one patient were only detected with phosphorylated
RNA polymerase I. The present data suggest that at least a significant fraction of the anti-
RNA polymerase I autoantibodies in the sera of
systemic lupus erythematosus patients might be directed against phosphorylated sites on the
enzyme and that phosphorylation may have a role in the production of this and other autoimmunogenic nuclear components which are hallmarks of this disease.