THE AUTOANTIGEN LA SSB IS A CALMODULIN-BINDING PROTEIN/

The work reported here has been directed to the identification of new nuclear calmodulin-binding proteins. To achieve this goal, nuclei from rat hepatocytes were purified and a fraction enriched in DNA- and RNA -binding proteins was extracted using DNase I and RNase A. Calmodulin- binding proteins present in this nuclear subfraction were purified by chromatography using first a DEAE-Sephacel column and subsequently a c almodulin-Sepharose column. Four major polypeptides of 118, 107, 48 an d 45 kDa were found to bind to the calmodulin column in a Ca2+-depende nt way. [I-125]-calmodulin overlay analysis confirmed that the protein s of 118, 48 and 45 kDa are calmodulin-binding proteins. These protein s bind single-stranded and also double-stranded DNA. A partial amino a cid sequence obtained from the 48 kDa protein revealed a 100% identity with the La/SSB protein, an autoantigen implicated in several autoimm une diseases, such as lupus erythematosus and Sjogren's syndrome. Two- dimensional gel electrophoresis, Western blot analysis and experiments of binding to poly(U), also supports the identity of p48 as La/SSB. C aM and La/SSB protein colocalize in the heterochromatinic regions with in the nucleus of rat hepatocytes. Preincubation of La/SSB with calmod ulin in the presence of Ca2+ resulted in an increase in the binding of ssDNA to La/SSB, suggesting that calmodulin can play a role in the re gulation of the association of La/SSB with DNA.