1245 Identification of a DNA-consensus sequence necessary for phosphorylation-dependent interaction with α-crystallin

m a-Crystallins were demonsl:rated recently to be involved fin interaction with a short element of the sense strand of the murine yDE/F-ctystallin genes immediately downstresm of the transcriptional initiation site @OTIS). The aim of the study was to demonstrate a consensus sequence and to test, if the interaction with a-crystallin is dependent on phospholylation. M&&& 3*P-labelled oligonucleotides including various alterations within the DOTIS element were used in gel-retention experiments ss binding partners for bovine a-crystallin containing fractions. Recombinant or bovine a-q&&n were phosphorylated by its autokinase activity or by the catalytic subunit of the protein kinase A (PKA). &g&c 1. A consensus sequence for the interaction of a-crystallin with DOTIS could be deduced as GGTC(N&GGANNG. 2. Autophosphorylation of a-crystallin prevents this particular interaction as well as an equal amount of r-crystallins. 3. It could be demonstrated for the first time that both, recpmbiiant and bovine a-crystallin can be phosphorylated by PKA very efficiently. This PKA-dependent phosphorylation has no influence on the interaction with DOTIS. Cmclusions The phosphorylation-dependent inhibition of the interaction of a-crystallin with the DOTIS element as well ss the inhibition by y-aystallin itself allows to understand, how the feedback mechanism between the proposed stimulating activity of a-crystallins on the y-crystallin expression might be explained.