Identification of cyclin A1/cdk2 phosphorylation sites in the C-terminal region of murine B-myb.

Wild-type or point-mutated B-myb fused to GST were phosphorylated in vitro by cyclin A1/cdk2 and subjected to 2-dimensional tryptic phosphopeptide mapping. (A) More than 20 different spots were identified when wild-type GST–B-myb was phosphorylated. Point mutations at potential phosphorylation sites were used to determine the identity of several of these spots. (B-D) Point mutations that changed the indicated threonine to an alanine (an amino acid that cannot be phosphorylated) led to the disappearance of single spots indicated by the arrow. (E) The mutation of S581 (serine to alanine) erased 3 radioactive spots. The reason for the absence of several spots is probably that S581 is located in a region of B-myb containing multiple basic amino acids, and trypsin cleavage after either arginine or lysine residues might lead to differently sized peptides containing the same S581.