Immunological Characterization of cdc2 and wee1 Proteins during the Growth and Differentiation of Dictyostelium discoideum.

Abstract

Two different antibody preparations, raised independently against the conserved EGVPSTAIREISLLKE sequence of the protein kinases encoded by the Schizosaccharomyces pombe cdc2 gene and its species homologs, immunoblotted a Dictyostelium protein of 32 kDa (p32). This polypeptide bound to p13suc1 -agarose beads, suggesting that it represents the Dictyostelium cdc2 and / or cdk2 products. The amounts of p32 detectable in cell free extracts and bound to p13suc1 -agarose were unaltered during the growth of cells synchronized for division. Although there was also essentially no change in the level of p32 during differentiation, the protein from the pseudoplasmodial stage of development did not bind to p13suc1 -agarose, implicating a developmentally regulated modification of the kinase. One of the EGVPSTAIREISLLKE antibodies also recognized a protein of 49 kDa (p49) that increased in amount dramatically during aggregation and then remained at elevated levels throughout the remainder of the differentiation process. This protein was present in low amounts throughout the growth of cells synchronized for division and was not absorbed by p13suc1 -agarose. A 103 kDa protein (p103) was detected by Western blot analysis using antibodies raised against two different peptides corresponding to sequences in the S. pombe protein kinase p105wee1 , which is a putative upstream negative regulator of p34cdc2 in fission yeast. The levels of p103 were constant during differentiation and during the growth of cells synchronized for division.