The physical association and phosphorylation of Cdc25C protein phosphataseby Prk

prk encodes a protein serine/threonine kinase involved in regulating M phas e functions during the cell cycle. We have expressed His6-Prk and His6-Cdc2 5C proteins using the baculoviral vector expression system. Purified recomb inant His6-Prk, but not a kinase-defective mutant His6-Prk(K52R), is capabl e of strongly phosphorylating His6-Cdc25C in vitro. Co-immunoprecipitation and affinity column chromatography experiments demonstrate that GST-Prk and native Cdc25C interact. When co-infected with His6-Prk and His6-Cdc25C rec ombinant baculoviruses, sf-9 cells produce His6-Cdc25C antigen with an addi tional slower mobility band on denaturing polyacrylamide gels compared with cells infected with His6-Cdc25C baculovirus alone. In addition, His6-Cdc25 C immunoprecipitated from sf-9 cells co-infected with His6-Prk and His6-Cdc 25C baculoviruses, but not with His6-Prk(K52R) and His6-Cdc25C baculoviruse s, contains a greatly enhanced kinase activity that phosphorylates His6-Cdc 25C in vitro. Moreover, phosphopeptide mapping shows that His6-Prk phosphor ylates His6-Cdc25C at two sites in vitro and that the major phosphorylation site co-migrates with the one that is phosphorylated in vivo in asynchoniz ed cells. Further studies reveal that His6-Prk phosphorylates Cdc25C on ser ine(216), a residue also phosphorylated by Chk1 and Chk2. Together, these o bservations strongly suggest that Prk's role in mitosis is at least partly mediated through direct regulation of Cdc25C.