The physical association and phosphorylation of Cdc25C protein phosphatase by Prk.
prk encodes a protein serine/threonine kinase involved in regulating M phase functions during the cell cycle. We have expressed His6-Prk and His6-Cdc25C proteins using the baculoviral vector expression system. Purified recombinant His6-Prk, but not a kinase-defective mutant His6-PrkK52R, is capable 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 recombinant baculoviruses, sf-9 cells produce His6-Cdc25C antigen with an additional slower mobility band on denaturing polyacrylamide gels compared with cells infected with His6-Cdc25C baculovirus alone. In addition, His6-Cdc25C immunoprecipitated from sf-9 cells co-infected with His6-Prk and His6-Cdc25C baculoviruses, but not with His6-PrkK52R and His6-Cdc25C baculoviruses, contains a greatly enhanced kinase activity that phosphorylates His6-Cdc25C in vitro. Moreover, phosphopeptide mapping shows that His6-Prk phosphorylates His6-Cdc25C at two sites in vitro and that the major phosphorylation site co-migrates with the one that is phosphorylated in vivo in asynchonized cells. Further studies reveal that His6-Prk phosphorylates Cdc25C on serine216, a residue also phosphorylated by Chk1 and Chk2. Together, these observations strongly suggest that Prk's role in mitosis is at least partly mediated through direct regulation of Cdc25C.
Ouyang
B
B
Division of Hematology/Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine; ML-508, K-pavilion, 231 Bethesda Avenue, Cincinnati, Ohio, OH 45267-0508, USA.
Li
W
W
Pan
H
H
Meadows
J
J
Hoffmann
I
I
Dai
W
W
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000821:virology; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; C061570:protein kinase N; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases