Activation of the cdc25C phosphatase in mitotic HeLa cells.
The cdc2-activator cdc25C was immunoprecipitated from HeLa cell extracts and assayed as tyrosine phosphatase (PTP) using tyrosine-phosphorylated myelin basic protein. The PTP activity was 12-fold higher in immunocomplexes from mitotic (nocodazole-arrested) than from asynchronous cells. This difference is due to enzyme activation, since the same amount of cdc25C was immunodetected in both conditions. However, mitotic cdc25C had M(r) 59,000, while a 56,000-59,000 doublet was detected in immunocomplexes from asynchronous cells. The PTP activity of mitotic cdc25C was decreased by treatment with Phosphatase-2A catalytic subunit (but not with Phosphatase-1), with re-appearance of the 56,000 polypeptide. cdc25C was also found associated with cdc2-p13-Sepharose complex and its PTP activity was 7-fold higher in samples from mitotic than from asynchronous cells. cdc25C and cdc2 co-migrated during gel filtration and the higher activity of mitotic cdc25C was retained through gel filtration.
Villa-Moruzzi
E
E
Department of Biomedicine, University of Pisa, Italy.
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008938:Mitosis / Q000502:physiology*; D004676:Myelin Basic Protein / Q000378:metabolism; D010762:Phosphorylase a / Q000378:metabolism; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020687:cdc25 Phosphatases
D004676:Myelin Basic Protein; D011506:Proteins; D010762:Phosphorylase a; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases