Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase.
Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression. In vitro, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C alpha phosphorylated at T320. Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 was reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1/PP-2A inhibitor, calyculin A. Immunofluorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase. Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions. Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.
Kwon
Y G
YG
Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021, USA.
Lee
S Y
SY
Choi
Y
Y
Greengard
P
P
Nairn
A C
AC
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002453:Cell Cycle; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D007701:Kinetin; D008322:Mammals; D008387:Marine Toxins; D051379:Mice; D008938:Mitosis; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011687:Purines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013912:Threonine; D014162:Transfection
D000906:Antibodies; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D011687:Purines; D011994:Recombinant Proteins; D013912:Threonine; C090046:olomoucine; C059041:calyculin A; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D007701:Kinetin