Phosphoprotein phosphatase 1 (PP1) is a component of the isolated sea urchin mitotic apparatus.
A protein component of isolated mitotic apparatus having a relative molecular mass of 62,000 (p62) is a substrate of a calcium/calmodulin dependent protein kinase, and the phosphorylation of p62 in vitro correlates directly with microtubule disassembly. In vivo experiments have determined the phosphorylation of p62 increases after fertilization; maximum incorporation of phosphate occurs during late metaphase/early anaphase and decreases thereafter. Because the level of p62 is constant throughout the cell cycle [Johnston and Sloboda, 1992: J. Cell Biol. 119:843-54] the decrease in phosphorylation of p62 observed after anaphase onset is most likely due to the action of a phosphatase. By examination of the relative amount of phosphorylated p62 which remained radiolabeled as a function of time using a standard in vitro phosphorylation assay, the activity of a phosphoprotein phosphatase capable of dephosphorylating p62 in the isolated mitotic apparatus was observed. To characterize the p62 phosphatase, okadaic acid and calyculin A were used to inhibit the dephosphorylation of p62 in vitro. It was found that specific concentrations of okadaic acid (50-500 nM) and of calyculin A (10-100 nM) were effective at inhibiting the dephosphorylation of p62 in vitro. Lower concentrations of either inhibitor had a negligible effect on dephosphorylation of p62. These data indicate the presence of phosphoprotein phosphatase type 1 activity associated with mitotic apparatus isolated from sea urchin embryos using the procedures described here. The implications of these findings relative to our understanding of the regulation of mitosis and cytokinesis are discussed.
Johnston
J A
JA
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire.
Sloboda
R D
RD
Silver
R B
RB
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002453:Cell Cycle; D004988:Ethers, Cyclic / Q000494:pharmacology; D005260:Female; D008387:Marine Toxins; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D019319:Okadaic Acid; D009865:Oocytes; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012617:Sea Urchins; D008941:Spindle Apparatus / Q000201:enzymology*
D004988:Ethers, Cyclic; D008387:Marine Toxins; D008869:Microtubule-Associated Proteins; D010080:Oxazoles; D019319:Okadaic Acid; C059041:calyculin A; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1