Role of phosphoprotein phosphatases in the corpus luteum: I identification and characterisation of serine/threonine phosphoprotein phosphatases in isolated rat luteal cells.
Although the role of protein kinases and phosphorylation in steroidogenesis has received much attention, very little is known about the activities of phosphoprotein phosphatases (PP) and dephosphorylation in steroidogenic tissues. The aims of the present study were therefore to identify which of those serine/threonine PPs more commonly involved in intracellular signalling are expressed in rat luteal cells; to quantify, in vitro, the effects of inhibitors on PP activity extracted from purified rat luteal cells; and to measure the effects of PP inhibitors on the phosphorylation of endogenous luteal cell proteins. Polyclonal antibodies raised against the catalytic subunits of PP types 1 and 2A, and a monoclonal antibody raised against the Ca(2+)-binding subunit of PP2B, were used to identify immunoreactive proteins that migrated on SDS-PAGE with approximate molecular masses of 37, 34 and 16 kDa, corresponding well with the reported molecular mass of PP1, PP2A and PP2B respectively. Five selective inhibitors of PP1/PP2A: okadaic acid, calyculin A, cantharidin, tautomycin and microcystin-RR, each caused a dose-dependent decrease in the activity of PPs in luteal cell homogenates, and also enhanced 32P incorporation into numerous luteal cell proteins; most notably, proteins with approximate molecular masses of 20 and 22 kDa. The results of this study suggest that PPs may play an important role in the regulation of rat luteal cell functions.
Ford
S L
SL
Biomedical Sciences Division, King's College London, UK.
Abayasekara
D R
DR
Persaud
S J
SJ
Jones
P M
PM
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000502:physiology; D002193:Cantharidin / Q000494:pharmacology; D002478:Cells, Cultured; D003338:Corpus Luteum / Q000187:drug effects / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D008387:Marine Toxins; D052998:Microcystins; D008970:Molecular Weight; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000502:physiology*; D010766:Phosphorylation; D011714:Pyrans; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D013141:Spiro Compounds
D000935:Antifungal Agents; D002148:Calmodulin-Binding Proteins; D008387:Marine Toxins; D052998:Microcystins; D010080:Oxazoles; D010456:Peptides, Cyclic; D011714:Pyrans; D013141:Spiro Compounds; C053079:tautomycin; D019319:Okadaic Acid; C059041:calyculin A; C063855:microcystin RR; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002193:Cantharidin