Interdependence of steroidogenesis and shape changes in Y1 adrenocortical cells: studies with inhibitors of phosphoprotein phosphatases.
Y1 adrenocortical cells respond to activators of the cyclic AMP-dependent protein kinase (PKA) signalling pathway not only with increases in steroid secretion but also with a characteristic change in cell morphology from flat and adherent to round and loosely attached. This change of shape, which may facilitate cholesterol transport to the mitochondrion, requires tyrosine dephosphorylation of the focal adhesion protein, paxillin, and can be blocked by inhibitors of phosphotyrosine phosphatase (PTP) activity. In a previous study we demonstrated that inhibition of phosphoserine/threonine phosphatase 1 and 2A (PP1/2A) activities caused a similar morphological response to PKA activation whilst opposing the effects on steroid production. We have now investigated the responses to PKA activation and inhibition of PP1/2A and used PTP inhibitors to examine the relationship between the morphological changes and enhanced steroid production. Both forskolin (FSK) and the PP1/2A inhibitor, calyculin A (CA), caused rapid and extensive rounding of Y1 cells. FSK-induced cell rounding was reversible and accompanied by a reduction in the tyrosine phosphorylation of paxillin. Rounding was prevented by the PTP inhibitors pervanadate (PV) and calpeptin (CP) and was associated with the maintained tyrosine phosphorylation of paxillin. In contrast, CA-induced cell rounding was not reversible over a 2-h period and was not affected by the presence of PTP inhibitors, and CA had no effect on the tyrosine phosphorylation of paxillin. Although neither CA nor FSK produced any gross changes in cell viability as judged by Trypan Blue exclusion or mitochondrial activity, CA-treated cells showed a marked reduction in total protein synthesis assessed by (35)S-incorporation. The effects of FSK and the PTP inhibitors on cell rounding were reflected in their effects on steroid production since PV and CP also inhibited FSK-stimulated steroid production. These results suggest that the mechanism through which inhibition of PP1/2A activities induces morphological changes in Y1 cells is fundamentally different from that seen in response to activation of PKA. They are consistent with PKA-induced shape changes in adrenocortical cells being mediated through increased PTP activity and the dephosphorylation of paxillin, and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are intimately linked.
Whitehouse
B J
BJ
Endocrinology and Reproduction Research Group, GKT School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL, UK. barbara.whitehouse@kcl.ac.uk
Gyles
S L
SL
Squires
P E
PE
Sayed
S B
SB
Burns
C J
CJ
Persaud
S J
SJ
Jones
P M
PM
D000302:Adrenal Cortex / Q000378:metabolism* / Q000473:pathology*; D000818:Animals; D048429:Cell Size / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008387:Marine Toxins; D051379:Mice; D010080:Oxazoles / Q000494:pharmacology*; D051419:Paxillin; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011284:Pregnenolone / Q000096:biosynthesis; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology
D003598:Cytoskeletal Proteins; D004151:Dipeptides; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; C076715:pervanadate; C071482:calpeptin; D005576:Colforsin; D014638:Vanadates; D011284:Pregnenolone; C059041:calyculin A; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases