Protein phosphatase-1 and -2a activities in cultured fetal chick neurons: differential regulation by insulin and insulin-like growth factor-I.
In this study, we examined the developmental expression and regulation by insulin and insulin-like growth factor-I (IGF-I) of protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) in cultured fetal chick neurons. Protein phosphatase activities were measured using 32P-labeled phosphorylase-a or 32P-labeled S6 kinase substrate peptide. In cell extracts from day 1-5 cultures, 40-45% of spontaneous protein phosphatase activity was due to PP-1. PP-2A accounted for the remaining 55-60% of enzyme activity. Spontaneous PP-1 activity increased by 100% in day 2 cultures and remained constant thereafter. PP-2A activity increased by 48% in day 2 cultures, with minimal increases in enzyme activity in later cultures. Under the assay conditions employed, at all times in culture a significant proportion (45-50%) of PP-1 was in an inactive form that could be reactivated by trypsin. PP-2A activity was not influenced by trypsin. Insulin stimulated neuronal PP-1 activity in day 4 and 5 cultures, but had no effect in earlier cultures. The activation of PP-1 by insulin was rapid, with a maximal effect (30-40% increase over basal levels) at 5 min with 10 ng/ml insulin. Insulin did not alter total (trypsin-released) PP-1 activity, the content of PP-1 catalytic subunit, or PP-2A activity at any time in culture. In contrast to insulin, IGF-I had no effect on PP-1 activity at any time in culture, but significantly increased PP-2A activity in day 5 cultures. Maximal stimulation of PP-2A activity by IGF-I was observed at 10 min, with an EC50 of 5 ng/ml. These results indicate that chick forebrain neurons contain both PP-1 and PP-2A activities and that neuronal PP-1 and PP-2A activities are differentially regulated by insulin and IGF-I. We conclude that although insulin and IGF-I share many steps in signal transduction, these growth factors have distinct actions on neuronal phosphatase activity that may impact on differences in their neurotropic actions.
Begum
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Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501.
Robinson
L J
LJ
Draznin
B
B
Heidenreich
K A
KA
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002642:Chick Embryo; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007700:Kinetics; D009474:Neurons / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D016548:Prosencephalon / Q000196:embryology / Q000201:enzymology; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D014357:Trypsin / Q000494:pharmacology
D007328:Insulin; D007334:Insulin-Like Growth Factor I; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D014357:Trypsin