Tyrosine hydroxylase dephosphorylation by protein phosphatase 2A in bovine adrenal chromaffin cells.
This study was undertaken to characterise the protein phosphatases in bovine adrenal chromaffin cells acting on tyrosine hydroxylase. Cells were pre-labelled with 32Pi and permeabilized with digitonin. The extent of dephosphorylation of Ser-8, Ser-19, Ser-31 and Ser-40 on tyrosine hydroxylase was found to be 30%, 38%, 37% and 71% respectively over 5 min. For Ser-19, Ser-31 and Ser-40 the dephosphorylation was entirely due to protein phosphatase 2A, as the dephosphorylation could be completely blocked by microcystin, but not by the protein phosphatase I inhibitory peptide. Permeabilization did not change the distribution of protein phosphatase 2A or tyrosine hydroxylase, or the activity of PP2A, from that occurring in intact cells. The dephosphorylation of Ser-8 was not altered by any inhibitor, suggesting the involvement of other protein phosphatases. The method developed here can be used to determine the protein phosphatases acting on substrates in conditions closely approximating those in situ, including the endogenous state of substrate phosphorylation and phosphatase location.
Leal
Rodrigo B
RB
The School of Biomedical Sciences, The University of Newcastle, Callaghan, New South Wales, Australia.
Sim
Alistair T R
AT
Gonçalves
Carlos A S
CA
Dunkley
Peter R
PR
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000201:enzymology*; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D054648:Protein Phosphatase 2; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D010710:Phosphates; D010750:Phosphoproteins; D014446:Tyrosine 3-Monooxygenase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2