Ecto-protein kinase and surface protein phosphorylation in PC12 cells: interactions with nerve growth factor.
The phosphorylation of surface proteins by ecto-protein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ecto-protein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [gamma-32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32Pi. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled Pi. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-alpha-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75-87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca(2+)-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Pawlowska
Z
Z
CSI/IBR Center for Developmental Neuroscience, City University of New York, College of Staten Island 10301.
Hogan
M V
MV
Kornecki
E
E
Ehrlich
Y H
YH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016716:PC12 Cells; D010710:Phosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013866:Thioguanine / Q000494:pharmacology
D008565:Membrane Proteins; D009414:Nerve Growth Factors; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C034454:ectoprotein kinase; D013866:Thioguanine; D008274:Magnesium; D002118:Calcium