Protein phosphorylation and dephosphorylation in type II pneumocytes.
Protein phosphorylation and dephosphorylation are a major mechanism for regulating cellular activity. Substantial evidence exists for ascribing a key role of protein phosphorylation and dephosphorylation in the regulation of surfactant secretion from type II pneumocytes, yet understanding of the specific molecular mechanisms is generally lacking. Herein, we report two-dimensional electrophoretic mapping of proteins phosphorylated in type II pneumocytes in primary culture, the response to protein kinase C simulation with phorbol ester, and the response to protein phosphatase inhibition with okadaic acid. Exposure of cells for 15 min to phorbol ester at a concentration (10(-4) M) which maximally stimulated both translocation of protein kinase C from cytosol to membranes and surfactant secretion increased phosphorylation (50-80% compared with control) of three specific proteins (50 kDa, pI 5.8 and 5.7; 25 kDa, pI 5.7). Exposure of cells for 2.5 h to okadaic acid (10(-6) M), a concentration that inhibited 90% of protein phosphatase activity, resulted in greatly increased phosphorylation (200-1,500% compared with control) of five specific proteins (50 kDa, pI 5.7 and 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). Combined treatment with okadaic acid and phorbol ester resulted in further increases (145-3,080% compared with control) in phosphorylation of four specific proteins (50 kDa, pI 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). We conclude that these respective proteins comprise major substrates for protein kinase C-dependent phosphorylation and for protein phosphatases in type II pneumocytes in primary culture. Furthermore, we speculate that these proteins will prove to play key roles in the regulation of type II pneumocyte function.
Warburton
D
D
Developmental Lung Cell and Molecular Biology Research Center, Childrens Hospital of Los Angeles, California.
Tayag
A
A
Buckley
S
S
Cosico
L
L
Seth
R
R
D000818:Animals; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004988:Ethers, Cyclic / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011479:Protamines / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D011663:Pulmonary Surfactants / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004988:Ethers, Cyclic; D010750:Phosphoproteins; D011479:Protamines; D011506:Proteins; D011663:Pulmonary Surfactants; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate