cAMP-dependent phosphorylation of betaig-h3 protein in human corneal endothelial cells.
OBJECTIVE
To determine the identity of a major membrane/ extracellular matrix (ECM)-associated 66-kDa protein in human corneal endothelial cells and its phosphorylation in vivo.


METHODS
A membrane/ECM-associated 66 kDa protein was purified from human corneal endothelial cells of 50-80 year-old donors by a three-step procedure that included preparative SDS-PAGE, preparative isoelectric focusing (IEF) and HPLC using a C-18 column. The phosphorylation of the 66-kDa protein was determined by both endogenous kinases and exogenous protein kinase A in the presence of endogenous or added cAMP, or added Walsh inhibitor (a specific inhibitor of protein kinase A). The phosphorylated proteins were analyzed by SDS-PAGE followed by autoradiography. The phosphoamino acids were identified following hydrolysis of the purified phosphorylated 66-kDa protein and thin layer chromatography with standard phosphoamino acids.


RESULTS
Following purification, the 66-kDa protein showed a single band on SDS-PAGE, a single species on two dimensional (2D)-gel electrophoresis and a single peak during C-18 HPLC. The partial N-terminal sequence of the 66-kDa protein matched with that of the 68-kDa beta ig-h3 protein (minus the signal peptide) of lung adenocarcinoma cells. Furthermore, on Western blot analysis, the 66-kDa protein immunoreacted with anti-beta ig-h3 past signal peptide (residue nos.24-32) antibody but not with the anti-beta ig-3 signal peptide (residue nos.1-9) antibody. During the phosphorylation of endothelial proteins by endogenous kinases or added protein kinase A in the presence of endogenous or added cAMP, the 66 kDa protein was the major substrate with its Ser residues phosphorylated in both cytosolic- and membrane/ECM-fractions.


CONCLUSIONS
The human corneal endothelial 66-kDa protein is identical to the 68-kDa beta ig-h3 protein (minus signal peptide) from lung adenocarcinoma cells. The corneal protein exists in a phosphorylated state in vivo with its Ser residues phosphorylated by a cAMP-dependent protein kinase A.
Srivastava
O P
OP
Department of Physiological Optics, University of Alabama at Birmingham, AL 35294, USA.
Srivastava
K
K
D000230:Adenocarcinoma / Q000737:chemistry; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein
D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D016326:Extracellular Matrix Proteins; D009363:Neoplasm Proteins; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein