Identification of a 50-kDa Ca-, cAMP-, and cGMP-dependent epithelial phosphoprotein as a cAMP regulatory protein.
Multiple second messengers, presumably acting via protein phosphorylation mechanisms, regulate flounder intestinal ion transport. We recently reported [C. Toskulkao, N. T. Nash, K. Leach, and M. C. Rao. Am. J. Physiol. 258 (Cell Physiol. 27): C879-C888, 1990] that this tissue possesses adenosine 3',5'-cyclic monophosphate (cAMP)- and guanosine 3',5'-cyclic monophosphate (cGMP)-specific protein kinases, types II and III Ca-calmodulin kinases, and very low levels of protein kinase C. These results correlate with ion transport studies in which cGMP and Ca were shown to inhibit salt absorption, cAMP to increase anion permeability, and phorbol esters to have no effect. In the present study we characterized in detail a 50-kDa protein the phosphorylation of which is regulated by more than one second messenger. The 50-kDa (pI 5.2) phosphoprotein is present in both the cytosol (50 kDa-C) and particulate (50 kDa-P) fractions and appears to be regulated by Ca, cAMP, and cGMP. Although the pI and Mr of the regulated proteins are identical, there are differences in the regulation of 50 kDa-P and 50 kDa-C. The phosphorylation of 50 kDa-P is high in the basal state, and Ca and cGMP enhance this. cAMP has a biphasic effect, increasing it at low and decreasing it at high protein concentrations. The isoquinoline derivatives H-8 [50% effective dose (ED50) approximately 2.3 microM] and H-7 (ED50 approximately 45 microM) inhibit basal 50 kDa-P phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Toskulkao
C
C
Department of Physiology and Biophysics, University of Illinois, College of Medicine, Chicago 60680.
Rao
M C
MC
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D002373:Cyclic AMP Receptor Protein; D006152:Cyclic GMP / Q000378:metabolism* / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D005432:Flounder; D007413:Intestinal Mucosa / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D015290:Second Messenger Systems; D013347:Subcellular Fractions / Q000378:metabolism
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D007546:Isoquinolines; D010750:Phosphoproteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D000242:Cyclic AMP; D006152:Cyclic GMP