Atrial natriuretic factor inhibits autophosphorylation of protein kinase C and A 240-kDa protein in plasma membranes of bovine adrenal glomerulosa cells: involvement of cGMP-dependent and independent signal transduction mechanisms.
To clarify the intracellular signalling mechanisms of atrial natriuretic factor (ANF), we studied its effect on protein phosphorylation in plasma membranes of bovine adrenal cortical cells. ANF (1 x 10(-7) M) inhibited phosphorylation of the 78-kDa protein kinase C (PKC) and a 240-kDa protein in specific manner. In parallel experiments, cGMP (0.5 mM) inhibited phosphorylation of only the 78-kDa PKC but it did not affect phosphorylation of the 240-kDa protein. Phosphorylation of the 78-kDa PKC was enhanced in a Ca(2+)-/phospholipid-dependent manner. However, after prolonged preincubation of plasma membranes with Ca2+ (0.5 mM), the incorporation of 32P-radioactivity rapidly decreased in the 78-kDa PKC and subsequently increased in the 45- and 48-kDa protein bands due to Ca(2+)-dependent proteolytic degradation of 78-kDa PKC. Polyclonal antibodies against brain PKC were used to immunoblot and immunoprecipitate the 78-kDa PKC. Preincubation of plasma membranes with Ca2+ for varying times, followed by immunoblotting revealed a gradual loss of the immunoreactive 78-kDa PKC band in a time-dependent manner. Immunoprecipitation of phosphorylated 78-kDa PKC in plasma membranes showed that its phosphorylation was significantly inhibited in the presence of ANF as compared to control membranes, phosphorylated in the absence of ANF. The results in this present study document a new signal transduction mechanism of ANF at molecular level which possibly involves dephosphorylation of the 78-kDa PKC and a 240-kDa protein in a cGMP-dependent and -independent manner in bovine adrenal glomerulosa cell membranes.
Pandey
K N
KN
Department of Biochemistry and Molecular Biology, Medical College of Georgia School of Medicine, Augusta 30912.
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D015384:Zona Glomerulosa / Q000166:cytology / Q000378:metabolism*
D008565:Membrane Proteins; D009320:Atrial Natriuretic Factor; D011493:Protein Kinase C; D006152:Cyclic GMP; D002118:Calcium