cAMP-mediated protein phosphorylation of microsomal membranes increases mannosylphosphodolichol synthase activity.
We have investigated the possible role of a cAMP-mediated protein-phosphorylation event(s) as the key regulatory mechanism in beta-adrenoreceptor-stimulated activation of mannosylphosphodolichol (Man-P-Dol) synthase (GDP-mannose:dolichyl-phosphate O-beta-D-mannosyltransferase, EC 2.4.1.83) in rat parotid acinar cells. Microsomal membranes isolated from these cells pretreated with 10 microM isoproterenol for 60 min showed approximately 40-80% enhanced Man-P-Dol synthase activity compared to the untreated controls. This change in enzyme activity was not associated with a significant alteration in apparent Km for GDP-mannose, but the Vmax was enhanced 2-fold. When microsomal membranes isolated from control cells were phosphorylated in vitro by a cAMP-dependent protein kinase, an increase in Man-P-Dol synthase activity, similar to that with membranes from isoproterenol-treated cells, was observed (i.e., a moderate change in Km for GDP-mannose but a 2-fold higher Vmax). Furthermore, treatment of in vitro phosphorylated microsomal membranes by alkaline phosphatase led to a substantial reduction in Man-P-Dol synthase activity. Increased Man-P-Dol synthesis (approximately 30-40%) was also observed in bovine brain and hen oviduct microsomal membranes after in vitro protein phosphorylation. In aggregate, these results strongly suggest that agents that increase cAMP in cells may modulate protein N-glycosylation in those cells by activating this key glycosyltransferase of the dolichol cascade by a cAMP-dependent protein kinase-mediated protein phosphorylation/dephosphorylation cycle.
Banerjee
D K
DK
Kousvelari
E E
EE
Baum
B J
BJ
D000469:Alkaline Phosphatase; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D006602:Hexosyltransferases / Q000378:metabolism*; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008364:Mannosyltransferases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D010306:Parotid Gland; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000502:physiology*
D002413:Cations, Divalent; D008565:Membrane Proteins; D010750:Phosphoproteins; D011943:Receptors, Adrenergic, beta; D000242:Cyclic AMP; D006602:Hexosyltransferases; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D011494:Protein Kinases; D000469:Alkaline Phosphatase; D007545:Isoproterenol