Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes.
The activities of enzymes involved in lipid metabolism--phospholipase A2 (PLA2) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)--were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA2 activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA2 activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA2 activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA2 activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA2 activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.
Castagnet
P I
PI
Instituto de Investigaciones Bioquímicas, Universidad Nacional del Sur, Blanca, Argentina.
Roque
M E
ME
Pasquaré
S J
SJ
Giusto
N M
NM
D000818:Animals; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000528:radiation effects; D005136:Eye Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D008027:Light; D050356:Lipid Metabolism; D008566:Membranes / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism* / Q000528:radiation effects; D050918:Phosphatidylethanolamine N-Methyltransferase; D010741:Phospholipases A / Q000378:metabolism* / Q000528:radiation effects; D054467:Phospholipases A2; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism* / Q000528:radiation effects
D005136:Eye Proteins; D008780:Methyltransferases; D050918:Phosphatidylethanolamine N-Methyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2