Abstract
Calium/phospholipid-dependent protein kinase (protein kinase C) was purified from bovine retinae rod outer segments (ROS). In the presence of 0.1-2 microM calcium protein kinase C binds tightly to ROS and phosphorylates rhodopsin in the absence or presence of illumination. This property of protein kinase C contrasts with that of rhodopsin kinase, which in vitro phosphorylates only bleached rhodopsin. Peptide maps of rhodopsin phosphorylated by protein kinase C or rhodopsin kinase were compared using limited Staphylococcus aureus V8 protease digestion or complete tryptic digestion. Phosphorylation sites map to serine and threonine residues on the cytoplasmic carboxylterminal domain of rhodopsin for both kinases. The functional consequence of protein kinase C phosphorylation of rhodopsin was a reduced ability to stimulate the light-dependent rhodopsin activation of [35S]guanosine 5'-O-(thiotriphosphate) binding to transducin, the GTP-binding regulatory protein present in ROS. Properties of the calcium-stimulated interaction of protein kinase C with membranes and in vitro phosphorylation of intrinsic proteins are discussed based upon the findings.
Highlights
Calium/phospholipid-dependentprotein kinase was purified from bovine retinae rod calculated for rhodopsin kinase
If Rhodopsin phosphorylation outersegments (ROS) membranes are prepared in the presence of calcium and the extrinsic membrane proteins exhaustively stripped with chelator, dialyzed,and subsequentlyreconstituted with the stripped ROS in the presence of 200 pM CaC12,phosphorylation is observed in the absence of light and further enhanced by rhodopsin bleaching (Fig.1B).Rhodopsin kinase is obviously present in the extract, but no rhodopsin phosphorylation is observed in the dark in the absence of added calcium (Fig. lA, lane 3)
Rhodopsin kinase readily phosphorylates bleached rhodopsin and does not require calcium for its activation [3,4,5,6,7]. These facts indicate that thecalcium-dependentrhodopsinphosphorylation observed in Fig. 1 was not due to the presence of bleached rhodopsin in the dark-adapted ROS, because rhodopsin kinase would readily phosphorylate the bleached photopigment
Summary
Calium/phospholipid-dependentprotein kinase (pro- present in ROS in approximately the same abundance as that tein kinase C) was purified from bovine retinae rod calculated for rhodopsin kinase. Fig. lA shows the results of such an experiment demonstrating that the extrinsic membrane protein fraction (referred to as extract) reconstitutes the light-dependent phosphorylation of rhodopsin.
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