Relocation of annexin V to platelet membranes is a phosphorylation-dependent process.
Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with agents that increase intracellular [Ca2+] induces the relocation of annexin V to membranes, and that this annexin V may be binding to a 50 kDa protein located within platelet membranes. We report here, using an in vitro reconstitution system, that the relocation of annexin V to membranes is enhanced by ATP. We also demonstrate that when adenosine 5'-[gamma-thio]-triphosphate, which can replace ATP in phosphorylation reactions, is substituted for ATP, the amount of annexin V that binds to membranes is further increased. In separate experiments using intact cells, we show that the protein phosphatase inhibitor okadaic acid mimics the action of the physiological agonist thrombin, in that it induces annexin V to bind to membranes and that the addition of the protein kinase inhibitor staurosporine inhibits A23187-induced relocation of annexin V. In addition, alkaline phosphatase, when added to isolated membranes, was found to remove endogenous annexin V from the membranes. Furthermore, immunoprecipitation of 33P-labelled proteins indicated that annexin V may form a multi-protein complex including phosphoproteins of 25, 50 and 83 kDa. Taken together these observations suggest that, following physiological activation, the phosphorylation of one or more proteins is responsible for the tight association of annexin V with platelet membranes and the subsequent regulation of membrane localized processes.
Trotter
P J
PJ
Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
Orchard
M A
MA
Walker
J H
JH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism*; D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002451:Cell Compartmentation; D006801:Humans; D008566:Membranes / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017304:Annexin A5; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D019319:Okadaic Acid; D000001:Calcimycin; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate