Characterization of Ca2+-dependent phospholipid binding and phosphorylation of lipocortin I.
Lipocortin I is a high affinity substrate for the epidermal growth factor receptor/kinase that can be purified by reversible Ca2+-dependent association with cellular particulate fractions. Purified human lipocortin I was shown by ultraviolet spectroscopy to undergo conformational changes in response to high concentrations of Ca2+ (30-700 microM). Equilibrium dialysis of lipocortin I alone against 45Ca2+ showed barely detectable binding at 5-60 microM Ca2+. However, in the presence of phosphatidylserine, binding was significantly enhanced, and Scatchard analysis of the binding data showed that lipocortin I contained four Ca2+-binding sites with an apparent Kd of 75 microM. Lipocortin I associated with phosphatidylserine vesicles and F-actin in a Ca2+-dependent manner with half-maximal association occurring at 22 and 180 microM Ca2+, respectively. Neither phosphatidylcholine vesicles nor intact cultured fibroblasts bound detectable amounts of lipocortin I. Phosphorylation of lipocortin I by the epidermal growth factor-stimulated kinase in A431 membranes required Ca2+ (half-maximum = 28 microM) in the presence of Mg2+, but phosphorylation was not Ca2+-dependent in the presence of Mn2+. The difference in Ca2+ requirement for phosphorylation probably is a reflection of the fact that Mn2+, but not Mg2+, promoted the association of lipocortin I with phospholipid. Although the physiological role of lipocortin I phosphorylation has not yet been determined, it was observed that phosphorylation of Tyr-21 reduced by 5-fold the amount of Ca2+ required for half-maximal association of the protein with phosphatidylserine vesicles.
D017302:Annexins; D002118:Calcium / Q000494:pharmacology*; D005260:Female; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D017302:Annexins; D006023:Glycoproteins; D008081:Liposomes; D010743:Phospholipids; D002118:Calcium