Enhancement of calcium sensitivity of lipocortin I in phospholipid binding induced by limited proteolysis and phosphorylation at the amino terminus as analyzed by phospholipid affinity column chromatography.
A phospholipid column was prepared by coating siliconized porous glass beads with phospholipids. The analysis of the Ca2+ requirement of lipocortin I and its derivatives in the binding to phospholipids was carried out with this column. The Ca2+ concentration required for 50% binding to the phospholipid column at room temperature was about 30 microM for lipocortin I, while that was reduced to 15 microM when lipocortin I was phosphorylated by the epidermal growth factor receptor/kinase, and a further reduction in the Ca2+ requirement was observed with proteolytic cleavage at the N-terminal region. Cathepsin D and calpain I (low calcium-requiring form of calcium-activated neutral protease) rapidly cleaved human placental lipocortin I at Trp-12 and Lys-26, respectively. These N-terminal-truncated proteins required only 5 microM Ca2+ for 50% binding to the phospholipid column. This enhancement of Ca2+ sensitivity by limited proteolysis was also observed for porcine lung lipocortin I. Essentially the same results were obtained when the Ca2+ sensitivities of the modified lipocortins I were analyzed using dispersed phospholipid vesicles instead of the phospholipid affinity column. Equilibrium dialysis indicated that the release of the N-terminal region markedly increased the affinity of lipocortin I for Ca2+ in the presence of phosphatidylserine, without any appreciable change of the number of Ca2+-binding sites. Limited proteolysis by endogenous proteases such as calpain may be an important regulatory mechanism for the Ca2+ sensitivity of lipocortin I in phospholipid binding.
Ando
Y
Y
Department of Dermatology, Faculty of Medicine, Kyoto University, Japan.
Imamura
S
S
Hong
Y M
YM
Owada
M K
MK
Kakunaga
T
T
Kannagi
R
R
D000595:Amino Acid Sequence; D000818:Animals; D017302:Annexins; D002118:Calcium / Q000502:physiology*; D002154:Calpain / Q000494:pharmacology; D002402:Cathepsin D / Q000494:pharmacology; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000378:metabolism*; D008168:Lung; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D013552:Swine
D017302:Annexins; D006023:Glycoproteins; D010743:Phospholipids; D002154:Calpain; D002402:Cathepsin D; D002118:Calcium