Activation of phospholipase C delta1 through C2 domain by a Ca(2+)-enzyme-phosphatidylserine ternary complex.
The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.
Feinberg Cardiovascular Research Institute, Departments of Pathology and Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
J W
JW
Lomasney
H F
HF
Cheng
S R
SR
Roffler
K
K
King
D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008823:Micelles; D008956:Models, Chemical; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism* / Q000494:pharmacology*; D051966:Phospholipase C gamma; D010743:Phospholipids / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
D007527:Isoenzymes; D008081:Liposomes; D008823:Micelles; D019269:Phosphatidylinositol 4,5-Diphosphate; D010718:Phosphatidylserines; D010743:Phospholipids; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium