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.
Lomasney
J W
JW
Feinberg Cardiovascular Research Institute, Departments of Pathology and Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Cheng
H F
HF
Roffler
S R
SR
King
K
K
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