Structural requirements of phospholipase C delta1 for regulation by spermine, sphingosine and sphingomyelin.
We studied the relationship between sphingomyelin, calcium, spermine and sphingosine in regulation of phospholipase C (PLC) delta1 activity. Inhibition of PLC delta1 by sphingomyelin was promoted by spermine and Ca2+ and was partially abolished by sphingosine. The effect of sphingosine and spermine entirely depended on Ca2+. In the absence of Ca2+, no effect of these substances on PLC delta1 activity was observed. Using deletion mutants and active fragments of PLC delta1 generated by limited proteolysis, we have studied the structural requirements of the enzyme for regulation by these compounds. The deletion mutant of PLC delta1 lacking the first 58 amino acids and the mutant lacking the entire pleckstrin homology (PH) domain were fully active in the detergent assay, and their activities were affected by spermine, sphingosine, Ca2+ and sphingomyelin to the same extent as the native enzyme. The limited proteolysis of PLC delta1 generated two fragments of 40 kDa and 30 kDa, which formed a stable active complex. The relationship between Ca2+ concentration and enzymatic activity was almost identical for the native PLC delta1 and the proteolytic complex. The activity of the proteolytic complex formed by the 40 kDa and 30 kDa peptides was not affected by spermine and sphingosine. Sphingomyelin inhibited the complex slightly less than the native PLC delta1, and this inhibition was not promoted by spermine. These observations suggest that for activation of PLC delta1 by spermine and sphingosine, the region spanning domains of high conservation, named X and Y, must be intact. In contrast, the PH domain and the intact spanning region of the X and Y domains are not essential for inhibition of PLC delta1 by sphingomyelin.
Pawelczyk
T
T
Department of Clinical Biochemistry, Medical University of Gdansk, Poland. tkpaw@amedec.amg.gda.pl
Matecki
A
A
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D007527:Isoenzymes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D054803:Phospholipase C delta; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013096:Spermine / Q000378:metabolism* / Q000494:pharmacology; D013109:Sphingomyelins / Q000378:metabolism* / Q000494:pharmacology; D013110:Sphingosine / Q000378:metabolism* / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D010738:Type C Phospholipases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D011994:Recombinant Proteins; D013109:Sphingomyelins; D013096:Spermine; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D014357:Trypsin; D013110:Sphingosine; D002118:Calcium