D-myo-inositol 1,4,5-trisphosphate inhibits binding of phospholipase C-delta 1 to bilayer membranes.

M.E Cifuentes,T Delaney,M.J Rebecchi

doi:10.1016/s0021-9258(17)42118-1

M.E Cifuentes, T Delaney + Show 1 more

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https://doi.org/10.1016/s0021-9258(17)42118-1

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Abstract

The binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) to bilayer membranes composed of phosphatidylcholine (PC) and phosphatidylinositol 4,5-bisphosphate (PIP2) was measured in the presence or absence of inositol phosphates. Binding was inhibited by the natural D-isomer of myo-inositol 1,4,5-trisphosphate (D-InsP3), but not by the L-isomer. The concentration of D-InsP3 required to decrease binding by 50% was 5.4 +/- 0.5 microM. 1-(alpha-Glycerophosphoryl)-D-myo-inositol 4,5-bisphosphate and D-myo-inositol 2,4,5-trisphosphate were nearly as effective as D-Ins(1,4,5)P3. D-myo-inositol monophosphate with phosphate esterified at either positions 1 or 2 of the myo-inositol ring, had no significant effect on binding. D-myo-inositol 1,4-bisphosphate weakly inhibited the binding, whereas the 4,5-isomer was nearly as potent as D-InsP3. Neither ATP nor inorganic phosphate significantly affected binding. As expected, D-Ins(1,4,5)P3 but not L-Ins(1,4,5)P3 decreased the initial rate of PIP2 hydrolysis in bilayer vesicles. The concentration required to decrease hydrolysis by 50% was 12.4 +/- 0.5 microM. A catalytic fragment of PLC-delta 1 that lacks a domain necessary for high affinity PIP2 binding was prepared as previously described (Cifuentes, M. E., Honkanen, L., and Rebecchi, M. J. (1993) J. Biol. Chem. 268, 11586-11593). In contrast to the native enzyme, the rate of PIP2 hydrolysis, catalyzed by the fragment, was not affected by D-Ins(1,4,5)P3. These data suggest that high affinity binding of the enzyme to PIP2 and processive catalysis, involve specific recognition of the 4- and 5-position phosphates of the inositol ring. Our results are consistent with feedback inhibition by the polar head group product, D-Ins(1,4,5)P3, at a step that precedes catalysis, namely interfacial recognition.

Highlights

The binding of phosphoinositide-specific phospholi- Taylor et al, 1991; Sternweis and Smrcka,19921, whereas the pase C-& (PLC-S1)to bilayer membranes composed of PLC-yisozymes are substratesfortyrosineproteinkinases phosphatidylcholine (PC) and phosphatidylinositol 4,5- (Meisenhelder et al, 1989; Todderud et al, 1990; Kim et a l . , bisphosphate (PIP,) was measured in the presence or 1990; Wahl et al, 1992)
In not affected by DhS(1,4,5)P~T. hese data suggest that the present study, we test the hypothesis that high affinity high affinity binding of the enzyme to PIPz and proces- membranebindingandprocessivecatalysisinvolvestereosive catalysis, involve specific recognition of the 4- and specific recognition of the PIP2 polar head group. 5-position phosphates of the inositol ring
Our results demonstrate that thDebut not theL enantiomer of Ins(1,4,5)P3 inhibits binding ofPLC-S1 to bilayer vesicles composed of PC and PIP, (98:2)

Summary

Introduction

The binding of phosphoinositide-specific phospholi- Taylor et al, 1991; Sternweis and Smrcka,19921, whereas the pase C-& (PLC-S1)to bilayer membranes composed of PLC-yisozymes are substratesfortyrosineproteinkinases phosphatidylcholine (PC) and phosphatidylinositol 4,5- (Meisenhelder et al, 1989; Todderud et al, 1990; Kim et a l . , bisphosphate (PIP,) was measured in the presence or 1990; Wahl et al, 1992). Immunoblots were performed using sequence-specificantibodies against PLC-S1as previously described(Cifuthe Kd (assuming a 1:l complex) of PLC-til for PIP, as measured previously (Rebecchei t al., 1992).Under these conditions, the naturalD isomer of Ins(1,4,5)P3(60p ~ )w, hich corresponds to the PIP, polar head group, nearly abolished binding to LUVs (TableI), whereas the L isomer of Ins(1,4,5)P3had littleeffect.2 entes et al.,1993).Catalytic activity of free PLC-8, was measured in a The presence of phosphates at the4 and 5 positions of the reaction with octylglucoside-PIPzmixedmicelles as previouslydescribed (Cifuentes et al, 1993). Measurementof PIP, Hydrolysis in LWS-The effect onthe catalytic activity of PLC-6, of various inositol phosphate compounds wasmeasured using PC/PIPz (95:5) L W s .

Results

Conclusion