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. 1998 Feb 15;330(Pt 1):461–468. doi: 10.1042/bj3300461

Activation of a phospholipase Cbeta2 deletion mutant by limited proteolysis.

P Schnabel 1, M Camps 1
PMCID: PMC1219161  PMID: 9461544

Abstract

All phosphoinositide-specific phospholipases C (PLC) identified until today exhibit a high degree of similarity within two regions of 170 and 260 residues, respectively, which are designated regions X and Y. The PLCbeta family, including four members designated PLCbeta1, PLCbeta2, PLCbeta3 and PLCbeta4, is regulated by heterotrimeric G proteins. In order to investigate structure-function relationships of PLCbeta2, we expressed PLCbeta2Delta, a deletion mutant of PLCbeta2 which lacks most of the sequence downstream of region Y, in the baculovirus/insect cell system. The mutant was present in both soluble and particulate fractions of Sf9 cells and was demonstrated to be catalytically active and sensitive to betagamma-subunits. Sf9 cytosol containing this mutant was subjected to limited proteolysis by trypsin and S. aureus protease V8, respectively. Immunochemical analysis revealed that both proteases cleaved the enzyme between the regions X and Y. Most interestingly, proteolytic cleavage at this site by both proteases stimulated the catalytic activity of PLC2beta2Delta. The proteolytically activated enzyme was still sensitive to betagamma-subunits and showed an unchanged concentration dependence on Ca2+. Gel filtration chromatography indicated that the fragments generated by cleavage between the regions X and Y were still connected and formed a functional heterodimeric complex. In order to visualize all fragments generated by protease V8, PLCbeta2Delta was purified to homogeneity from Sf9 cytosol. Limited proteolysis of the purified enzyme by S. aureus protease V8 and subsequent SDS/PAGE and silver staining revealed that several cuts take place between the regions X and Y and that the resulting fragments remain intact. We hypothesize that the activating proteolytic cut induces a conformational change of the enzyme which might facilitate hydrolysis of the phospholipid substrate.

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Selected References

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