Requirements for distinct steps of phospholipase C gamma 2 regulation, membrane-raft-dependent targeting and subsequent enzyme activation in B-cell signalling

Studies of PLCgamma (phospholipase Cgamma) have identified a number of regulatory components required for signalling; however, molecular mechanisms and the relationship between events leading to translocation and an increase of substrate hydrolysis have not been well defined. The addition of a membrane-targeting tag to many signal transducers results in constitutive activation, suggesting that these processes could be closely linked and difficult to dissect. The present study of PLCgamma2 regulation by cross-linking of the BCR (B-cell antigen receptor) or H2O2 stress in DT40 B-cells, demonstrated that the membrane targeting is a separate step from further changes that result in enzyme activation and substrate hydrolysis. Furthermore. we have defined the roles of different domains of PLCgamma2 and, using a panel of cell lines deficient in components linked to PLCgamma2 regulation, the involvement of signalling molecules with respect to each of the steps. We have found that only the lipid-raft-targeted Lyn-PLCgamma2 construct, unlike nonspecific membrane targeting, overcame the requirement for the adapter protein BLNK (B-cell linker). The stable expression of Lyn-PLCgamma2 was not accompanied by an increase in substrate hydrolysis in resting cells, which followed stimulation and specifically required the presence and/or activation of Syk, Btk, phosphoinositide 3-kinase but not BLNK, as established using deficient cell lines or specific inhibitors. Based on mutational analysis of the specific tyrosine residues [Tyr(753) –> Phe (Y753F)/ Y759F] and SH2 (Src homology 2) domains (R564A/R672A) in the context of Lyn-PLCgamma2, we found that Tyr(753)/Tyr(759) were essential, whereas the PLCgamma2 SH2 domains did not have an important role in the transient activation of Lyn-PLCgamma2 but may serve to stabilize an activated form in sustained activation