Requirements for distinct steps of phospholipase Cγ2 regulation, membrane-raft-dependent targeting and subsequent enzyme activation in B-cell signalling

Studies of PLCγ (phospholipase Cγ) 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 PLCγ2 regulation by cross-linking of the BCR (B-cell antigen receptor) or H₂O ₂ 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 PLCγ2 and, using a panel of cell lines deficient in components linked to PLCγ2 regulation, the involvement of signalling molecules with respect to each of the steps. We have found that only the lipid-raft-targeted Lyn-PLCγ2 construct, unlike non-specific membrane targeting, overcame the requirement for the adapter protein BLNK (B-cell linker). The stable expression of Lyn-PLCγ2 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 [Tyr⁷⁵³ → Phe (Y753F)/Y759FJ and SH2 (Src homology 2) domains (R564A/R672A) in the context of Lyn-PLCγ2, we found that Tyr⁷⁵³/Tyr⁷⁵⁹ were essential, whereas the PLCγ2 SH2 domains did not have an important role in the transient activation of Lyn-PLCγ2 but may serve to stabilize an activated form in sustained activation.