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 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 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)/Y759F] 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.
Abbreviations used: BCR, B-cell antigen receptor; BLNK, B-cell linker; EGF, epidermal growth factor; Fluo 3/AM, Fluo-3 acetoxymethyl ester; PH, pleckstrin homology; PHδ1PLCγ2, chimera of PLCγ2 containing PLCδ1 PH domain; PI3K, phosphoinositide 3-kinase; PI-PLC, phosphoinositide-specific phospholipase C; SH2 and SH3, Src homology 2 and 3 respectively; TCR, T-cell antigen receptor; Y753F etc., Tyr753→Phe substitution etc.