Requirements for distinct steps of phospholipase Cgamma2 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 non-specific 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 [Tyr753-->Phe (Y753F)/Y759F] and SH2 (Src homology 2) domains (R564A/R672A) in the context of Lyn-PLCgamma2, we found that Tyr753/Tyr759 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.
Rodriguez
Rosie
R
Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK.
Matsuda
Miho
M
Storey
Amy
A
Katan
Matilda
M
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D021962:Membrane Microdomains / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D051966:Phospholipase C gamma; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D051966:Phospholipase C gamma