Functional Independence and Interdependence of the Src Homology Domains of Phospholipase C-γ1 in B-Cell Receptor Signal Transduction

Abstract

B-cell receptor (BCR)-induced activation of phospholipase C-gamma1 (PLCgamma1) and PLCgamma2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCgamma activation, the mechanism coupling PLCgamma to the BCR remains undefined. The role of PLCgamma1 SH2 and SH3 domains at different steps of BCR-induced PLCgamma1 activation was examined by reconstitution in a PLCgamma-negative B-cell line. PLCgamma1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCgamma1 with the adapter protein, BLNK. Forcing PLCgamma1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCgamma1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCgamma1 activation.

Intramolecular Regulation of Phospholipase C-γ1 by Its C-Terminal Src Homology 2 Domain

“…Constitutive, tyrosine phosphorylation-independent in vitro PLC-␥1 catalytic activity was observed when the SH2C domain was functionally disrupted (6). The activity of this SH2C domain mutant was, in fact, greater than that of receptor-activated, wild-type PLC-␥1.…”

“…The SH2C domain has been suggested to interact intramolecularly with phosphorylated tyrosine 783 (26) and intermolecularly with Grb2 (4) and to cooperate with the PLC-␥1 SH3 domain in an interaction with c-Cbl (28). However, in many cases, the in vitro interactions observed between the isolated SH2C domain and other phosphoproteins have not been shown to occur with intact PLC-␥1 (6,33). When tested as glutathione S-transferase (GST) fusion proteins, both PLC-␥1 SH2 domains pulled down the key adaptor proteins, LAT and BLNK, from activated T-and B-cell lysates, respectively.…”

“…This area is composed of two Src homology 2 (SH2) domains and an Src homology 3 (SH3) domain. The N-terminal SH2 domain (SH2N) is critical for binding to either tyrosine-phosphorylated receptor kinases or adaptor molecules that mediate PLC-␥1 relocation to the plasma membrane (6,14,25). The function of the C-terminal SH2 domain (SH2C) is less clear but is also essential for PLC-␥1 activation (1,6,14,33).…”

“…The N-terminal SH2 domain (SH2N) is critical for binding to either tyrosine-phosphorylated receptor kinases or adaptor molecules that mediate PLC-␥1 relocation to the plasma membrane (6,14,25). The function of the C-terminal SH2 domain (SH2C) is less clear but is also essential for PLC-␥1 activation (1,6,14,33). Interestingly, the tyrosine residues that are essential for PLC-␥1 activity (Y775 and Y783 in PLC-␥1 and Y753 and Y759 in PLC-␥2) are also located in this region (29,30).…”

“…When tested as glutathione S-transferase (GST) fusion proteins, both PLC-␥1 SH2 domains pulled down the key adaptor proteins, LAT and BLNK, from activated T-and B-cell lysates, respectively. However, the SH2N domain but not the SH2C domain interacted with these adapter proteins in intact cells (6,33). The isolated PLC-␥1 SH2C domain was also shown by nuclear magnetic resonance to bind a phosphorylated peptide encompassing tyrosine 1021 of the platelet-derived growth factor (PDGF) receptor, but a role for this interaction in PDGF-activated cells was not identified (15,25).…”