Differential Roles of the Src Homology 2 Domains of Phospholipase C-γ1 (PLC-γ1) in Platelet-derived Growth Factor-induced Activation of PLC-γ1 in Intact Cells

Abstract

Upon stimulation of cells with platelet-derived growth factor (PDGF), phospholipase C-␥1 (PLC-␥1) binds to the tyrosine-phosphorylated PDGF receptor through one or both of its Src homology 2 (SH2) domains, is phosphorylated by the receptor kinase, and is thereby activated to hydrolyze phosphatidylinositol 4,5-bisphosphate. Association of PLC-␥1 with the insoluble subcellular fraction is also enhanced in PDGF-stimulated cells. The individual roles of the two SH2 domains of PLC-␥1 in mediating the interaction between the enzyme and the PDGF receptor have now been investigated by functionally disabling each domain. A critical Arg residue in each SH2 domain was mutated to Ala. Both wild-type and mutant PLC-␥1 proteins were transiently expressed in a PLC-␥1-deficient fibroblast cell line, and these transfected cells were stimulated with PDGF. The mutant protein in which the COOH-terminal SH2 domain was disabled bound to the PDGF receptor. Accordingly, it was phosphorylated by the receptor, catalyzed the production of inositol phosphates, and mobilized intracellular calcium to extents similar to (but slightly less than) those observed with the wild-type enzyme. In contrast, the mutant in which the NH 2 -terminal SH2 domain was impaired did not bind to the PDGF receptor and consequently was neither phosphorylated nor activated. These results suggest that the NH 2 -terminal SH2 domain, but not the COOH-terminal SH2 domain, of PLC-␥1 is required for PDGF-induced activation of PLC-␥1. Functional impairment of the SH2 domains did not affect the PDGF-induced redistribution of PLC-␥1, suggesting that recruitment of PLC-␥1 to the particulate fraction does not involve the SH2 domains.

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

“…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). Taken together, these studies suggest that access to the SH2C domain is tightly regulated by the overall structure of PLC-␥1.…”

“…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 mechanism of phospholipase C-γ1 regulation

“…In contrast to the EGF receptor-signaling, there is one specific autophosphorylated tyrosine that specifies an association with the PLC-γ1 SH2 domains for platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and nerve growth factor (NGF) receptors (for example, the Tyr1021 of the β-type PDGF receptor) (Valius et al, 1993). Both SH2 domains of PLC-γ1 have been shown to be required for growth factor-induced receptor association and Ca 2+ mobilization and also for association with the EGF receptor (Chattopadyay et al, 1999;Poulin et al, 2000). The N-terminal SH2 domain of PLC-γ1 is known to play a major role in the association with the activated growth factor receptor, while the C-terminal SH2 domain is considered to play a minor role (Chattopadyay et al, 1999;Poulin et al, 2000).…”

“…Both SH2 domains of PLC-γ1 have been shown to be required for growth factor-induced receptor association and Ca 2+ mobilization and also for association with the EGF receptor (Chattopadyay et al, 1999;Poulin et al, 2000). The N-terminal SH2 domain of PLC-γ1 is known to play a major role in the association with the activated growth factor receptor, while the C-terminal SH2 domain is considered to play a minor role (Chattopadyay et al, 1999;Poulin et al, 2000). However, recently, it has been reported that the C-terminal SH2 domain of PLC-γ1 is involved in interactions with synaptojanin (Ahn et al, 1998), the actin-cytoskeleton (Pei et al, 1996), PIP 3 (Bae et al, 1998;Rameh et al, 1998) and FAK (Zhang et al, 1999).…”

“…These domains are known to facilitate the PLC-γ1 association with other proteins. The SH2 domains recognize phosphotyrosine sequences in other proteins (Gergel et al, 1994;Pawson 1994;Sillman and Monroe, 1995;Valius et al, 1995;Yablonski et al, 1998;Poulin et al, 2000), while the SH3 domain mediates interactions with proteins containing prolinerich sequences (PXXP motif) (Pawson and Gish, 1992;Pawson and Nash, 2000). Cbl, SOS1, and dynamin are considered to bind to the SH3 domain of PLC-γ1 (Seedorf et al, 1994;Graham et al, 1998;Kim et al, 2000).…”