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

Abstract

Phosphoinositide-specific phospholipase C-␥1 (PLC-␥1) is a key enzyme that governs cellular functions such as gene transcription, secretion, proliferation, motility, and development. Here, we show that PLC-␥1 is regulated via a novel autoinhibitory mechanism involving its carboxy-terminal Src homology (SH2C) domain. Mutation of the SH2C domain tyrosine binding site led to constitutive PLC-␥1 activation. The amino-terminal split pleckstrin homology (sPHN) domain was found to regulate the accessibility of the SH2C domain. PLC-␥1 constructs with mutations in tyrosine 509 and phenylalanine 510 in the sPHN domain no longer required an intact amino-terminal Src homology (SH2N) domain or phosphorylation of tyrosine 775 or 783 for activation. These data are consistent with a model in which the SH2C domain is blocked by an intramolecular interaction(s) that is released upon cellular activation by occupancy of the SH2N domain.Phosphoinositide-specific phospholipase C-␥1 (PLC-␥1) is activated in response to ligand binding by a variety of receptors (2). PLC-␥1 hydrolyzes phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], leading to the generation of the second messengers, inositol triphosphate (IP 3 ) and diacylglycerol, which release Ca 2ϩ from intracellular stores and activate Ras through the RasGRP/protein kinase C pathway, respectively (8, 13). PLC-␥1 has also been shown to play a role in the activation of NF-B (7). These pathways in turn initiate signaling cascades that culminate in cytokine production, activation of effector function, and cell proliferation (2, 5). The central role of PLC-␥1 in cellular function is further illustrated by the fact that inactivation of the PLC-␥1 gene in mice is embryonic lethal (16).Regulation of PLC-␥1 activity is complex. In quiescent cells, cytosolic sequestration of PLC-␥1 limits access to PI(4,5)P 2 in the plasma membrane. Cell stimulation induces membrane recruitment of PLC-␥1 and its tyrosine phosphorylation, essential steps in PLC-␥1 activation (2,23,36). Key components of PLC-␥ that are involved in these activation events are located within a region unique to PLC-␥ between the X and Y portions of the catalytic domain. 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). 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).Components of the SH region not only are essential for PLC-␥1 activation but also may participate in its autoregulation. The individual X or Y elements of the catalytic domain alone are inactive. When mixed together in vitro, the hydrolytic activity of the combined X and Y elements was found...

A double point mutation in PCL-γ1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation

“…Phosphorylated PLC-γ1 intramolecularly binds the C-terminal SH2 domain, promoting higher accessibility to the substrate, PIP2 (DeBell et al, 1999(DeBell et al, , 2007Poulin et al, 2005). Data of our present study show that the Y509A/F510A mutant protein exhibits a remarkable increase in PIP2-hydrolyzing activity, without a need for Y783 phosphorylation.…”

“…In contrast, our current results show that the Y509A/F510A mutant protein produced elevated levels of IP 3 , both in the presence and absence of agonist in vivo. When it is considered that PIP 2 hydrolysis by inactive PLC-γ1 is inhibited in quiescent cells by effective removal of substrate via intramolecular interaction (Poulin et al, 2005;DeBell et al, 2007), a possible explanation for the current finding is that the Y509A/F510A mutation results in an open-state conformation that retains activity with respect to substrate hydrolysis. This theory is supported by the existence of a PLC catalytic-inhibitory peptide (PCI peptide) encoded immediately downstream of the C-terminal SH2 domain of PLC-γ1 (Homma and Takenawa, 1992;Homma et al, 1997).…”

“…In a previous study, we demonstrated, for the first time, that the Y509 and F510 residues within the nPH2 domain were critical for protein-protein interaction, and a double point mutant (Y509A/F510A) completely lost binding affinity with EF-1α (Chang et al, 2002). Based on this finding, DeBell and colleagues (DeBell et al, 2007) further reported that a Y509A/F510A mutant protein modulated cellular calcium signaling and NF-AT …”

Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase Cγ2

“…3B and C). In a previous study, a similar deletion of the spPH domain in PLC␥1 resulted in higher phosphorylation in the basal state that was further enhanced above the wild-type levels upon stimulation of the B-cell receptor (8). Deletion of the SH2n domain did not affect basal activity but, like the point mutation in this domain, reduced the activation of PLC␥2 by EGF (Fig.…”

Stimulation of phospholipase Cβ by membrane interactions, interdomain movement, and G protein binding — How many ways can you activate an enzyme?

“…In support of this idea, Roberts and collaborators point out that initiation of ligand binding to the active site could shift the PLC-δ1 to more active form of the enzyme, possibly through a conformational change involving the X/Y linker [20]. In the case of PLC-γ, the X/Y linker contains a split PH-domain, two SH2 domains, and SH3 domain which, by binding specifically to specific lipids and adaptors, can induce an intramolecular rearrangement gathering the separated X and Y region and reconstituting a fully-active catalytic site [27].…”