Dynamics of allosteric regulation of the phospholipase C-γ isozymes upon recruitment to membranes
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Evaluation Summary:
This work provides insight into how phospholipase C gamma (PLCγ1) becomes activated upon binding to phosphorylated receptor tyrosine kinase, with an analysis of PLC γ1 bound to the soluble kinase domain of FGFR1 (FGFR1K) and/or liposomes containing PIP2. The most interesting finding is that regions of the protein far from the FGFR1K binding site increase in exchange upon binding. This is new information for a large protein that is arguably difficult to study, but it conforms to what has been observed in many other autoinhibited systems with similar SH2 and SH3 domains such as kinases. The results will be of interest to structural biologists and cell biologists with interest in the mechanisms leading to the regulation of phospholipase C activity on membranes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Numerous receptor tyrosine kinases and immune receptors activate phospholipase C-γ (PLC-γ) isozymes at membranes to control diverse cellular processes including phagocytosis, migration, proliferation, and differentiation. The molecular details of this process are not well understood. Using hydrogen-deuterium exchange mass spectrometry, we show that PLC-γ1 is relatively inert to lipid vesicles that contain its substrate, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), unless first bound to the kinase domain of the fibroblast growth factor receptor (FGFR1). Exchange occurs throughout PLC-γ1 and is exaggerated in PLC-γ1 containing an oncogenic substitution (D1165H) that allosterically activates the lipase. These data support a model whereby initial complex formation shifts the conformational equilibrium of PLC-γ1 to favor activation. This receptor-induced priming of PLC-γ1 also explains the capacity of a kinase-inactive fragment of FGFR1 to modestly enhance the lipase activity of PLC-γ1 operating on lipid vesicles but not a soluble analog of PIP 2 and highlights potential cooperativity between receptor engagement and membrane proximity. Priming is expected to be greatly enhanced for receptors embedded in membranes and nearly universal for the myriad of receptors and co-receptors that bind the PLC-γ isozymes.
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Evaluation Summary:
This work provides insight into how phospholipase C gamma (PLCγ1) becomes activated upon binding to phosphorylated receptor tyrosine kinase, with an analysis of PLC γ1 bound to the soluble kinase domain of FGFR1 (FGFR1K) and/or liposomes containing PIP2. The most interesting finding is that regions of the protein far from the FGFR1K binding site increase in exchange upon binding. This is new information for a large protein that is arguably difficult to study, but it conforms to what has been observed in many other autoinhibited systems with similar SH2 and SH3 domains such as kinases. The results will be of interest to structural biologists and cell biologists with interest in the mechanisms leading to the regulation of phospholipase C activity on membranes.
(This preprint has been reviewed by eLife. We include the …
Evaluation Summary:
This work provides insight into how phospholipase C gamma (PLCγ1) becomes activated upon binding to phosphorylated receptor tyrosine kinase, with an analysis of PLC γ1 bound to the soluble kinase domain of FGFR1 (FGFR1K) and/or liposomes containing PIP2. The most interesting finding is that regions of the protein far from the FGFR1K binding site increase in exchange upon binding. This is new information for a large protein that is arguably difficult to study, but it conforms to what has been observed in many other autoinhibited systems with similar SH2 and SH3 domains such as kinases. The results will be of interest to structural biologists and cell biologists with interest in the mechanisms leading to the regulation of phospholipase C activity on membranes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors have performed extensive and well-controlled HDX-MS experiments using recombinant PLCγ, phosphorylated receptor tyrosine kinase (FGFR1K) and PIP2-containing liposomes to investigate the mechanisms of PLCγ activation. The data supports a model in which PLCγ, which is basally inactive, is initially primed by the interaction of its nSH2 domain with tyrosine-phosphorylated FGFR1K and therefore recruited to the membrane, after which it gets phosphorylated, leading to a situation that favors a fully open PLCγ that can efficiently catalyze membrane-embedded PIP2, and thus trigger downstream signalling cascades. Interestingly, the authors also studied an oncogenic PLCγ mutation (D1165H), showing that this mutant, which seems to be conformationally more flexible, mimics RTK engagement.
The experiments are …
Reviewer #1 (Public Review):
The authors have performed extensive and well-controlled HDX-MS experiments using recombinant PLCγ, phosphorylated receptor tyrosine kinase (FGFR1K) and PIP2-containing liposomes to investigate the mechanisms of PLCγ activation. The data supports a model in which PLCγ, which is basally inactive, is initially primed by the interaction of its nSH2 domain with tyrosine-phosphorylated FGFR1K and therefore recruited to the membrane, after which it gets phosphorylated, leading to a situation that favors a fully open PLCγ that can efficiently catalyze membrane-embedded PIP2, and thus trigger downstream signalling cascades. Interestingly, the authors also studied an oncogenic PLCγ mutation (D1165H), showing that this mutant, which seems to be conformationally more flexible, mimics RTK engagement.
The experiments are performed using catalytically inactive PLCγ mutant (H335A), so the phospholipase can interact with PIP2-containing liposomes but not catalytically hydrolyze PIP2 during the course of the HDX experiments.
Finally, the experiments with the liposomes were all performed for a given lipid composition (90% PE+10% PIP2).
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Reviewer #2 (Public Review):
This manuscript provides HDX-MS analysis of PLCg1 bound to the soluble kinase domain of FGFR1 (FGFR1K) and/or liposomes containing PIP2. PLCg1 is autoinhibited as shown by the crystal structure of the full-length protein which includes multiple regulatory domains as well as the TIM barrel lipase domain. The FGFR1K binds to the N-terminal SH2 domain and this is verified by decreased exchange upon binding. The authors use a catalytically dead mutant in order to study the complexes at a constant concentration of PIP2 in the liposomes. The interesting story of the paper is that regions of the protein far from the FGFR1K binding site increase in exchange upon binding. While this is new information for this large protein that is arguably difficult to study, it is rather expected at this point having been observed …
Reviewer #2 (Public Review):
This manuscript provides HDX-MS analysis of PLCg1 bound to the soluble kinase domain of FGFR1 (FGFR1K) and/or liposomes containing PIP2. PLCg1 is autoinhibited as shown by the crystal structure of the full-length protein which includes multiple regulatory domains as well as the TIM barrel lipase domain. The FGFR1K binds to the N-terminal SH2 domain and this is verified by decreased exchange upon binding. The authors use a catalytically dead mutant in order to study the complexes at a constant concentration of PIP2 in the liposomes. The interesting story of the paper is that regions of the protein far from the FGFR1K binding site increase in exchange upon binding. While this is new information for this large protein that is arguably difficult to study, it is rather expected at this point having been observed in many other autoinhibited systems with similar SH2 and SH3 domains such as kinases.
The authors are able to study the ternary complex between liposomes containing PIP2 substrate, the FGF receptor kinase domain and the protein of interest, Phospholipase C gamma, which is a large multidomain protein. The crystal structure of the protein is in an autoinhibited form, and the authors are able to show using HDX-MS how opening might occur to an active state.
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