Structural insights and activating mutations in diverse pathologies define mechanisms of deregulation for phospholipase C gamma enzymes

Abstract

A B S T R A C TBackground: PLCg enzymes are key nodes in cellular signal transduction and their mutated and rare variants have been recently implicated in development of a range of diseases with unmet need including cancer, complex immune disorders, inflammation and neurodegenerative diseases. However, molecular nature of activation and the impact and dysregulation mechanisms by mutations, remain unclear; both are critically dependent on comprehensive characterization of the intact PLCg enzymes. Methods: For structural studies we applied cryo-EM, cross-linking mass spectrometry and hydrogen-deuterium exchange mass spectrometry. In parallel, we compiled mutations linked to main pathologies, established their distribution and assessed their impact in cells and in vitro. Findings: We define structure of a complex containing an intact, autoinhibited PLCg1 and the intracellular part of FGFR1 and show that the interaction is centred on the nSH2 domain of PLCg1. We define the architecture of PLCg1 where an autoinhibitory interface involves the cSH2, spPH, TIM-barrel and C2 domains; this relative orientation occludes PLCg1 access to its substrate. Based on this framework and functional characterization, the mechanism leading to an increase in PLCg1 activity for the largest group of mutations is consistent with the major, direct impact on the autoinhibitory interface. Interpretation: We reveal features of PLCg enzymes that are important for determining their activation status. Targeting such features, as an alternative to targeting the PLC active site that has so far not been achieved for any PLC, could provide new routes for clinical interventions related to various pathologies driven by PLCg deregulation. Fund: CR UK, MRC and AstaZeneca.

Severe Autoinflammatory Manifestations and Antibody Deficiency Due to Novel Hypermorphic PLCG2 Mutations

“…Therefore, the molecular mechanisms that lead to an increase in PLC activity in diverse pathologies could be similar. Considerable supporting evidence is consistent with a model where PLCγ2 is kept in an inactive state by extensive intramolecular interactions between the regulatory domains (including cSH2 and spPH domains) and the PLC-core domains [11,31]. Release of this autoinhibition by physiological stimulation (via phosphorylation) or by mutations represents an important step leading to an increase in PLC activity.…”

“…Proteins were detected with a 1:150 dilution of the anti-PLCγ2 antibody sc5283 (Santa Cruz) and a 1:150 dilution of the anti-β-actin antibody 13E5 (Cell Signaling Technology). For the comparison of PLC activity of different PLCγ2 variants, the expression levels were quantitated and points with the same protein expression used, as previously described [11]. The differences were confirmed using the 2 tailed t test.…”

Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane

“…This PLC is sperm-specific and is the physiological trigger responsible for generating I(1,4,5)P 3 -mediated Ca 2+ oscillations that induces oocyte activation during mammalian fertilisation [43,44]. A substantial number of 3D structures for PLC enzymes provide a valuable basis for the understanding of various functional properties at the molecular level, including their PLC activity and regulatory mechanisms [45][46][47][48][49]. Notably, despite the diversity of their interacting proteins, the general molecular mechanism for regulation of PLCs is centred on intramolecular interactions that maintain PLCs in their inactive form, also referred to as autoinhibition, that becomes released in the process of activation.…”

“…One example that illustrates this concept is provided by recent structural insights into PLCγ1, primarily regulated by RTKs ( Figure 3 B). In the inactive form, two domains within the regulatory region (cSH2 and sPH) directly contribute to autoinhibition by interacting with the PLC-core, preventing membrane interactions required for the access to the PLC substrate, PI(4,5)P 2 [ 47 , 48 ]. Following phosphorylation of PLCγ1, the critical pTyr residue in PLCγ1 binds to its cSH2 domain; this intramolecular interaction is required for repositioning of the regulatory region and release of the autoinhibition.…”

A novel somatic PLCG2 variant associated with resistance to BTK and SYK inhibition in chronic lymphocytic leukemia

“…19 Though the pathogenic potential of EF hand mutations is not fully understood, aberrant expression of EF-associated calcium-binding proteins has been associated with malignant cell proliferation in a number of disease states. 20,21 The physiologic consequences of the novel variant described in this report are unknown, though it may augment calcium-dependent BCR pathway signaling, enabling resistance to BTK inhibition.…”