Characterisation of molecular mechanisms for PLCγ2 disease-linked variants.

The phospholipase C enzyme PLCγ2 is best characterised in the context of immune cell regulation. Furthermore, many mutations discovered in PLCγ2 have been linked to the development of complex immune disorders as well as resistance to ibrutinib treatment in chronic lymphocytic leukaemia. Importantly, it has also been found that a rare variant of PLCγ2 (P522R) has a protective role in Alzheimer's disease (AD). Despite initial characterisation of these disease-linked variants, a comprehensive understanding of their differences and underpinning molecular mechanisms, needed to facilitate therapeutic efforts, is lacking. Here, we used available structural insights for PLCγ enzymes to further analyse PLCγ2 M1141K mutation, representative for mutations in immune disorders and cancer resistance, and the AD-protective variant, PLCγ2 P522R. Together with several other mutations in the autoinhibitory interface, the PLCγ2 M1141K mutation was strongly activating in a cell-based assay, under basal and stimulated conditions. Measurements of PLC activity in various in vitro assays demonstrated enhanced activity of PLCγ2 M1141K while the activity of PLCγ2 P522R was not significantly different from the WT. Similar trends were observed in several other assays, including direct liposome binding. However, an enhanced rate of phosphorylation of a functionally important tyrosine by Btk in vitro was observed for PLCγ2 P522R variants. To further assess implications of these in vitro findings in a cellular context relevant for the PLCγ2 P522R variant, microglia (BV2) stable cell lines were generated and analysed under growth conditions. The PLC activity in cells expressing PLCγ2 P522R at physiologically relevant levels was clearly enhanced compared to the WT, and differences in cell morphology observed. These data, combined with the structural insights, suggest that the PLCγ2 P522R variant has subtle, localised structural changes that do not directly affect the PLC activity by compromising autoinhibition, as determined for PLCγ2 M1141K. It is also likely that in contrast to the PLCγ2 M1141K, the functional impact of the P522R substitution completely depends on further interactions with upstream kinases and other regulatory proteins in a relevant cellular context, where changes in the PLCγ2 P522R variant could facilitate processes such as phosphorylation and protein-protein interactions.

A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells.

BACKGROUND: Development of adult T-cell leukemia/lymphoma (ATL) involves human T-cell leukemia virus type 1 (HTLV-1) infection and accumulation of somatic mutations. The most frequently mutated gene in ATL (36 % of cases) is phospholipase C gamma1 (PLCG1). PLCG1 is also frequently mutated in other T-cell lymphomas. However, the functional consequences of the PLCG1 mutations in cancer cells have not been characterized. METHODS: We compared the activity of the wild-type PLCγ1 with that of a mutant carrying a hot-spot mutation of PLCγ1 (S345F) observed in ATL, both in cells and in cell-free assays. To analyse the impact of the mutation on cellular properties, we quantified cellular proliferation, aggregation, chemotaxis and apoptosis by live cell-imaging in an S345F+ ATL-derived cell line (KK1) and a KK1 cell line in which we reverted the mutation to the wild-type sequence using CRISPR/Cas9 and homology-directed repair. FINDINGS: The PLCγ1 S345F mutation results in an increase of basal PLC activity in vitro and in different cell types. This higher basal activity is further enhanced by upstream signalling. Reversion of the S345F mutation in the KK1 cell line resulted in reduction of the PLC activity, lower rates of proliferation and aggregation, and a marked reduction in chemotaxis towards CCL22. The PLCγ1-pathway inhibitors ibrutinib and ritonavir reduced both the PLC activity and the tested functions of KK1 cells. INTERPRETATION: Consistent with observations from clinical studies, our data provide direct evidence that activated variants of the PLCγ1 enzyme contribute to the properties of the malignant T-cell clone in ATL. FUNDING: MRC (UK) Project Grant (P028160).

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover.

Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2ß, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2ß. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch.