Abstract
Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.
Highlights
German Research Center for Environmental Health, Institute of Experimental Genetics, D-85764 Munich/Neuherberg, Germany, the ¶¶Lehrstuhl fur Experimentelle Genetik, Technische Universitat Munchen, D-85350 Freising-Weihenstephan, Germany, the §§Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 259-1193 Kanagawa, Japan, and the ʈʈInstitute for Immunology, Philipps-Universitat Marburg, 35032 Marburg, Germany
Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine phospholipase C (PLC)␥2 that lead to spontaneous inflammation and autoimmunity
We suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state
Summary
PLC␥, phospholipase C␥; ENU, N-ethyl-N-nitrosourea; DMEM, Dulbecco’s modified Eagle’s medium; BCR, B-cell receptor; EGF, epidermal growth factor; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate. Previous studies of PLC␥2, have demonstrated the first gainof-function mutation in a PLC molecule in the context of an organism, and shown that, in principle, PLC activity can be greatly enhanced by point mutations (13). Changes that lead to PLC activation in response to different input signals, or due to point mutations, are not well understood and require further studies. We describe further analysis of the two gain-of-function mutations, ALI5 and ALI14, obtained using ENU mutagenesis These mutations map to different regions in PLC␥2, and we performed detailed analysis of these regions in both PLC␥ isozymes. We have found that ALI5- and ALI14-type point mutations lead, by distinct mechanisms, to an enhancement of responses to a variety of input signals while their combination results in a constitutively active PLC enzyme
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