A kinetic model of phospholipase C-γ1 linking structure-based insights to dynamics of enzyme autoinhibition and activation.

Phospholipase C-γ1 (PLC-γ1) is a receptor-proximal enzyme that promotes signal transduction through PKC in mammalian cells. Because of the complexity of PLC-γ1 regulation, a two-state (inactive/active) model does not account for the intricacy of activation and inactivation steps at the plasma membrane. Here, we introduce a structure-based kinetic model of PLC-γ1, considering interactions of its regulatory Src homology 2 (SH2) domains and perturbation of those dynamics upon phosphorylation of Tyr783, a hallmark of activation. For PLC-γ1 phosphorylation to dramatically enhance enzyme activation as observed, we found that high intramolecular affinity of the C-terminal SH2 (cSH2) domain-pTyr783 interaction is critical, but this affinity need not outcompete the autoinhibitory interaction of the cSH2 domain. Under conditions for which steady-state PLC-γ1 activity is sensitive to the rate of Tyr783 phosphorylation, maintenance of the active state is surprisingly insensitive to the phosphorylation rate, since pTyr783 is well protected by the cSH2 domain while the enzyme is active. In contrast, maintenance of enzyme activity is sensitive to the rate of PLC-γ1 membrane (re)binding. Accordingly, we found that hypothetical PLC-γ1 mutations that either weaken autoinhibition or strengthen membrane binding influence the activation kinetics differently, which could inform the characterization of oncogenic variants. Finally, we used this newly informed kinetic scheme to refine a spatial model of PLC/PKC polarization during chemotaxis. The refined model showed improved stability of the polarized pattern while corroborating previous qualitative predictions. As demonstrated here for PLC-γ1, this approach may be adapted to model the dynamics of other receptor- and membrane-proximal enzymes.

Mechanistic models of PLC/PKC signaling implicate phosphatidic acid as a key amplifier of chemotactic gradient sensing.

Chemotaxis of fibroblasts and other mesenchymal cells is critical for embryonic development and wound healing. Fibroblast chemotaxis directed by a gradient of platelet-derived growth factor (PDGF) requires signaling through the phospholipase C (PLC)/protein kinase C (PKC) pathway. Diacylglycerol (DAG), the lipid product of PLC that activates conventional PKCs, is focally enriched at the up-gradient leading edge of fibroblasts responding to a shallow gradient of PDGF, signifying polarization. To explain the underlying mechanisms, we formulated reaction-diffusion models including as many as three putative feedback loops based on known biochemistry. These include the previously analyzed mechanism of substrate-buffering by myristoylated alanine-rich C kinase substrate (MARCKS) and two newly considered feedback loops involving the lipid, phosphatidic acid (PA). DAG kinases and phospholipase D, the enzymes that produce PA, are identified as key regulators in the models. Paradoxically, increasing DAG kinase activity can enhance the robustness of DAG/active PKC polarization with respect to chemoattractant concentration while decreasing their whole-cell levels. Finally, in simulations of wound invasion, efficient collective migration is achieved with thresholds for chemotaxis matching those of polarization in the reaction-diffusion models. This multi-scale modeling framework offers testable predictions to guide further study of signal transduction and cell behavior that affect mesenchymal chemotaxis.

A Reaction-Diffusion Model Explains Amplification of the PLC/PKC Pathway in Fibroblast Chemotaxis.

During the proliferative phase of cutaneous wound healing, dermal fibroblasts are recruited into the clotted wound by a concentration gradient of platelet-derived growth factor (PDGF), together with other spatial cues. Despite the importance of this chemotactic process, the mechanisms controlling the directed migration of slow-moving mesenchymal cells such as fibroblasts are not well understood. Here, we develop and analyze a reaction-diffusion model of phospholipase C/protein kinase C (PKC) signaling, which was recently identified as a requisite PDGF-gradient-sensing pathway, with the goal of identifying mechanisms that can amplify its sensitivity in the shallow external gradients typical of chemotaxis experiments. We show that phosphorylation of myristoylated alanine-rich C kinase substrate by membrane-localized PKC constitutes a positive feedback that is sufficient for local pathway amplification. The release of phosphorylated myristoylated alanine-rich C kinase substrate and its subsequent diffusion and dephosphorylation in the cytosol also serves to suppress the pathway in down-gradient regions of the cell. By itself, this mechanism only weakly amplifies signaling in a shallow PDGF gradient, but it synergizes with other feedback mechanisms to enhance amplification. This model offers a framework for a mechanistic understanding of phospholipase C/PKC signaling in chemotactic gradient sensing and can guide the design of experiments to assess the roles of putative feedback loops.