PKCδ Localization at the Membrane Increases Matrix Traction Force Dependent on PLCγ1/EGFR Signaling

Abstract

IntroductionDuring wound healing, fibroblasts initially migrate into the wound bed and later contract the matrix. Relevant mediators of transcellular contractility revealed by systems analyses are protein kinase c delta/myosin light chain-2 (PKCδ/MLC-2). PKCδ is activated by growth factor-driven PLCγ1 hydrolysis of phosphoinositide bisphosphate (PIP2) hydrolysis when it becomes tranlocated to the membrane. This leads to MLC-2 phosphorylation that regulates myosin for contractility. Furthermore, PKCδ n-terminus mediates PKCδ localization to the membrane in relative proximity to PLCγ1 activity. However, the role this localization and the relationship to its activation and signaling of force is not well understood. Therefore, we investigated whether the membrane localization of PKCδ mediates the transcellular contractility of fibroblasts.MethodsTo determine PKCδ activation in targeted membrane locations in mouse fibroblast cells (NR6-WT), two PKCδ constructs were generated; PKCδ-CaaX with farnesylation moiety targeting PKCδ to the membrane and PKCδ-SaaX a non-targeting control.ResultsIncreased mean cell force was observed before and during EGF stimulation in fibroblasts expressing membrane-targeted PKCδ (PKCδ-CaaX) when analyzed with 2D cell traction force and 3D compaction of collagen matrix. This effect was reduced in cells deficient in EGFR/PLCy1 signaling. In cells expressing non-membrane targeted PKCδ (PKCδ-SaaX), the cell force exerted outside the ECM (extracellular matrix) was less, but cell motility/speed/persistence was increased after EGF stimulation. Change in cell motility and increased force exertion was also preceded by change in cell morphology. Organization of actin stress fibers was also decreased as a result of increasing membrane targeting of PKCδ.ConclusionFrom these results membrane tethering of PKCδ leads to increased force exertion on ECM. Furthermore, our data show PLCγ1 regulation of PKCδ, at least in part, drives transcellular contractility in fibroblasts.

PKCδ Regulates Force Signaling during VEGF/CXCL4 Induced Dissociation of Endothelial Tubes

“…Although PKCδ has been shown in previous literature to mediate apoptosis [24] , [25] }, it seems that these cells may actually alter their mesenchymal phenotype to incorporate into the cords and to actively mediate tension at junctions ( Fig 4b , Movie S4 ). This would be consistent with our finding that PKCδ directing cell contractility [7] . These data imply that activation of PKCδ is associated with force generation and possibly force mechanics in endothelial cord dissociation.…”

“…Tube dissociation involves the separation of cell-cell contacts, we therefore investigated whether transcellular contractility involves PKCδ, a key signaling nexus in contractility and tension [7] , [8] . Dissociation was blunted by downregulation of PKCδ.…”

Predictive data-driven modeling of C-terminal tyrosine function in the EGFR signaling network

“…Other sites on the receptor (Y703, Y803, Y845, Y954, Y992, Y1101) were detected and also demonstrated significantly increased phosphorylation levels upon EGF induction (Fig 1D). Although NR6 cells do not naturally express EGFR or other ErbB receptors, they are signaling competent, in agreement with multiple previous studies (Pruss & Herschman, 1977;Masui et al, 1991;Glading et al, 2000;Jamison et al, 2013). Signaling pathways downstream of the receptor in our wtEGFRexpressing NR6 cells are intact and responsive to EGFR stimulation, as shown by a strong temporal response of several wellcharacterized proximal adaptor protein phosphorylation sites and the ERK 1/2 mitogen-activated protein kinases (MAPK1 and MAPK3) (Fig 1E).…”

Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells

“…In iPSC-RPE, aphidicolin enhanced ciliogenesis activated PKCδ, translocating it to the cell membrane and to the base of the primary cilium ( Figures 4A–4C ), likely through direct protein-protein interactions with the ciliary trafficking protein BBS8, as confirmed by co-immunoprecipitation between BBS8 and PKCδ ( Figure 4D ; Figure S3A ). Phospho-Myosin Light Chain 2 (pMLC2), a downstream effector of PKCδ ( Jamison et al, 2013 ), was increasingly organized along cell boundaries in aphidicolin-treated cells ( Figures 4E and 4F ), suggesting an increased activity of PKCδ in cells with enhanced ciliogenesis. Rottlerin, a specific PKC δ inhibitor, completely blocked aphidicolin-induced organization of pMLC2 along cell boundaries ( Figure 4G ), suggesting a prominent role of this kinase in inducing RPE polarization downstream of primary cilia.…”