The black box illuminated: signals and signaling

Abstract

Unraveling the signaling pathways that transmit information from the cell surface to the nucleus has been a major accomplishment of modern cell and molecular biology. The benefit to humans is seen in the multitude of new therapeutics based on the illumination of these pathways. Although considerable insight has been gained in understanding homeostatic and pathological signaling in the epidermis and other skin compartments, the translation into therapy has been lacking. This review will outline advances made in understanding fundamental signaling in several of the most prominent pathways that control cutaneous development, cell-fate decisions, and keratinocyte growth and differentiation with the anticipation that this insight will contribute to new treatments for troubling skin diseases.

Figure 1.. Integration of the calcium signaling circuitry:

The major regulators of calcium homeostasis in keratinocytes are depicted. Plasma membrane pumps and channels (PMCA, NCX, SOCE) regulate flux in and out of the cytosol. G protein coupled receptors (CaR and others not shown) initiate signals that modify compartmentalized calcium stores (e.g. IP3). Calcium ATPases on organelles (SERCA, SPCA1) monitor and replenish intracellular storage sites. The figure is modified from (Savignac et al, 2011) with permission from the publisher.

Figure 2.. Integration of PKC signaling circuitry:

Protein kinase C isoforms can be activated by the second messenger inputs calcium (Ca²⁺) and DAG generated by the activity of PLC. Intracellular calcium activates PKCα, β and γ via their C2 domains, while DAG and TPA activate PKCα, β, γ, δ, ε, η and θ via C1 domains. Src family kinases can also tyrosine phosphorylate and alter the expression of PKCδ. The outputs of PKC signaling are isoform dependent as indicated: PKCα (differentiation-associated growth arrest and cytokine-mediated inflammation), PKCβ (melanogenesis), PKCδ (squamous differentiation, growth arrest and apoptosis), PKCη (squamous differentiation and growth arrest), PKCε (hyperplasia).

Figure 3.. Integration of EGFR signaling circuitry:

EGFR and other ERBB receptors dimerize after engagement with their ligands activating multiple signaling cascades through recruitment of adaptor and signaling proteins to the intracellular portion of the receptor. Phospholipase Cγ (PLC γ) activity triggers the activation or downmodulation of protein kinase C isoforms (PKCs) and increases intracellular calcium leading to the regulation and expression of proteins that control differentiation. Phosphoinositol 3’-OH kinase (PI3K) promotes survival responses through AKT activity. Activation of Ras regulates JNK1/2 and ERK1/2 MAPKs activity causing changes in gene transcription and mRNA stability. As a consequence, cell cycle progression and inflammatory responses are fine tuned by EGFR activity.

Figure 4:. Integration of Notch signaling circuitry:

The Notch pathway generates a short-range communication channel utilized throughout skin development and elsewhere in the adult to regulate multiple cellular processes (e.g., proliferation, stem cell and stem cell niche maintenance, cell fate specification, differentiation and cell death). Notch signals in a unique mechanism mediated by proteolysis and without any secondary messengers. Post-translational modifications and trafficking of the ligands and receptors can regulate the amplitude and timing of Notch activity. 1–2) Receptor Maturation. Upon translation, the Notch protein is fucosylated by the Pofut1. The fucose can be extended by the glycosyltransferase activity of Fringe. This impacts the ability of specific ligands to activate Notch (see below). During exocytosis the Notch receptor is cleaved by PC5, a protein convertase, at Site 1 (S1). At the cell surface Notch is a heterodimer (HD) held together by non-covalent interactions within the HD domain which, together with three Lin-Notch repeats (LNR) forms the negative regulatory region (NRR). 2) The steady-state levels of the Notch receptor at the cell surface are regulated by several proteins, many of which are E3 ubiquitin ligases (Dtx, Nedd4). After recycling, inappropriate receptor activation within endosomes can occur in the absence of ligand binding (NICD*). The ESCRT complex proteins are involved in Notch down-regulation, and mutations in this complex may contribute to pathogenesis in different cellular contexts. 3) Ligand maturation. Notch ligands are also Type I transmembrane proteins. The two major classes of ligands are Delta and Jagged (Serrate in Drosophila), the latter containing a cysteine rich domain. To signal, ligands must undergo ubiquitinylation by E3 ubiquitin ligases Neur and Mib which trigger Epsin-mediated endocytosis, and an undefined modification produces an active ligand which recycles to the cell surface in a Rab11 dependent process. Current models explaining the nature of ligand modification include ligand clustering, post-translational modifications and/or recycling into specific membrane domains. 4–6) Activation. Productive receptor-ligand interactions occur between neighboring cells (in TRANS) whereas negative interactions occur between receptor-ligand proteins coexpressed in the same cell (in CIS). Fringe-modified Notch1 receptors favor Delta binding (shown). Ligand endocytosis is thought to generate sufficient force to unfold the NRR, exposing Notch to cleavage at site S2 by ADAM10 metalloproteases. The membrane-anchored NEXT (Notch extracellular truncation) fragment is recognized by γ-secretase, an enzymatic complex composed of PS, NCT, PEN2 and APH1. γ-secretase then cleaves the Notch transmembrane domain sequentially starting near the cytosolic surface (sites S3 and S4) to release the Notch intracellular domain (NICD) and Nβ peptides, respectively. 5) In the absence of NICD, the DNA-binding protein CSL associates with ubiquitous co-repressor (Co-R) proteins, HDACs and Sirt1 to repress transcription of target genes. NICD binding to CSL recruits the adaptor protein Mastermind (MAM), which recruits the mediator complex and assembles an active transcription complex on target promoters. 6) During the transcriptional activation process, NICD is phosphorylated on its PEST domain by kinases such as CDK8 and targeted for proteasomal degradation by E3 ubiquitin ligases such as Sel10/Fbw7. This terminates the Notch signal and resets the cell for the next round of signaling. This figure is reproduced from (Ilagan and Kopan, 2007) with permission from the publisher.