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Figure 3

Figure 3. Generation of protein half-life biosensors and their potential use in signalling integration studies. From: Endocytic control of growth factor signalling: multivesicular bodies as signalling organelles.

a | A generic biosensor of intracellular glycogen synthase kinase 3 (GSK3) activity is shown. It consists of a fluorescent protein (green fluorescent protein (GFP) or red FP (RFP)) with a carboxy-terminal peptide containing phosphorylation sites. Three artificial GSK3 sites are placed in front of a ‘priming kinase’ site (which could correspond to mitogen-activated protein kinase (MAPK), casein kinase 1 (CK1), CK2, protein kinase A (PKA) or many others); further downstream, an epitope tag (flag; which is useful for biochemical analyses) and a stop codon complete the biosensor. The presence of GSK3 phosphorylation sites in the protein destabilizes it, forming a short phosphodegron that promotes polyubiquitylation by endogenous E3 ligases and its proteasomal degradation. b | Phosphorylation sites that promote degradation in human β-catenin and in the artificial GFP biosensor GFP–CK1–GSK3, derived from their sequences12. CK1 primes three phosphorylation events by GSK3, and the presence of such priming kinase phosphorylation sites may promote specificity in signalling responses, helping to ‘insulate’ different signalling pathways from one another.

Radek Dobrowolski, et al. Nat Rev Mol Cell Biol. ;13(1):53-60.
Figure 4

Figure 4. Hypothesis: the dorsal determinants of the Xenopus laevis egg may be endosomal components. From: Endocytic control of growth factor signalling: multivesicular bodies as signalling organelles.

The vegetal pole of the frog egg contains ‘maternal determinants’ of unknown composition. We propose here that they may correspond to wnt-containing early endosomes that become incorporated into multivesicular bodies (MVBs) on the dorsal side of the zygote. This would sequester glycogen synthase kinase 3 (gsk3) and axis inhibition protein (axin) inside MVBs, triggering the earliest wnt signal in vertebrate embryonic differentiation, which induces the Nieuwkoop signalling centre. a | In the unfertilized egg, membrane vesicles are observed in the vegetal pole61. b | The sperm brings with it the centriole, giving rise to a centrosome that organizes a cortical network of microtubules. Early endosomes from the vegetal pole are transported along microtubules to the dorsal side, which forms opposite the sperm entry point. The new embryonic axis forms where vegetal material and animal cytoplasm mix55. Cortical microtubules cause a rotation of the egg cortex towards the sperm entry point, displacing the superficial pigment of the egg53. This forms a lighter ‘dorsal crescent’ that marks the dorsal side. One possibility is that maternal wnt is secreted by the oocyte and internalized into early endosomes at the vegetal pole. Upon fertilization, the formation of MVBs at the dorsal side might promote gsk3 sequestration and thereby allow persistent wnt signalling to promote axis formation. dvl, dishevelled; lrp6, low-density lipoprotein receptor-related 6.

Radek Dobrowolski, et al. Nat Rev Mol Cell Biol. ;13(1):53-60.
Figure 1

Figure 1. Current models for the intersection between endocytosis and signalling pathways. From: Endocytic control of growth factor signalling: multivesicular bodies as signalling organelles.

There are three major models for the crosstalk between endocytosis and cell signalling. a | Downregulation of signals through the degradation of receptor complexes. Receptor Tyr kinases (RTKs) and G protein-coupled receptors (GPCRs) signal at the plasma membrane through their effectors, such as SRC (for RTKs) and β-arrestin or activated G proteins (for GPCRs), which are then released as the receptors become internalized into early endosomes that undergo acidification. After trafficking of receptors into the intraluminal vesicles (ILVs) of multivesicular endosomes, these organelles fuse with lysosomes and the receptors are degraded. b | The mitogen-activated protein kinase (MAPK) pathway provides an example in which signalling downstream of RTKs or GPCRs can be maintained by early endosomes. The signal is generated at the plasma membrane and continues signalling in early endosomes through formation of RAS–RAF–MEK–ERK (RAS–RAF–MAPK/ERK kinase (also known as MAPKK)–extracellular signal-regulated kinase) complexes bound to the growth factor receptor-bound 2 (GRB2) adaptor for RTKs or β-arrestin for GPCRs. Activated ERK is released from the endosome and translocates to the nucleus to phosphorylate its targets. c | For signalling downstream of transforming growth factor-β receptor (TGFβR), as well as other receptors, the signal is actually generated on signalling endosomes. The ligand-bound TGFβR is internalized into endosomes upon phosphorylation of its cytoplasmic tail. SMAD anchor for receptor activation (SARA) is an FYVE domain (phosphatidylinositol-3-phosphate-binding) protein that recognizes this activated receptor and recruits the SMAD2 transcription factor to signalling endosomes. Phosphorylated SMAD2 is then released, entering the nucleus to promote transcriptional activation.

Radek Dobrowolski, et al. Nat Rev Mol Cell Biol. ;13(1):53-60.
Figure 2

Figure 2. Endocytosis is required for canonical WNT signalling. From: Endocytic control of growth factor signalling: multivesicular bodies as signalling organelles.

Two WNT co-receptors, Frizzled (FRZ) and low-density lipoprotein receptor-related 6 (LRP6), are required in the plasma membrane for canonical WNT signalling. When cells are exposed to the antagonist Dickkopf homologue 1 (DKK1), LRP6 is directed to endocytosis by clathrin-coated vesicles, becoming inactive and subsequently degraded through multivesicular body (MVB)-mediated delivery to lysosomes27. Conversely, when the cell receives a WNT signal, WNT binds to both LRP6 and FRZ. This recruits Dishevelled (DVL), which is required for polymerization of the co-receptors in lipid rafts and endocytosis through caveolin-containing vesicles13. This internalization also requires the endocytosis effector Dynamin (DYN) and vacuolar ATPase (v-ATPase)21,84. The cytoplasmic tail of LRP6 is phosphorylated by casein kinase 1γ (CK1γ) and glycogen synthase kinase 3 (GSK3), activating endocytosis85 and binding components of the β-catenin ‘destruction’ complex (axis inhibition protein (AXIN) and GSK3). Early endosomal vesicles mature into multivesicular endosomes (in a process that requires hepatocyte growth factor-regulated Tyr kinase substrate (HRS) and vacuolar protein sorting-associated 4 (VPS4)) and WNT receptor complexes are internalized, sequestering GSK3 inside intraluminal vesicles (ILVs). Sequestration of the GSK3 enzyme in multivesicular endosomes is an essential step and is required for sustained WNT signalling. The decrease in active GSK3 causes newly synthesized β-catenin to accumulate and then be transported to the nucleus, where it activates gene transcription by TCF–LEF. APC/C, anaphase-promoting complex, also known as the cyclosome.

Radek Dobrowolski, et al. Nat Rev Mol Cell Biol. ;13(1):53-60.

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