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Signal Transduction Through the Epidermal Growth Factor Receptor
Produced by Steven Pelech, Ph.D. Professor, Department of Medicine, University of British Columbia President & C.S.O., Kinexus Bioinformatics Corp. Copyright 2012 Steven Pelech Produced by Steven Pelech, Ph.D. Professor, Department of Medicine, University of British Columbia President & Chief Scientific Officer, Kinexus Bioinformatics Corporation. Copyright 2012 Steven Pelech
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EGFR EGF EGFR EGF 1. Epidermal growth factor (EGF) binds to the extracellular domain of the EGF receptor monomer.
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EGFR EGF EGFR EGF 2. Upon binding of EGF to its receptor, it inducesthe dimerization of the receptor in a head to tail conformation.
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EGFR EGF EGFR EGFR 3. The dimerization of the EGF receptor allows the juxaposition of its intracellular catalytic domains.
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EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 EGF 4. The ligand occupied EGF receptor kinase catalytic domain catalyzes the phosphorylation of tyrosine residues located on the other EGF receptor chain. Beside those phospho-tyrosine sites shown above, mass spectrometry studies have revealed the additional tyrosine phosphorylation sites including Tyr-270, Tyr-316, Tyr-978, Tyr-998, Tyr-1081, and Tyr It is not clear that these are catalyzed directly by the EGF receptor, nor has the functional roles of these phospho-sites been determined.
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EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 EGF PLCg1 SH2 Vav2
5. The cross phosphorylation of the EGF receptor chains activates docking sites for a variety of downstream signalling proteins. Many of these proteins are cytoplasmic and feature SH2 domains, which have a specific affinity for tyrosine phosphorylation sites on the EGF receptor. These SH2 domain mediate interaction only when these tyrosine phosphorylation sites are phosphorylated. A notable exception appears to be the p85 subunit of the phosphatidylinositol 3-kinase (PI3K), which does not appear to interact directly with the EGF receptor. It is unclear whether the Tyr-869 site autophosphorylates, but it is phosphorylated by the Src protein-tyrosine kinase and this increases binding of mitochondrial protein cytochrome c oxidase subunit II to the EGF receptor. The Tyr-869 site is located within the sequence LGAEEKEYHAEGGKV. PLCg1 SH2 Vav2 PTPN11 Ras-GAP Shc1 SOS GrbB2 PTPN6 Cbl Cbl-N
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EGFR EGF Y869 EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 Y1016
PLCg1 SH2 Vav2 SH2 PTPN11 SH2 Ras-GAP SH2 SOS GrbB2 SH2 6. The Tyr-1016 site has been found to bind several different proteins, including phospholipase C-gamma1 (PLCg1, which cleaves phosphatidylinositol-4,5-bisphosphate to release inositol-1,4,5-trisphosphate and diacylglycerol), the Vav2 GTP for GDP exchange factor for the small G protein Rac, the cytoplasmic protein-tyrosine phosphatase PTPN11 (also known as SHP2, PTP1D and PTP2C), the Ras GTPase activating protein GAP, and the adaptor protein GrbB2. This site is located within the sequence DVVDADEYLIPQQGF. Shc1 SH2 PTPN6 SH2 Cbl Cbl-N
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EGFR EGF Y869 EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 Y1016
PLCg1 SH2 Vav2 SH2 PTPN11 SH2 Ras-GAP SH2 SOS GrbB2 SH2 Cbl Cbl-N 7. The Tyr-1069 site binds the E3 ubiquitin protein ligase Cbl at its N-terminus. This site is located within the sequence EDSFLQRYSSDPTGA. Binding of Cbl induces the degradation of the EGF receptor to terminate its signalling. Shc1 SH2 PTPN6 SH2
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EGFR EGF Y869 EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 Y1016
PLCg1 SH2 Vav2 SH2 PTPN11 SH2 Ras-GAP SH2 SOS GrbB2 SH2 PTPN6 SH2 Cbl Cbl-N 8. The Tyr-1092 site has been found to bind several different proteins, including phospholipase C-gamma1 (PLCg1, which cleaves phosphatidylinositol-4,5-bisphosphate to release inositol-1,4,5-trisphosphate and diacylglycerol), the Ras GTPase activating protein GAP, the adaptor protein GrbB2, and the cytoplasmic protein-tyrosine phosphatase PTPN6 (also known as SHP1, and PTP1C). This site is located within the sequence TFLPVPEYINQSVPK. Shc1 SH2
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EGFR EGF Y869 EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 Y1016
PLCg1 SH2 Vav2 SH2 PTPN11 SH2 Ras-GAP SH2 SOS GrbB2 SH2 PTPN6 SH2 Cbl Cbl-N 9. The Tyr-1110 site has been found to bind the Ras GTPase activating protein GAP. This site is located within the sequence GSVQNPVYHNQPLNP. The recruitment of Ras-GAP is thought to lead to subsequent downregulation of Ras signalling. Shc1 SH2
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EGFR EGF Y869 EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 Y1016
PLCg1 SH2 Vav2 SH2 PTPN11 SH2 Ras-GAP SH2 SOS GrbB2 SH2 PTPN6 SH2 Cbl Cbl-N 10. The Tyr site has been found to bind several different proteins, including the Vav2 GTP for GDP exchange factor for the small G protein Rac, the cytoplasmic protein-tyrosine phosphatase PTPN11 (also known as SHP2, PTP1D and PTP2C), the Ras GTPase activating protein GAP, and the adaptor protein GrbB2. This site is located within the sequence ISLDNPDYQQDFFPK. Shc1 SH2
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EGFR EGF Y869 EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 Y1016
PLCg1 SH2 Vav2 PTPN11 Ras-GAP Shc1 SOS GrbB2 PTPN6 Cbl Cbl-N 11. The Tyr-1197 site has been found to bind several different proteins, including phospholipase C-gamma1 (PLCg1, which cleaves phosphatidylinositol-4,5-bisphosphate to release inositol-1,4,5-trisphosphate and diacylglycerol), the Ras GTPase activating protein GAP, the adaptor protein Shc1, and the cytoplasmic protein-tyrosine phosphatase PTPN6 (also known as SHP1, and PTP1C). This site is located within the sequence STAENAEYLRVAPQS.
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EGFR EGF Y869 EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 Y1016
SH2 SH2 SH2 SH2 SH2 SH2 SH2 Cbl-N Y771 Y142 Y542 Ras-GAP Y349 Y209 Y654 Y371 12. The binding of many of these proteins to the EGF receptor permits their subsequent phosphorylation by the EGF receptor. Solid green lines with arrows indicate direct phosphorylation and apparent activation of substrates that bind to the EGF receptor by the catalytic domain of the receptor. Dotted green arrows indicate suspected phosphorylation by the EGF receptor, but these are not yet firmly established. Red lines indicate phosphorylation which appears to be inhibitory. Note that PTPN11 and Shc1 both can bind to Grb2 when they become tyrosine-phosphorylated. Y775 Y159 S576 Y350 GrbB2 Y350 Y552 Y783 Y172 Y580 Y427 PTPN6 Y674 Y1253 Vav2 S591 Shc1 SOS Y700 PLCg1 PTPN11 Y731 Y774 Cbl
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EGFR EGF Y869 EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 Y1016
SH2 SH2 SH2 SH2 SH2 SH2 SH2 Cbl-N Y771 Y142 Y542 Ras-GAP Y349 Y209 Y654 Y371 13. Phosphorylation by the EGF receptor can regulate the signalling from most of these effector proteins. Y775 Y159 S576 Y350 GrbB2 Y350 Y552 Y783 Y172 Y580 Y427 PTPN6 Y674 Y1253 Vav2 S591 Shc1 SOS Y700 PLCg1 PTPN11 Y731 Y774 Cbl
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EGFR EGF EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 EGFR PLCg1 Vav2
PTPN11 Ras-GAP Shc1 GrbB2 PTPN6 Cbl 14. The following slides provide a more detailed account of the downstream signalling from each of these proteins that directly interact with the EGF receptor. SOS
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PIP2 DAG + IP3 CALCISOMES Ca2+ EGF T678 Y869 Y1016 Y1069 EGFR Y1092
PLCg1 EGF Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 T678 PKCa PKCb S1070 S1071 PIP DAG + IP3 Ca2+ CALCISOMES CaMK2 15. The EGF receptor catalyzes the phosphorylation of bound phospholipase C-gamma1 (PLCg1) at Tyr-771, Tyr-775, Tyr-783 and Tyr With the exception of Tyr-783. all of the other tyrosine phosphorylation events appear to lead to increased catalytic activity of PLCg1 to cleave phosphatidylinositol-4,5-bisphosphate in the plasma membrane to release inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Phosphorylation of the Tyr-783 sites appears to induce the binding of Grb2, which inhibits the catalytic activity of PLCg1. The released IP3 travels to calcisome vesicles in the cytoplasm, where calcium is stored. IP3 binds to receptors on the surface to calcisomes to induce the release of calcium into the cytoplasm. The released calcium can bind to calmodulin subunits of the multi-subunit calmodulin-dependent kinase 2 (CaMK2) to permit the autophosphorylation (at Thr-286) and activation of the catalytic activity of this kinase. CaMK2 is one of the protein kinases that has been implicated in the phosphorylation of Ser-1070 and Ser-1071 to induce the internalization of the EGF receptor and its desensitization. The released DAG and the calcium are activators of the alpha, beta and gamma isoforms of protein kinase C (PKC). PKC-alpha (and probably the beta and gamma isoforms) can phosphorylate Thr-678 on the EGF receptor and inhibits its calmodulin binding activity and catalytic kinase activity.
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EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 T678 S1070 S1071 EGF T693 Vav2 16. The EGF receptor catalyzes the phosphorylation of the bound guanine nucleotide exchange protein Vav, which activates the small Rho family G protein Rac. While Tyr-142, Tyr-159 and Tyr-172 are all directly phosphorylated by the EGF receptor, this does not appear to directly activate its guanine nucleotide exchange. However, it would permit the recruitment of Rac to the plasma membrane, where it could activate the p21-activated kinase-alpha (PAKa). Autophosphorylation of PAKa at Thr-423 facilitates the stimulation of its catalytic activity toward other targets, including a MAP kinase kinase kinase that phosphorylates and activates the MEK3 protein kinase. Phosphorylation of MEK3 at Ser-189 and Thr-193 in the activation loop of this kinase stimulates its catalytic activity towards p38 MAP kinase isoforms, such as p38-alpha at Thr-179 and Tyr-181, which activates this kinase. P38-alpha has been shown to phosphorylate Thr-693 of the EGF receptor, which triggers internalization and inhibition of the EGF receptor. T193 MEK3 S189 p38a Y181 T179 Rac T423 PAKa MEKK?
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EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 T693 T678 S1070 S1071 Shc1 PTPN11 SOS GrbB2 EGF Ras-GAP Src S695 S1026 S1190 ? S499 S497 PKCa PKCb Y341 Y340 S339 S338 PAKa 17. The EGF receptor can trigger the recruitment of the Grb2-SOS complex either directly by binding to the receptor or indirectly via the protein-tyrosine phosphatase PTPN11 or the adapter protein Shc1 upon tyrosine phosphorylation of these proteins. Shc1 phosphorylation at Tyr-349 and Tyr-350 by Src (and likely by the EGF receptor) and Tyr-427 by Src and the EGF receptor permits the binding of Grb2. Grb2 can also be directly phosphorylated by the EGF receptor, and this appears to inhibit its interaction with SOS. Normally, Grb2 is associated with SOS. SOS is a guanine nucleotide exchange protein for members of the Ras small G protein family (H-, K- and N-Ras). GTP-bound Ras, which is located in the plasma membrane, is able to permit the recruitment of the Raf1 protein kinase to the plasma membrane, where it is phosphorylated and activated by other several other protein kinases. These include PAKa, PKC-alpha and beta, and Src. Activated Raf1 phosphorylates and activates the Mek1 protein kinase (and its isoform Mek2). Phosphorylation of Mek1 at Ser-218 and Ser-222 in the activation loop of this kinase stimulates its catalytic activity towards the Thr-184 and Tyr-186 activation sites of MAP kinase Erk2 (and Thr-202 and Tyr-204 activation sites of its related isoform Erk1). pErk2 (and Erk1) has been shown to phosphorylate Thr-693 of the EGF receptor, which triggers internalization and inhibition of the EGF receptor. PKC-alpha (and probably the beta and gamma isoforms) can phosphorylate Thr-678 on the EGF receptor and inhibits its calmodulin binding activity and catalytic kinase activity. The Ser-695, Ser-1026, Ser-S1070, Ser-1071 and S1190 can also be phosphorylated in response to EGF via the activation of as yet uncharacterized protein-serine kinases. These phosphorylation lead to inhibition of the EGF receptor signalling. CaMK2 is one of the protein kinases that has been implicated in the phosphorylation of Ser-1070 and Ser-1071 to induce the internalization of the EGF receptor and its desensitization. Ser-695 phosphorylation causes inhibition and internalization of the EGF receptor.Ser-695 and Ser-1026 phosphorylation also causes inhibition of the EGF receptor, and Ser-695 and Ser-1190 phosphorylation cause its internalization. Ras S222 Mek1 S218 Erk2 Y186 T184 Raf1
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EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 T693 T678 S1070 S695 S1026 S1071 S1190 EGF PKCa PKCb Src PTPN11 PTPN6 18. At least two SH2 domain-containing protein-tyrosine phosphatases, PTPN6 and PTPN11, associate with the tyrosine phosphorylated EGF receptor.
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PKCa PKCb Src PTPN6 Y536 Y654 PTPN11 Y580 S591 S576 Y542 EGFR Y1016
JAK2 Y813 Stat1 SH2 Y701 Stat3 Y705 S727 19. PTPN6 is known to be phosphorylated at Tyr-536 by Src and the insulin receptor (and possibly by the EGF receptor). Src and the related protein-tyrosine kinase Lck can also phosphorylate Tyr-654. This appears to regulate binding of 3BP2 and the PI3-kinase. PTPN11 is known to be phosphorylated at Tyr-542 and Tyr-580 by the PDGF receptor-beta (and possibly by the EGF receptor). Phosphorylation at these sites stimulates PTPN11 interaction with Grb2. The Ser-576 and Ser-591 can by phosphorylated by the alpha, beta and eta isoforms of PKC, but the functional effect is unclear. PTPN11 is likely to dephosphorylate several of the tyrosine phosphorylation sites in the EGF receptor, and this has been clearly established for Tyr-1016. JAK2 has been shown to associate with the EGF receptor, and it can be directly phosphorylated at Tyr-813 by the EGF and PDGF receptors. This phosphorylation increases the association of JAK2 with SH2-B-beta. JAK2 may also phosphorylate the EGF receptor. JAK1 and JAK2 activation by EGF has been correlated with phosphorylation of the JAK substrates Stat1 at Tyr-701 and Stat3 at Tyr These phosphorylation events induce the homodimerization of these Stat proteins through internal SH2-phospho-tyrosine interactions, which permits the activation of these transcription factors. The Stat3 Ser-727 site is also phosphorylated in response to EGF, and can be directly phosphorylated by the Erk1, Erk2 and p38-alpha MAP kinases, as well as by CDK1, GNA14 and NOR1.
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EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 T693 T678 S1070 S695 S1026 S1071 S1190 EGF Src Cbl 20. Cbl is an E3 ubiquitin protein ligase that binds to the EGF receptor (and other growth factor receptors such as the PDGF receptor) to terminate its signalling by inducing its ubiquitination and degradation.
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Ubiquitination + Degradation by 20S Proteosome
EGFR Y1016 Y1069 Y1092 Y1110 Y1172 Y1197 Y869 T693 T678 S1070 S695 S1026 S1071 S1190 EGF Cbl Cbl-N Y674 Y700 Y552 Y774 Y731 Y371 Ubiquitination + Degradation by 20S Proteosome Src 21. Cbl is phosphorylated by the EGF receptor at the Tyr-371, Tyr-552 and Tyr-674 sites, and possibly at the Tyr-700, Tyr-731 and Tyr-774 sites. Phosphorylation of the Tyr-371 site by the EGF receptor and the insulin receptor has been shown to induce degradation of these receptors and the binding of Crk to Cbl. The Tyr-700 site can be phosphorylated by the insulin receptor for its degradation and promoting the assocation of Crk and Vav1. The Tyr-731 can be phosphorylated by Src to induce association and activation of PI3-kinase and its degradation. The Tyr-774 site can be phosphorylated by both the insulin receptor and Src to induce association with Crk and protein degradation. The ubiquitinated proteins are recognized for degradation by the 20S protetosome.
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