EGF Pathway

EGF (Epidermal Growth Factor) is a small 53 amino acid residue protein that is involved in normal cell growth, oncogenesis, and wound healing. This protein shows both strong sequential and functional homology with hTGF-Alpha (human type-Alpha Transforming Growth Factor), which is a competitor for EGF receptor sites. EGF binds to a specific high-affinity, low-capacity receptor on the surface of responsive cells known as EGFR (Epidermal growth factor receptor). EGFR is a member of the ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) family receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB1), Her2/c-neu (ErbB2), Her3 (ErbB3) and Her4 (ErbB4). In response to toxic environmental stimuli, such as ultraviolet irradiation, or to receptor occupation by EGF, the EGFR forms Homo- or Heterodimers with other family members. Binding of EGF to the extracellular domain of EGFR leads to receptor dimerization, activation of the intrinsic PTK (Protein Tyrosine Kinase) activity, tyrosine autophosphorylation, and recruitment of various signaling proteins to these autophosphorylation sites located primarily in the C-terminal tail of the receptor. Tyrosine phosphorylation of the EGFR leads to the recruitment of diverse signaling proteins, including the Adaptor proteins GRB2 (Growth Factor Receptor-Bound Protein-2) and Nck (Nck Adaptor Protein), PLC-Gamma (Phospholipase-C-Gamma), SHC (Src Homology-2 Domain Containing Transforming Protein), STATs (Signal Transducer and Activator of Transcription), and several other proteins and molecules. The evolutionary conservation of all the components of the EGFR signaling pathway in Nematode, Fruit fly, Mouse, and Man underscores the biological significance of this signaling pathway. Furthermore, aberrant regulation of the activity or action of EGFR and other members of the RTK family have been implicated in multiple cancers, including those of brain, lung, mammary gland, and ovary (Ref.1 & 2).

GRB2 is an essential component of EGFR signaling to Ras. The SH2 (Src Homology-2) domain of GRB2 can bind directly to phosphotyrosines 1068 and 1086 of the activated EGFR or indirectly through the tyrosine-phosphorylated adaptor protein SHC. The SH3 domains of GRB2 are constitutively associated with SOS (Son of Sevenless), an exchange factor of Ras GTPase. Besides interaction with SOS, GRB2 SH3 domains are capable of association with several proteins, including Dynamin and Cbl (c-Cbl, Cbl-b, and Cbl-3), both implicated in the regulation of EGFR endocytosis. Binding of the GRB2 and SOS complex to the EGFR places SOS in proximity to Ras, thus leading to GTP-loading of Ras and subsequent activation of Ras effectors, such as Raf kinases and PI3K (Phosphatidylinositol 3-Kinase). Raf initiates a cascade of phosphorylation events including the phosphorylation and activation of the MEKs (MAPK/ERK Kinases) and ERKs (Extracellular Signal-Regulated Kinases). EGF stimulation of PI3K may also be mediated by the docking protein GAB1 (GRB2-Associated Binder-1). GAB1 is a docking protein that recruits PI3K and other effector proteins in response to the activation of many RTKs (Receptor Tyrosine Kinases). PI3K once activated, phosphorylates membrane bound PIP2 (Phosphatidylinositol (4,5)-bisphosphate) to generate PIP3 (Phosphatidylinositol-3,4,5-trisphosphate). The binding of PIP3 to the PH domain anchors Akt to the plasma membrane and allows its phosphorylation and activation by PDK1 (Phosphoinositide-Dependent Kinase-1). Akt then phosphorylates several substrates and take part in cell survival (Ref.3 & 4). One of the prominent enzymes activated by EGFR is the G1 isoform of PLC (Phospholipase-C-Gamma1). This enzyme, which has two SH2 domains, catalyses the hydrolysis of PIP2, generating the second messengers DAG (1,2-Diacylglycerol) and IP3 (Inositol Trisphosphate). IP3 diffuses through the cytosol and releases stored Ca2+ (Calcium) ions from the ER (Endoplasmic Reticulum). DAG is the physiological activator of PKC (Protein Kinase-C), which in turn lead to phosphorylation of various substrate proteins that are involved in an array of cellular events. PKC also leads to the activation of IKKs (I-KappaB-Kinases), and finally nuclear factor NF-KappaB (Nuclear Factor-KappaB)-dependent transcription (Ref.5).

Dok2 (Docking Protein-2) also associates with the EGFR and become tyrosine phosphorylated in response to EGF stimulation. The recruitment of Dok2 to the EGFR, which is mediated through its PTB (Phosphotyrosine Binding) domain, results in attenuation of MAPK (Mitogen-Activated Protein Kinase) activation. Dok2's ability to attenuate EGF-driven MAPK activation is independent of its ability to recruit RasGAP, a known attenuator of MAPK activity, suggesting an alternate Dok2-mediated pathway. Dok2 associate with c-Src and with the SFK (Src Family Kinase)-inhibitory kinase, Csk. Dok2 associates constitutively with c-Src through an SH3-dependent interaction and that this association is essential to Dok2's ability to attenuate c-Src activity and diminish MAPK and Akt/PKB activity (Ref.6). One of the important signaling events activated by EGFR involves tyrosine phosphorylation of STAT. Stimulation of EGFR induces Tyrosine phosphorylation of STAT1 and STAT3 and initiates complex formation of STAT1 and STAT3 with JAK1 (Janus Kinase-1) and JAK2 (Janus Kinase-2). JAKs are essential to mediate interaction of EGFR with STAT1 and STAT3. Thereafter, the STATs translocate to the nucleus, where they are active in gene transcription. EGFR also activate STAT3 in a manner largely independent of JAKs but dependent upon the activation of Src kinases. c-Src is activated by EGF-induced EGF receptor activation. Activated c-Src phosphorylates EGF receptor on tyrosine 845 that plays an important role in tyrosine phosphorylation and activation of STAT proteins (Ref.2 & 7).

E3B1 (EPS8 Binding Protein)/ABI1 (Abl-Interactor-1) is a protein involved in the EGFR signalling pathway. E3B1/ABI1 interacts with EPS8 (Epidermal Growth Factor Receptor Pathway Substrate-8), a protein which is phosphorylated in fibroblasts in response to EGF. Remarkably, E3B1/ABI1 is a negative regulator of the cellular response mediated by growth factor receptors. EPS8 binds, through its SH3 domain, to either E3B1 or RNTRE (Related to the N-terminus of tre). EPS8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3B1 and SOS1. SOS1, a bifunctional GEF (Guanine nucleotide Exchange Factor), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with EPS8 and E3B1. On the other hand, by entering in a complex with EPS8, RNTRE acts on Rab5A (Rab5A, member RAS oncogene family) and inhibits internalization of the EGFR (Ref.8). Cbl is another major substrate for the EGFR that associates with this receptor upon EGF stimulation, and forms complexes with several signaling proteins that play key roles in EGF-mediated cell growth. Since Nck is known to bind to activated EGFR through its SH2 domain and to Cbl via its SH3 domains, Nck represented another potential adaptor that could mediate Cbl-EGFR association. The SH3-SH3-SH3-SH2 adapter protein Nck links EGFR to downstream signaling pathways, among which p21CDC42 (Cell Division Cycle-42) /Rac-activated kinase cascade, SOS-activated Ras signaling and the human WASP (Wiskott-Aldrich Syndrome protein)-mediated actin cytoskeleton changes, have been implicated. Nck also activates PAK1 (p21/CDC42/Rac1-Activated Kinase-1). The association between Nck and PAK1 has been shown to occur through the first N-terminal polyproline domain of PAK1 and an SH3 domain of Nck. PAK activated by Nck activates JNKs (c-Jun Kinases) via MEKK1 (MAP/ERK Kinase Kinase-1) and MKK4/7 (MAP Kinase Kinase-4/7) respectively. JNKs once activated enter the nucleus and causes phosphorylation of transcription factors such as c-Fos and c-Jun. EGFR also activates Vav (Oncogene Vav). Vav proteins are guanine nucleotide exchange factors for Rho family GTPases which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Each Vav protein co precipitated with activated EGF and multiple phosphorylated tyrosine residues on the EGFR were able to mediate Vav2 tyrosine phosphorylation. Vav activates Rho or Rac. The Rho family GTPase Rac initiates a cascade leading to JNK/SAPK, presumably by binding and activating the protein kinase, a kinase that phosphorylates and promotes activation of MEKK1. Rho, activated by Vav, is responsible for actin cytoskeletal rearrangement (Ref.9 & 10).

EGFR also activates several other proteins including FAK (Focal Adhesion Kinase), Paxillin, Caveolin, E-Cadherin and Ctnn-Beta (Catenin-Beta). FAK1 take part in cell motility, whereas Caveolin, Cadherin and Ctnn-beta are involved ibn Cytoskeletal Regulation. Activation of Ctnn-Beta occurs via Muc1 (Mucin-1). Activated EGFR phosphorylates the Muc1 cytoplasmic tail on tyrosine at a YEKV motif that functions as a binding site for the c-Src SH2 domain. EGFR also increases binding of Muc1 and Ctnn-Beta. Thus, EGFR regulates interactions of Muc1 with c-Src and Ctnn-beta. EGFR is overexpressed or activated by autocrine growth factors in many types of tumors, including breast, thyroid, ovarian, colon, head and neck, and brain. Furthermore, EGFR overexpression has been linked to a poor prognosis in breast cancer and may promote proliferation, migration, invasion, and cell survival as well as inhibition of apoptosis. EGFR-targeted therapies offer the promise of better treatment for many types of solid tumors, including non-small cell lung cancer. Anti-EGFR agents include mAbs (Monoclonal Antibodies) targeting the EGFR extracellular receptor domain and small-molecule TKIs (Tyrosine Kinase Iinhibitors) targeting the EGFR intracellular kinase domain. Both mAbs and TKIs have demonstrated encouraging results as monotherapies and in combination with chemotherapy and radiotherapy (Ref.11 & 12).