Receptor protein tyrosine kinases (RPTKs) are transmembrane proteins that initiate mitogenic signaling pathways and promote cellular processes such as cell growth, size, adhesion, and migration in response to activation by growth factors. Following protein synthesis, RPTKs are post-translationally modified in the Golgi by N-linked glycosylation (Lane et al., 1985; Slieker et al., 1986), after which they are translocated to the plasma membrane. Here the receptors bind specific extracellular or cell associated ligands. Ligand binding stimulates receptor homo- and heterodimerization. This triggers activation of kinase activity and trans- phosphorylation of intracellular tyrosine residues. These phosphorylated tyrosine residues then act as binding sites for intracellular signal transduction proteins resulting in activation and the initiation of mitogen signaling pathways including PI3K/AKT/mTORC1. RPTK activation by growth factors also causes receptor internalization, which may occur via clathrin-mediated endocytosis or through a clathrin-independent mechanism, depending on the receptor. RPTK internalization is important for their activation as the affinity of some ligands for their receptor is high enough such that the RPTK signaling lifespan is extended following clathrin-mediated endocytosis.
1.2.1 ErbB/HER activation and signal transduction – EGFR
Over 20 different ligands have been described for ErbB RPTKs, including epidermal growth factor (EGF). Ligand binding and dimerization stimulate ErbB tyrosine kinase activity
ligands have been identified to date (reviewed in (Yarden and Sliwkowski, 2001)), it is generally accepted that ErbB2 is activated by heterodimerization with other ligand activated ErbB family members. Ligand binding to EGFR induces the trans-phosphorylation of at least 7 carboxyl- terminal, intracellular tyrosine residues (Fernandes et al., 2001). EGFR tyrosine phosphorylation serves as a binding site for adaptor proteins through their Src homology (SH2) domains, causing the activation of downstream signaling cascades. The adaptor protein Grb2 directly associates with EGFR following Y1068 autophosphorylation, activating both the PI3K and Ras/MAPK signaling pathways (Rojas et al., 1996). Shc binds EGFR following Y1148 and Y1173 autophosphorylation, activating MAPK signaling, and EGFR autophosphorylation at Y992 promotes PLCγ binding and signal transduction (Emlet et al., 1997; Zwick et al., 1999). Tyrosine phosphorylation also mediates EGFR stability; Y1045 phosphorylation causes c-Cbl binding and subsequent targeting of EGFR for ubiquitin mediated degradation (Levkowitz et al., 1999) (Fig 1.4). EGFR dimerization-dependent internalization through clathrin-mediated endocytosis is associated with receptor activation and can either lead to receptor recycling to the cell surface or receptor degradation (Wang et al., 2005). Robust EGFR activation is only obtained upon receptor internalization, although clathrin mediated internalization is biased towards receptor recycling rather than degradation, sustaining receptor activation (Sigismund et al., 2008).
1.2.2 IR/ IGFR1-R activation and signal transduction
Insulin receptors (IR) and insulin-like growth factor receptors (IGFR) are also activated upon dimerization followed by ligand binding. While insulin and IGF-I/IGF-II are the
physiological ligands for IR and IGFR, respectively, IR/IGFR heterodimers bind both insulin and IGF with reduced affinity for insulin and with comparable affinity for IGF (reviewed in
Figure 1.4. EGFR Activation and Regulation. EGFR is activated by ligand binding and
dimerization, which induces the autophosphorylation of several carboxyl-terminal tyrosine residues. Autophosphorylation promote the association of adaptor proteins via SH2 domains with the receptor and activate downstream signaling cascades. SHC associates with EGFR following phosphorylation of Y1148 and Y1173, activating MAPK signaling; Grb2 binds after EGFR Y1068 phosphorylation and causes the activation of MAPK and PI3K signaling; EGFR Y992 phosphorylation promotes PLCγ association and the activation of PKC; C-Src binds EGFR following Y845 phosphorylation, which activates FAK signaling. EGFR stability is regulated by the phosphorylation of Y1045, promoting the association with c-Cbl and the targeted EGFR degradation. EGFR activation is abrogated by protein tyrosine phosphatases including PTP1B and SHP2.
(Belfiore et al., 2009)). Following ligand binding, IRβ and IGFR1β-R are autophosphorylated in a conserved kinase activation loop at residues Y1146/1150/1151 or Y1131/1135/1136,
respectively (Hernandez-Sanchez et al., 1995; White et al., 1988) (Fig 1.5). Autophosphorylation causes full receptor activation and initiates downstream effector signaling. Similar to ErbB RPTKs, autophosphorylation induces clathrin mediated receptor internalization. There is also evidence for caveolin dependent internalization. IR/IGFR1-R phosphorylation is regulated by the adaptor proteins Grb10 and Grb14. Grb10 and Grb14 may maintain IR/IGFR1-R activation by protecting the receptors from dephosphorylation by tyrosine phosphatases. Alternatively, Grb10 regulates the stability of IGFR1-R by targeting it for NEDD4 mediated ubiquitination and proteasomal degradation (Vecchione et al., 2003). Grb10 is also a downstream target of
mTORC1. mTORC1 mediated Grb10 phosphorylation increases its stability, causing feedback inhibition with the lipid kinase phosphoinositide 3-kinase (PI3K) , AKT, and MAPK/ERK signaling pathways. As a result, Grb10 may also participate in IR/IGFR1-R negative feedback inhibition (Hsu et al., 2011; Yu et al., 2011). The major downstream effectors of IR and IGFR1- R signaling are the PI3K and Ras/MAPK signaling pathways. PI3K and Ras/MAPK activation is dependent on IR/ IGFR1-R mediated phosphorylation of insulin receptor substrates 1 and 2 (IRS) at YxxM motifs. Phosphorylation of IRS1/2 at this motif allows for recognition and association of adaptor proteins Grb2 and Shc, or class Ia PI3K regulatory subunits, which in turn activate PI3K and Ras/MAPK signaling pathways. Protein phosphatases also regulate IR and IGFR1-R activity, including SHP2/PTPN11, which associates with, dephosphorylates, and inactivates IRS1/2 through its SH2 domain (Goldstein et al., 2000). The protein tyrosine
Figure 1.5. IR Activation and Regulation. IR is activated by ligand binding and dimerization,
which induces the autophosphorylation of the carboxyl-terminus at several tyrosine residues. IR is phosphorylated at Y1146/50/51, with the phosphorylation of analogous residues occurring for IGFR. Phosphorylation promotes the association of IR and insulin receptor substrate 1 (IRS1). This predominantly results in the activation of PI3K signaling, although other adaptor proteins including Grb2 and SHC can associate indirectly with the receptor through binding IRS-1 and activate downstream signaling networks. IR activation is abrogated by protein tyrosine
1.3 mTOR SIGNALING: REGULATION AND THE ROLE OF mTORC1