Malak Benkhadra BMB507 Fall 2017 Honor code: Abstract. The mammalian target of rapamycin (mTOR), a serine threonine kinase belongs to the phosphoinositide-3-kinase (PI3K). The PI3K family plays many roles in important processes such as regulation for cell growth, metabolism, glucose regulation, proliferation, aging, and apoptosis. mTOR kinase is formed of two complexes, mTOR complex 1(mTORC1) and mTOR complex 2 (mTORC2).
mTORC1 responds to growth factors, nutrients, and cellular energy. The best-characterized mTORC1 substrates are the initiation factor 4E (eIF4E) binding proteinl and the ribosomal S6 kinase (S6K1). mTORC2 signaling is less known, however recent literatures states that the function of mTORC2 maybe related to directly phosphorylating AKT, a serine threonine specific protein kinase, which play an important role in many cellular processes. Now, there are many attempts to better understand the mechanism of mTOR kinase in order to synthesize specific inhibitors that target the ATP binding site leading to a diminish feedback of activation. This paper will highlight the different structural and biological aspects of mTOR as well as its signaling networks, which promotes biochemical signaling, cell growth, and cell progression. I. Introduction.
Protein kinases are a large family of enzymes that catalyze the transfer of phosphate from ATP to serine, threonine, and tyrosine residues of their substrate proteins. Protein kinases are found in all eukaryotes from yeast to mammals. They are involved in many aspect of cell as they play a critical role in signaling and other major cellular processes. While each specific kinase is thought to have a specific function, there are many conserved domains among kinases regarding their structures and catalytic mechanisms. The Phosphoinositide 3-kinases (PI3Ks) related protein kinases (PIKKs) are a family of protein kinases with a large range of important cellular functions. PI3Ks phosphorylate the inositol ring on the 3 position, which creates a docking site for proteins (Okkenhaug et al.
, 2013).. There are eight catalytic PI3K subunits that are divided into three classes based on the sequence alignment and domain structures (Fig.1a,b). The class III PI3K is the oldest PI3K and is the only one found in yeast and plants with a Vps34 domain structure that phosphorylates phosphatidylinositol to generate PI3P (Okkenhaug et al.
, 2013). The class II PI3Ks with a CII domain structure are localized in endosomes, but their function is still not well understood. The class I PI3Ks are heterodimeric proteins with a p110 domain structure. The class I PI3K subunits are further subdivided into class IA and IB (Okkenhaug et al., 2013).
the class IA subunits are associated with a SH2-containing regulatory subunits of PIP3. PIP3 acts as a membrane tether for a subset of proteins with one or more pleckstrin homology (PH) domains. PH domains need to have enough affinity for the PIP3 in order for it to be selectively regulated by class I PI3Ks. In mammals, about 40 of the 200+ proteins with PH domains can be controlled by PIP3 (Okkenhaug et al., 2013). However most of the focus is contributed to the regulation of the Akt pathway and its role in controlling the activation of the mammalian target of rapamycin (mTOR). mTOR is a serine/threonine kinase in the PI3K-related kinase (PIKK).
It is a central regulator of cellular metabolism, growth and survival in response to hormones, growth factors, nutrients, energy, and stress signals (Saxton et al., 2017). mTOR directly or indirectly regulates the phosphorylation of many proteins. It functions as part of two structurally and functionally distinct signaling complexes mTOR complex 1 (mTORC1) and mTOR complex2 (mTORC2). Activated mTORC1 upregulates protein synthesis and is mainly involved in cell growth (Fig.
2.a). It is defined by its three core components: mTOR, Raptor (regulator associated with mTOR), and mLST8 (Saxton et al., 2017). Raptor facilitates substrate recruitment to mTORC1 while the mLST8 associate with the catalytic domain of mTORC1, which stabilize the kinase activation loop that is essential for mTORC1 function (Fig. 2b).
mTORC2 may regulate other cellular processes including the organization of the cytoskeleton (Saxton et al., 2017). It plays a critical role in the phosphorylation of AKT1, a pro-survival effector of PI3K (Fig. 2.a). mTOR2 also contains mTOR and mSLT8, but instead of Raptor mTORC2 contains Rictor, a rapamycin insensitive companion of mTOR (Fig.
2.c). · Overall structure of mTOR kinase. The mTOR protein is a 289-KDa that belongs to the PI3K-related kinase family and is conserved throughout evolution with a kinase domain similar to the PI3Ks (Baretic et al.
, 2014). The conserved N-terminal of the mTOR kinase domain, long helical repeats, is shared among all PIKKs. Recently, a crystal structure of the mTOR kinase domain in complex with mST8 has been resolved at 3.2 resolution (Fig.3).
The structure shows the two-lobe catalytic core found in both mTORC1 and mTORC2. It also shows the FRB (FK506-rapamycin binding) domain, the FATC (FRAPP, ATM, TOR at C-terminus) domain, the LBE (LST8 binding element), and the KAL (activation loop helix) are all PIKK specific features (Baretic et al., 2014). Also, the crystal structure (Fig.3) shows a potion of he N-teminal helical repeats with the FAT domain.
The interaction between the Kinase domain (KD) and the FAT domain is established through hydrogen bonds, which is thought to be important for the kinase domain structure and activity, and are common features of all PIKKs (Yang et al., 2013). There are three distinct clusters of activating mutation located within the kinase domain and in the interface between the kinase domain and the FAT domain (Baretic et al., 2014). Mutations in this area are believed to make the end of the catalytic cleft less protected by either weakening the interaction between the helices or decreasing the kinase domain interactions. This allows mTOR to become more active toward the physiological substrates 4E-BP1 and S6K1 as well as increases its kinase activity by having more access to the catalytic site (Baretic et al.
, 2014). One side of the activation loop packs with the k9b insertion, and the other side packs with FATC (Fig.2b). The FATC’s N-terminal half forms a helix (kthat is present in the PI3K structures, but its C-terminal half is absent from the PI3Ks. The FATC’s C-terminal forms three short helices that pack with the activation loop on one side and with the LBE on the other side (Fig.2b).
The FATC and activation loop sequences are conserved among the PIKKs, but not the LBE (Baretic et al., 2014). However, all PIKK family members contain an LBE-like insertion that may similarly pack with FATC. · Overview of mTOR signaling pathway.mTOR interacts with many proteins to form at least the two distinct multiprotein mTORC1 and mTORC2 . The mTOR complexes have differences in their sensitivities to rapamycin, in the upstream signals they integrate, in the substrates they regulate, and in the biological process they control (Saxton et al.
, 2017). . mTORC1 activity is controlled by the small GTPase Rheb. The GTPase-activating domain of Tuberin (Tsc2) increases the rate of hydrolysis of Rheb-bound GTP, rendering Rheb to the inactive GDP-bound form. Tsc2 is inhibited when Tsc2 phosphorylates it, which allow Akt to release Rheb from the inhibition and allows GTP-Rheb to activate mTORC1 (Fig. 4a).
When there is sufficient amino acid and ATP available in the cell, mTOR is activated (Saxton et al., 2017). However, when AMPK (plays a role in cellular homeostasis) is active it inhibits mTOR (Fig.4a). The activation of mTOR involves the assembly of proteins of the Rag GTPase family at the lysosome. The best-characterized substrates for mTORC1 are ribosomal S6 kinase (S6K) and the initiation factor 4E binding protein1 (4E-BP1). The phosphorylated form of S6K and 4E-BP1 promotes protein synthesis. S6K1phosphorylates and activates several substrates that promote mRNA translation initiation (Fig.
4b) (Saxton et al., 2017). mTORC1 also facilitates growth by promoting a shift in glucose metabolism from oxidative phosphorylation to glycolysis, which facilitates the incorporation of nutrients into new biomass (Fig.4b).
Furthermore, mTORC1 leads to increased flux through the oxidative pentose phosphate pathway (PPP), which use carbons from glucose to generate the NADPH and other intermediary metabolites needed for proliferation and cell growth (Saxton et al., 2017). In addition, mTORC1 also promotes growth by suppressing protein catabolism (Fig.
2a), precisely autophagy. When the cell is under starving conditions, mTORC1 phosphorylates ULK1, a kinase that drives autophagosome formation, which prevents its activation by AMPK (Fig4.b). mTORC1 can also negatively regulate class I PI3K signaling via different mechanisms, including phosphorylation of receptors (Saxton et al.
, 2017). While mTORC1 regulates cell growth and metabolism, mTORC2 instead controls proliferation and survival primarily by phosphorylating several members of the AGC (PKA/PKG/PKC) family protein kinases (Fig. 4c) (Saxton et al.
, 2017). Recently, it has been shown that mTORC2 can also phosphorylate different types of PKCs, which regulates different aspects of cytoskeletal remodeling and cell migration. Moreover, mTORC2 most important role is to phosphorylate and activate Akt. Activated Akt plays important roles in the cell such as cell survival, proliferation, and growth through the phosphorylation and inhibition of different substrates like FOXO and the mTORC1 inhibitor TSC2 (Fig.
1a). Even-though the signaling pathways that lead to mTORC2 activation are not well characterized, it is considered that mTORC2 kinase activity and AKT phosphorylation at Ser473 (Fig.4a) increases activity due to the growth factors (Saxton et al., 2017). With the growth factors simulation, AKT is phosphorylated at the cell membrane through the binding of ptdINS to its pleckstrin homology (PH) domain (Okkenhaug et al., 2013). Under these conditions, PDK1 is also recruited to the membrane through its PH domain and phosphorylate AKT at Ser308 (Fig.