7
1. PI3K/Akt/PDK1 pathway
Restoring the ability of carcinogenic cells to undergo apoptosis may provide much safer and effective treatment of cancer. Traditionally, cancer treatment has involved surgical removal of malignant tumors, followed by radiation and chemotherapy. The radiation and chemotherapy treatments that target rapidly dividing cells (i.e., tumor cells, epithelial cells, hair, etc.) are very toxic and can cause severe side effects.
Current research indicates that more effective treatment would result from the use of chemotherapeutic agents that bind and inactivate oncogenic cell-cycle control factors, so that the ratio of damage incurred by normal cells over tumor cells is dramatically decreased and side effects become much less severe.
The current list of known cancer genes includes 70 genes associated with germline mutations and 342 genes associated with somatic mutations. Generally speaking, however, mutations in two basic classes of genes proto-oncogenes and tumor suppressor genes are what lead to cancer.
Proto-oncogenes are a group of genes that cause normal cells to become cancerous when they are mutated1,2. Mutations in proto-oncogenes are typically dominant in nature, and the mutated version of a proto-oncogene is called an oncogene. Often, proto-oncogenes encode proteins that function to stimulate cell division, inhibit cell differentiation, and halt cell death. All of these processes are important for normal human development and for the maintenance of tissues and organs. Oncogenes, however, typically exhibit increased production of these proteins, thus leading to increased cell division, decreased cell differentiation, and inhibition of cell death;
taken together, these phenotypes define cancer cells. Thus, oncogenes are currently a major molecular target for anti-cancer drug design.
A majority of the oncogenic cell-cycle control factors belong to one of the several families of protein kinases, which are involved in a number of key cell survival, growth, and proliferation signal transduction pathways.
The cell’s ability to regulate normal cell-cycle behavior relies on its ability to detect and respond properly to extracellular chemical and physical stimuli.
8
Figure 1.0: Disruption of intracellular signalling by alterations in the cancer genome. A simplified signaling pathway highlights the known examples of bona fide oncogenes that are subjected to dysregulation by various mechanisms. It is clear that a signaling pathway can be disrupted at multiple points, and a variety of genomic and epigenomic alterations can contribute to this, ultimately leading to cancer.
The signal transduction pathways, which mediate the cell’s ability to respond to chemical and physical stimuli, can roughly be divided into two major types of protein kinases, the tyrosine protein kinases and the serine-threonine protein kinases.
Many of the serine-threonine protein kinases belong to one of two major families, the AGC family and the MAPK family. The AGC protein kinase family includes such members as the cyclic AMP-dependent protein kinase (PKA), Ca2+-activated protein kinase (PKC), phosphoinositide-dependent protein kinase-1 (PDK1), and protein kinase B (PKB or Akt), which are all known to be activated by small second- messenger molecules generated either by GPCR’s or RTK’s. The mitogen-activated protein kinase (MAPK) family includes such members as RAF and MEKK
9 (MAPKKK), MEK and MKK (MAPKK), and ERK and JNK/p38 (MAPK), which are all known to respond to the mitogen-activated G protein, RAS. In many cancers, oncogenic transformation has been found to correlate with increased activation of many of the protein kinases found in these two families, resulting from either an increased gene copy number or mutation to a constitutively active form. Hence, potent and selective inhibitors of protein kinases may be effective, in combination with other anticancer drugs, for the treatment of tumors.
Figure 1.1: Overview of cell signaling.
10
1.1. PI3K
During the past two decades it has been established that phosphoinositide 3- kinases (PI3Ks) play a central role in the control of metabolism, cell growth, proliferation, survival and migration, and membrane transport and secretion.
The discovery of PI3Ks by Lewis Cantley and colleagues began with their identification of a previously unknown phosphoinositide kinase associated with the polyoma middle T protein3. They observed unique substrate specificity and chromatographic properties of the products of the lipid kinase, leading to the discovery that this phosphoinositide kinase had the unprecedented ability to phosphorylate phosphoinositides on the 3' position of the inositol ring. Subsequently, Cantley and colleagues demonstrated that in vivo the enzyme prefers PtdIns(4,5)P2 as a substrate, producing the novel phosphoinositide PtdIns(3,4,5)P3.
Figure 1.2: The structure of a mammalian PI 3-kinase
1.1.1 Structure
Inositol-containing lipids consist of a glycerol backbone with fatty acids attached at positions 1 and 2, and an inositol 1-phosphate group at position 3. If this inositol ring carries no additional phosphates, this lipid is called phosphatidylinositol(PtdIns).
11 In cells, all free -OH groups of the inositol ring of PtdIns, apart from those at the 2' and 6' position, can be phosphorylated, in different combinations. A phosphorylated derivative of PtdIns is referred to as a phosphoinositide (PI).
PI3Ks phosphorylate the 3'-OH position of the inositol ring in inositol phospholipids, generating 3'-PIs. Inside cells, they produce three lipid products, namely PtdIns3P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3.4
Figure 1.3: Simplified representation of PtdIns and the point of action of PI3K
1.1.2. Classification
The phosphoinositol-3-kinase family is divided into three different classes:
Class I, Class II, and Class III. The classifications are based on primary structure, regulation, and in vitro lipid substrate specificity. Class I PI3Ks can be segregated into two groups: those able to associate with p85 will be directed to phosphorylated tyrosine motifs (class IA), while PI3Kγ interacts with trimeric G proteins and the p101 protein (class IB)5.
12
Figure 1.4: The phosphoinositide 3-kinase (PI3K) family and related proteins.
1.1.2.1. Class IA
PI3Ks are heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit. In mammals, there are three genes, PIK3CA, PIK3CB and PIK3CD, encoding p110 catalytic isoforms: p110α, p110β and p110δ, respectively.
While the expression of p110δ is largely restricted to the immune system, p110α and p110β are ubiquitously expressed.
The p110 catalytic isoforms are highly homologous and share five distinct domains:
an N-terminal p85-binding domain (p85BD) that interacts with the p85 regulatory subunit;
a Ras-binding domain (RasBD) that mediates activation by members of the Ras family of small GTPases;
a putative membrane-binding domain C2;
the helical domain;
the C-terminal kinase catalytic domain.
13
Figure 1.5: Ribbon diagram of PI3Kα (showing the four domains: RBD (magenta), C2 (blue), helical (green) and catalytic with N-lobe (red) and C-lobe (yellow). The N-terminal region preceding the RBD and the ordered portion between the RBD and C2 domain are white.
There are also three genes, PIK3R1, PIK3R2 and PIK3R3, encoding p85α (and its splicing variants p55α and p50 α), p85β and p55γ regulatory subunits, respectively, collectively called p85. These regulatory subunits share three core domains including a p110-binding domain (denoted as inter-SH2 or iSH2) flanked by two Src- homology 2 (SH2) domains (N-terminal nSH2 and C-terminal cSH2).
The two longer isoforms, p85α and p85β, have a Src-homology 3 (SH3) domain and a BCR homology (BH) domain located in their extended N-terminal regions. In the basal state, p85 binds to the N-terminus of the p110 subunit via its iSH2 domain, inhibiting its catalytic activity6.
1.1.2.2. Class IB
PI3K is a heterodimer composed of a catalytic subunit p110γ and a regulatory subunit p101. Two new regulatory subunits, p84 and p87PIKAP, have been recently described. p110γ is activated directly by GPCRs through interaction of its regulatory subunit with the Gβγ subunit of trimeric G proteins. p110γ is mainly expressed in leukocytes but is also found in the heart, pancreas, liver and skeletal muscles6.
14
Figure 1.6.: Subunit composition and mechanism of activation of class IA and IB isoforms of phosphoinositide 3-kinase (PI3K).
1.1.2.3. Class II
PI3Ks are monomers with only a single catalytic subunit.The first member of a new class of PI3Ks, characterized by a C-terminal C2 domain, was the Drosophila PI 3-kinase PI3K_68D7.
Members of this class are 170-210 kDa in size and phosphorylate exclusively PtdIns and PtdIns 4-P in vitro, although PI3-K-C2α/PI3K-IIK was claimed to phosphorylate PtdIns(4,5)P2 as well. Class II PI3Ks (PI3KII) has a divergent N-terminus followed by a Ras binding domain (RasBD), C2 domain, helical domain, and catalytic domain with PX and C2 domains at the C-termini.
Primary sequence information (e.g., the presence of a putative, N-terminal SH3 domain-binding domain), and the sensitivity to the PI3K inhibitor wortmannin, class II PI3K could be subdivided in three isoforms: PI3KC2α, PI3KC2β and PI3KC2γ.
PI3KC2α and PI3KC2β are expressed through the body, however expression of PI3KC2γ is limited to hepatocytes6.
The distinct feature of Class II PI3Ks is the C-terminal C2 domain. This domain lacks critical Asp residues to coordinate binding of Ca2+, which suggests class II PI3Ks bind lipids in a Ca2+-independent manner5.
15 1.1.2.4. Class III
PI3Ks produces only PtdIns(3,4,5)P3 from PI but are more similar to Class I in structure. This class consists of a single catalytic Vps34 subunit (homolog of the yeast vacuolar protein-sorting defective 34) and of a regulatory (Vps15/p150) subunits. Vps34 only produces PtdIns(3,4,5)P3, an important regulator of membrane trafficking, and has been shown to function as a nutrient-regulated lipid kinase mediating signaling through mTOR (mammalian target of mTOR), indicating a potential role in regulating cell growth.
Interestingly, it has also been implicated as an important regulator of autophagy, a cellular response to nutrient starvation6.
1.1.3. PI3K signal pathway
PI3K downstream signalling and resulting cell responses PI3K regulates cellular functions by recruiting PtdIns(3,4,5)P3-binding proteins to the plasma membrane Phosphoinositide-binding PH domains are found in numerous proteins, including the protein-serine/threonine kinases, phosphoinositide-dependent kinase- 1(PDK-1) and Akt/protein kinase B (PKB), both detrimental for the transforming effects of deregulated PI3K activity.
Figure 1.7: RPTK-induced PI3K signalling through PDK-1 and Akt.
16 1.1.4. Physiological role of PI3K
1.1.4.1.Immunology
The p110δ and p110γ isoforms differ from the other PI3K subunits in that they have a 'tissue-restricted expression'. The p110γ isoform is expressed only in white blood cells, whilst p110δ is expressed in white blood cells, breast tissue, and melanocytes. The expression of p110δ in white blood cells led one LICR (Ludwig Institute for Cancer Research) team to investigate PI3K signaling in immunity. By specifically deactivating the p110δ PI3K, the team was able to show that PI3K signaling plays an important role in the development and function of B and T lymphocytes. These results, taken with results from other investigators, suggest that too much PI3K activity could lead to autoimmunity and leukemia, whilst a reduction in PI3K activity could lead to immunodeficiency. Recently, LICR investigators found that p110δ plays an essential role in the allergic response. Inactivation of p110δ appears to protect against anaphylactic (allergic) responses, thus p110δ may be a new target for allergy therapies8.
1.1.4.2.Regulation of glucose transport
The effects of insulin on glucose uptake are mediated via the insulin receptor which, following insulin binding, undergoes autophosphorylation on tyrosine residues activating its tyrosine kinase activity. The activated IR then phosphorylates IRS-1 and other substrates. Tyrosine phosphorylated IRS-1 then serves as a docking protein for PI3K, which is activated by this interaction. The necessity of PI3K activation in the insulin effect is demonstrated by the complete ablation of the stimulatory effect of insulin on glucose transport when cells are incubated with specific inhibitors of this serine kinase. The serine phosphorylation cascade initiated by PI3K involves activation of PI3K-dependent serine/threonine kinases (PDK), and, in turn, Akt and results in the translocation of intracellular GLUT4 to the cell surface. It is the increased amount of GLUT4 on the cell plasma membrane that results in an increased rate of glucose transport into the cell. In muscle cells, stimuli other than insulin can produce a stimulation of the glucose transport system of a similar magnitude, albeit via a separate signaling route9.
17
Figure 1.8: Insulin signaling pathways involved in stimulating glucose transport.
18 1.1.5. Alteration of PI3K
1.1.5.1. Cancer
PI3K promotes cell growth, cell-cycle progression, and adhesion-independent survival and migration. Together these drive the progression of malignant tumours.
Oncogenes, such as mutant Ras and growth factor receptors, and loss of regulatory sites on the p85 subunit constitutively upregulate PI3K activity. Mutation and silencing of PTEN result in the accumulation of PtdIns(3,4,5)P3 and have been identified in several human malignancies10.
The recognition of the importance of this class of enzymes in cancer was cemented with the discovery that the PIK3CA gene, which encodes p110α. This gene was frequently mutated in a number of the most common forms of cancer, including colon, breast, prostate, liver, and brain tumors11.
PI3K also plays a major role in endothelial and immune cells that support tumor growth, intravasation and invasion. Inhibition of PI3K may thus have the potential to inhibit formation of secondary-site metastases.
The tumor microenvironment is a heterogeneous compartment comprised of stromal fibroblasts, immune cells, adipocytes and blood vessels. During chronic inflammation, wounding and cancer, the complex communication between these cell types is disrupted through persistent overproduction of chemokines and matrix metalloproteinases, leading to structural disorder of the tissue and abnormal proliferation. PI3K plays a critical role in signaling within endothelial and immune cells, and inhibition of PI3K may help to normalize the stroma, thereby indirectly inhibiting tumor growth.
Angiogenesis is critical in tumorigenesis because de novo blood vessel formation must occur to maintain oxygen and nutrient exchange between the tumor periphery and the hypoxic core. As critical signaling intermediaries, endothelial cells express numerous cell-surface receptors to integrate the vascular growth factors secreted by tumor and stromal cells. PI3K acts as a master regulator of angiogenic signaling in the endothelium. Several studies have recently elucidated the role of PI3K signaling in normal and tumor angiogenesis and provide encouraging support for targeting PI3K for antiangiogenic therapy13.
19
Figure 1.9: PI3K in the tumor microenvironment. In tumor cells, PI3K plays an important role in tumor initiation, growth and proliferation. PI3K also plays a major role in endothelial and immune cells that support tumor growth, intravasation and invasion. Inhibition of PI3K may thus have the potential to inhibit formation of secondary-site metastases. PI3K, phosphoinositide 3-kinase.
Immune cell infiltration Initiation of secondary site tumor formation
Maintenance of Vascular Integrity
Growth, Proliferation,
Survival, Motility
20
1.2 PDK1
Since it was originally discovered over a decade ago, PDK1 has emerged as a master regulator of the AGC family of protein kinases. It acts as an upstream protein kinase phosphorylating and activating several AGC-family members implicated in the control of cell growth, proliferation, survival and metabolism regulation. These include protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), p90 ribosomal S6 kinase (RSK), the serum and glucocorticoid-induced protein kinase (SGK), as well as several protein kinase C (PKC) isoforms. These enzymes are stimulated by hormones and growth factors, and phosphorylate regulatory proteins mediating the various physiological effects of these agonists13.
Figure 1.10.: Role of PDK1 in insulin signaling.
PDK1 stands for 3-phosphoinositide-dependent protein kinase 1. PDK1 functions downstream of PI3K through PDK1's interaction with membrane phospholipids including PtdIns(3,4)P2 and PtdIns(3,4,5)P3. PI3K indirectly regulates PDK1 by phosphorylating phosphatidylinositol which in turn generates PtdIns(3,4)P2 and PtdIns(3,4,5)P3.
21 PDK1 is ubiquitously expressed in cells and surprisingly for an enzyme that regulates at least 23 agonist stimulated AGC kinases, it is constitutively active. Much effort has been devoted to understand how an enzyme that is always active is capable of inducibly phosphorylating its substrates in response to specific stimuli. Data from many studies have suggested that agonists target the downstream AGC kinases converting them into substrates that can be recognized and phosphorylated by PDK1.
1.2.1 Structure
PDK1, which is 63 kDa, consists of an N-terminal kinase domain (amino acids 71-359) and a C-terminal PH domain (amino acids 459-550), which binds PtdIns(3,4,5)P3 and PtdIns(3,4)P2.
Figure 1.11: Secondary structure of PDK1
Identification of the PH domain as a specialized lipid-binding module has been a crucial clue in understanding the mechanism by which membrane-bound lipids convey signals to the cytoplasm. Deletion of the PH domain prevents PDK1 recruitment to the plasma membrane and affects the activation and membrane localization of Akt. Binding of PDK1 to PtdIns(3,4,5)P3 induces a major conformational change that is likely required for the activation of substrates.
However, PtdIns(3,4,5)P3 binding to the PH domain of PDK1 does not affect the activity of PDK1 directly.
22 As an AGC protein kinase, PDK1 belongs to the same subfamily of protein kinases as its substrates. Like all members of this family, the catalytic core of PDK1 possesses an N-terminal lobe that consists mainly of a β-sheet and a predominantly α-helical C-terminal lobe.
Figure 1.12: Phosphorylation sites of 3-phosphoinositide-dependent protein kinase-1.
Unlike other AGC kinases, PDK1 does not possess a hydrophobic motif (HM) C- terminal in its catalytic domain. Instead, it has been proposed that PDK1 possesses an HM pocket in the small lobe of its catalytic motif 14. The αC-helix (residues 124- 136), located in the small lobe of the kinase domain, is a key regulatory domain because it links a substrate-interacting site (HM pocket) with Ser-241 in the activation loop. The HM pocket in the kinase domain of PDK1 has been termed the PIF pocket after the first discovery that the C terminus of PKC-related kinase-2, which contains an HM motif, interacts with the kinase domain of PDK1.
Subsequent studies have indicated that this PIF pocket in PDK1 functions as a docking site, which enables the kinase to interact with some of its physiological substrates. The crystal structure of PDK1 reveals that phosphorylation of Ser-241 results in a hydrogen bond interaction with four residues, namely Arg-204 and Lys- 228 from the C-terminal lobe, and Tyr-126 and Arg-129 from the αC-helix in the N- terminal lobe. The highly conserved Arg-204, which immediately precedes the catalytic Arg-205, is connected directly to the catalytic machinery due to its position within the catalytic loop. Arg-204 controls the folding of the activation loop after
23 interaction with phosphorylated Ser-241. Lys-228 might also play a role in aligning catalytic site residues including Arg-223, which interacts with Mg2+.15
1.2.2. PDK1 regulation
Protein phosphorylation, which plays a key regulatory role in nearly every aspect of eukaryotic cell biology, is a reversible and dynamic process that is mediated by kinases and phosphatases. PDK1 is thought to be a constitutively active kinase that can use distinct mechanisms to phosphorylate different substrates within cells. PDK1 undergoes autophosphorylation and growth-factor induced phosphorylation at different sites, and its activity is correlated with its phosphorylation status. Therefore, understanding the mechanism of PDK1 phosphorylation could lead to greater knowledge of its function.
Figure 1.13: Mechanism for the regulation of PDK1 phosphorylation and stability
The phosphorylation level of each serine is unaffected by stimulation with insulin growth factor-1 (IGF-1). PDK1 itself is activated by phosphorylation in the activation loop (Ser241 for human and Ser244 for mouse PDK1), and this phosphorylation has been shown to be mediated by dimerization and transphosphorylation. In addition to phosphorylation, PDK1 function has also been
24 shown to be regulated by protein–protein interaction and its subcellular localization16.
PDK1 is phosphorylated on Tyr-9 and Tyr-373/376 with the help of Src and heat shock protein 90 (Hsp90). Tyrosine phosphorylation further increases PDK1 catalytic activity. In the absence of Hsp90 interaction, PDK1 is promoted towards proteasome-dependent degradation17.
25 1.2.3. Alteration of PDK1
1.2.3.1. Cancer
Hyperactivation of the PI3K/PDK1/Akt pathway is found in many human cancers. The hypomorphic PDK1 mice model was a useful tool to investigate the role of PDK1 under an increased PI3K signalling pathway.
PDK1 plays a pivotal role in breast cancer development. Increased copy number of PDK1 is often present in human breast cancer compared to normal breast epithelia and correlates with upstream PI3K pathway activation. In particular increased copy number of PDK1 correlates with the presence of activating mutation of PI3K classI p110α or with ERBB2 overexpression. Furthermore, overexpression of PDK1 is sufficient to transform mammary epithelial cells.
Increased levels of PDK1 have been reported in 45% of patients with acute myeloid leukemia, and a role for PDK1 in ovarian cancer progression has also been proposed.
Whether PDK1 is essential for tumourigenesis initiation or is required in later stages is still unclear.
PDK1 has an essential role in regulating cell migration especially in the context of PTEN deficiency. More importantly, down-regulation of PDK1 levels inhibits migration and experimental metastasis of human breast cancer cells. Furthermore, it has been shown that PDK1 controls migration and malignant transformation but not cell growth and proliferation in PTEN-null lymphocytes18.
Clinical observations together with recent preclinical data, suggest that PDK1 is a valid target for highly invasive and metastatic cancers.
Given the crucial role played by angiogenesis in cancer progression, it is worth of note that PDK1 has a key role in regulating endothelial directional migration. Indeed, endothelial cells differentiated from mouse embryonic stem cells lacking PDK1 completely lose their ability to migrate in vitro in response to vascular endothelial growth factor-A.
26 1.3. AKT
In 1991, three independent research teams identified genes corresponding to Akt/PKB (Protein kinase B), based on its homology to protein kinases A (PKA) and C (PKC) or as the cellular homolog to the retroviral oncogene viral akt (v-Akt)19. Together with the discovery of a second form, Akt2/ PKBβ, the three cloning papers established Akt as a novel phospho-protein kinase that was widely expressed, and paved the way for future experiments into the role of Akt in diverse cellular processes. Akt is now classified as a family of kinases, in mammalian cells, Akt has three closely related and highly conserved (>80% sequence identity) cellular homologues, designated as Akt1/PKBα, Akt2/PKBβ, and Akt3/ PKBγ. The three isoforms of human (mouse) Akt are located at chromosomes 14q32 (12F1-2), 19q13 (7B1), and 1q44 (1H4-6), respectively20.
Figure 1.14.: Akt domains and comparison of Akt isoforms (% of homology). Chromosome location of each Akt isoform in human as well as reported phosphorylation sites in Akt1 are also depicted.
In mouse tissues, both the Akt1 and Akt2 isoforms are ubiquitously expressed, whereas the Akt3 isoform is relatively highly expressed in tissues such as brain and testis. Akt2 is expressed predominantly in insulin target tissues, such as fat cells, liver and skeletal muscle.
27
Figure 1.15: Overlapping and specific functions of the Akt family members. Summarized are common and distinct Akt isoform function.
1.3.1. Structure
All three Akt isoforms consist of a conserved domain structure: an amino terminal pleckstrin homology (PH) domain, a central kinase domain and a carboxyl- terminal regulatory domain that contains the hydrophobic motif, a characteristic of AGC kinase.
.
Figure 1.16:Schematic representation of Akt1. (CTD: C-terminal domain; HG: Hydrophobic groove;
P: Phosphate; PH: Pleckstrin homology domain).
1.3.1.1. PH domain
The PH domain is a protein module comprised of ~ 100 amino acid residues in the N-terminal region that shows homology to the major PKC substrate pleckstrin in platelets. PH domain is found in many signaling proteins. One of its major functions is to bring an enzyme to the cell membrane by binding directly to phospholipids. The crystal structures of the PH domain of Akt1 unbound or bound to inositol(1,3,4,5) tetraphosphate (InsP4) or its surrogate have been solved by X-ray crystallography. Despite a lack of conserved primary structures, the three-
28 dimensional structures of the PH domains are remarkably conserved. The phospholipid binding pocket of the PH domain is a barrel-like structure consisting of two nearly orthogonal β-sheets formed by seven antiparallel β-strands, which are capped on one end of the β-sheets by the C-terminal α-helix. At the opening of the barrel lie three loops that are variable in sequence and length among different enzymes. The surfaces of these three loops are positively charged by basic residues of Lys, Arg and His, into which the negatively charged PtdIns(3,4,5)P3 is sandwiched and bound. The size and structure of the variable loops (VL1-3) likely accounts for the specificity toward different phospholipids among different kinases.
Point mutants for residues responsible for interaction with the D3 and D4 phosphate groups reduce the potential to interact with PtdIns(3,4,5)P3and PtdIns(3,4)P2, and these mutants could not be activated by phosphoinositide-dependent kinase 1 (PDK1). These observations suggest that the PH domain of Akt plays a significant role in recognition by upstream kinases, as well as membrane translocation.
1.3.1.2. Catalytic domain (kinase domain)
Connected to the PH domain is a structurally conserved kinase domain or catalytic domain comprised of ~ 260 amino acid residues. The crystal structures of the kinase domain of Akt in both active (Fig. 1.17 B) and inactive (Fig. 1.17 C) forms have been resolved.
The Akt kinase domain consists of nine β-strands, seven α-helices and numerous connecting loops, which are grouped into one small and one large lobe. The two lobes are connected through a very well conserved single peptide strand called the hinge, about which the lobes can rotate with respect to each other without disruption of the secondary structure of the kinase until it is activated by phosphorylation of both Thr308 (Akt1, same below) in the activation loop and Ser473 in the hydrophobic motif (HM) in the C terminus. By this time, the two lobes are locked by numerous interactions of the residues between them, including, among other things, an anchoring salt bridge between Lys179 from the small lobe and Glu198 of the C- helix and an anchoring hydrogen bond between His194 of the C-helix and the phosphate of Thr308. This also enables the phosphate of Thr308 to form a salt-bridge with Lys297 and, subsequently, a hydrogen bond to Arg273, which in turn forms a
29 salt bridge with Asp331 and a hydrogen bond with the backbone N-H of Lys296.
This in turn brings together all the function loops – the catalytic, the magnesium binding and the substrate binding loops.
Figure 1.17: Structure of active and inactive PKB and PKA. (A) Structure of the PKA. (B, C) Structure of inactive catalytic domain of Akt.
1.2.1.3. ATP-binding site
Central to the kinase domain is the ATP-binding site, which is located inside the deep cleft between the two lobes. ATP binds to the hinge via two hydrogen bonds, one with the N-hydrogen of Ala230 and one with the carbonyl group of Glu228.
The homology between the Akt and PKA (80%) or PKC (67%) is lower in the ATP pocket as compared with Akt isoforms, it is still considered to be high and may pose a challenge to designing ATP-site binders with real and meaningful selectivity over PKA, PKC or other members of AGC family of kinases. The C-terminal domain contains mostly hydrophobic residues (~ 50 – 70 residues) and many members of the AGC family have high degree of homology in this region. Sequence alignment of AGC kinases show the conserved and important Phe-X-X-Phe named hydrophobic motif in the C-terminal domain. The X-ray structure of Akt reveals that the hydrophobic motif (HM), which is comprised of Phe469 and Phe472 of the C terminus, binds intramolecularly to a hydrophobic groove (HG) on top of the small lobe.
The hydrophobic groove is located under the β 4- β 5 loop, behind the α B helix and above the α C helix on top of the small lobe. The HG is made up of hydrophobic residues and forms extensive hydrophobic interactions with Phe469 and Phe472. The
A) B) C)
30 phosphorylation of Ser473 of HM adds additional interactions between the phosphate of Ser473 and Arg144/Glun218, thus increasing the affinity of the HM for the HG and concomitantly activating the kinase.
There is increasing evidence that Akt may interact with its activators, inactivators and substrates via its HG or HM. Because of the association between HM of the C terminus and HG of the kinase domain, part of the C-terminal chain wraps over the surface of both lobes of the kinase domain and consequently blocks the entrance to the ATP-binding pocket by 326 – 418 residues. This structural feature will most likely have impact on the structures of ATP-site binders.
1.3.2. Akt regulation
Activation of receptor tyrosine kinases by ligands such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) leads to autophosphorylation of specific tyrosine residues on the intracellular portion of the receptor. Recruitment of phosphoinositol 3-kinase (PI3K) then occurs, via binding of the SH2 domains of the regulatory subunit (p85) to the phosphotyrosine residues on the receptor, which leads to a conformational change in the kinase, and consequently to activation.
3′-phosphoinositides generated by activated PI3K mediate the membrane recruitment Akt from the cytosol to the plasma membrane via its PH domain, and the kinase conformational change from an inactive to an active state.
Once Akt translocated from the cytoplasma to the plasma membrane, it binding via its PH domain to PtdIns(3,4,5)P3 and/or its hydrolyzed product PtdIns(3,4)P2 by 5´- specific inostitol phosphatase SHIP1 or SHIP2. Membrane association brings Akt in the proximity of PDK1, which is also recruited to the membrane by PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2.
Then PDK1 phosphorylate Thr308 in the binding site of Akt. An additional phosphorylation at Ser473 of the HM of the C terminus by a putative PDK2 (which is most likely the rictor-target of rapamycin (mTOR) complex) stabilizes the kinase domain and fully activates Akt. Finally, the activated Akt dissociates from the plasma membrane and enters the cytoplasm and nucleus where the substrates are located.
31
Figure 1.18.: Akt inhibition. It is thought that the N-terminal PH domain precludes kinase access to phosphorylation of the activation-loop at Thr 308 by PDK-1. Allosteric inhibitors stabilized the
‘closed’ Akt complex. PH-domain inhibitors devoid the recruitment of Akt to the membrane by binding to its PH domain. ATP-binding pocket inhibitors interact with the activated Akt isoform by precluding its kinase activity (but not its activation).
32 1.3.3. Physiological role of Akt
Akt isoforms contribute to a variety of cellular responses, including cell growth, cell survival and metabolism. This multiplicity of Akt functions might be due to the variation and specificity of its substrates. Up to now, more than 50 proteins have been identified as putative substrates for Akt. The minimal substrate consensus sequence for Akt, RXRXXS/T, where X is any amino acid and S/T is the phosphorylation site, is based on the sequence around the phosphorylation site on GSK3, the first identified substrate. Almost all the reported Akt substrates have this minimal sequence21.
Figure 1.19: Crystallographic structure of human GSK-3 beta (rainbow colored, N-terminus = blue, C-terminus = red).
1.3.3.1. Cell Growth and Cell Proliferation
One of the best-conserved functions of Akt is its role in promoting cell growth (i.e., an increase in cell mass). The predominant mechanism appears to be through activation of mTOR complex 1 (mTORC1 or the mTOR-raptor complex), which is regulated by both nutrients and growth factor signaling. mTORC1 is a critical regulator of translation initiation and ribosome biogenesis and plays an evolutionarily conserved role in cell growth control.
Akt positively regulates the pathway via phosphorylation and inhibition of two negative regulators: TSC2 (tuberous sclerosis complex 2) and PRAS40 (proline rich Akt substrate of 40 kDa) 22.
33 Although Akt is best known for promoting cell survival and growth through pathways parallel to Erk’s control over cell proliferation, Akt activation can also stimulate proliferation through multiple downstream targets impinging on cell-cycle regulation23.
Akt-dependent phosphorylation of target such as GSK3, TSC2, and PRAS40, is also likely to drive cell proliferation through regulation of the stability and synthesis of proteins involved in cell-cycle entry. GSK3-mediated phosphorylation of the G1 cyclins cyclin D and cyclin E and the transcription factors c-jun and c-myc, which all play a central role in the G1-to-S-phase cell-cycle transition, targets them for proteasomal degradation 24.
1.3.3.2. Cell Survival
Akt promotes cell survival via the negative regulation of the proapoptotic Bcl-2 homology domain (BH3)-only proteins, which carry out their function by binding to and inactivating the prosurvival Bcl-2 family members. Phosphorylation by Akt creates a binding site for 14-3-3 proteins, which sequester the phosphorylated substrate and thereby prevent association with target proteins (such as Bcl-2 and Bcl- XL) at the mitochondrial membrane25.
Pro-caspase-9 was also a substrate of Akt; phosphorylation was found to block the intrinsic protease activity of the enzyme induced following the release of cytochrome c. This evidence suggests a role for caspase-9 in the antiapoptotic effects of Akt, however there is some dispute as to whether caspase-9 is a direct substrate of the kinase or whether phosphorylation is induced by modifying an unknown cytosolic factor26.
34
Figure 1.20: Signaling by the Akt pathway regulates members of the Bcl-2 family, which promote cell survival by inhibiting the release of cytochrome c from mitochondria.
1.3.3.3. Metabolism
GSK3 phosphorylates and inactivates glycogen synthase in response to insulin stimulation27.Both isoforms (GSK3α and β) have the phosphorylation site in the amino-terminal region (Ser21 and Ser9, respectively), and they are phosphorylated and inactivated by Akt in a PI3-K-dependent manner.
GSK3 is implicated in signalling pathways other than insulin signalling, such as the wnt signalling pathway.
Phosphodiesterase 3B (PDE3B) is phosphorylated by Akt on Ser273. This causes enzymatic activation of PDE3B, which contributes to regulating the intracellular level of cyclic nucleotides such as cAMP and cGMP in response to insulin28.
The cardiac-specific isoform of 6-phosphofructo-2-kinase (6-PF2-K) is phosphorylated byAkt on Ser466, which results in the activation of this enzyme and promotion of glycolysis29.
1.3.3.4. Vascular Effects
Akt plays important roles in physiological angiogenesis through effects in both endothelial cells and cells producing angiogenic signals. In endothelial cells, the
35 Akt is robustly activated by vascular endothelial growth factor, and phosphorylation of the Akt targets discussed above is likely to contribute to endothelial cell survival, growth, and proliferation30.
In addition, Akt activates endothelial nitric oxide (NO) synthase (eNOS) through direct phosphorylation of S1177. The release of NO produced by activated eNOS can stimulate vasodilation, vascular remodeling, and angiogenesis31.
Akt signaling also leads to an increased production of the hypoxiainducible factor α (HIF1α and HIF2α) transcription factors, at least in part, through mTORC1- dependent translation. HIFα activation in endothelial cells and other cells leads to expression and subsequent secretion of VEGF and other angiogenic factors, thereby stimulating angiogenesis through both autocrine and paracrine signaling32.
36 1.3.4. Alteration of Akt
1.3.4.1. Cardiovascular disease
Akt has a clearly defined role in the functional behavior of cells in the cardiovasculature such as cardiomyocytes, endothelial cells and thrombocytes.
Disruption of Akt signaling homeostasis (either enhancement or reduction) would give rise to cardiac remodeling that, in a long run, lead to cardiomyopathy and heart failure. Modulation of Akt signaling could regulate the switch between hypertrophic (HCM) and dilated cardiomyopathy (DCM)33.
Figure 1.21: Akt signaling homeostasis is critical for normal heart function and its disruption regulates the switch between HCM (hypertrophic cardiomyopathy) and DCM (dilated cardiomyopathy).
Of the three isoforms, Akt1 seems to be the most relevant in regulating cardiovascular functions. In the heart Akt1 activity regulates cardiac growth, contractile function and coronary angiogenesis. Akt1 is therefore beneficial to the heart under physiological conditions such when it is acutely upregulated by factors such as IGF-134.
1.3.4.2. Diabetes
Initial reports on Akt signaling suggested a role for this kinase in insulin regulation of glucose metabolism. There is strong evidence for the involvement of
37 Akt in insulin-stimulated glucose uptake into muscle and adipocytes, and an impairment of Akt function in insulin resistance and diabetes35.
Akt regulates glucose uptake into muscle and fat cells by stimulating the translocation of GLUT4 glucose transporter to the plasma membrane. Akt also plays a role in the repression of liver gluconeogenesis by insulin through its ability to suppress the expression of two key enzymes in this process, phosphoenolpyruvate carboxykinase and glucose 6-phosphatase36.
Akt deregulation has been implicated in diabetes. The discovery of a dominant negative Akt2 mutation (R274H) causing severe hyperinsulinaemia and diabetes in humans, while an extremely rare event, confirmed the role of Akt in metabolic regulation. Akt not only plays an important role in glucose transport into insulin target tissues but also in stimulating survival and proliferation of insulin-secreting β- cells in the pancreas37.
1.3.4.3. Neurological disease
It is well established that Akt plays an important role in the ability of growth and neurotrophic factors to suppress neuronal cell death. Akt-mediated suppression of neuronal cell death occurs via multifarious mechanisms including alterations in gene expression, inhibition of caspase-9 and suppression of cytochrome-c release by mitochondria. Numerous Akt substrates have been implicated in these effects including Bad, TSC2, Foxo-family members and caspase-9 itself38.
In dopamine neurons of the substantia nigra, the death of which cause Parkinson's disease, over-expression of a constitutively active Akt (Myr-Akt) prevents neurotoxin-induced cell death and maintenance of axonal sprouting. Akt also plays a role in survival and nerve regeneration in neonatal and adult motor neurons38.
Furthermore, the antipsychotic drug haloperidol stimulates Akt phosphorylation on Thr308 and Ser473, and a genetic polymorphism linked with the Akt1 gene is associated with reduced Akt1 expression and increased risk of schizophrenia. It is also of particular interest that Akt phosphorylates the type A gamma aminobutyric acid receptor (GABA(A)R) which increases the number of GABA(A)Rs at the synaptic membrane which, in turn, increases synaptic strength. Taken together with the ability of lithium, a clinically proven antidepressant, to inhibit the activity of the
38 Akt substrate GSK3, suggests an important role for Akt in synaptic transmission, memory and psychosis39.
1.3.4.4. Cancer
Many human cancers, including carcinomas, glioblastoma multiforme, and various hematological malignancies, exhibit frequent activation of Akt.
In particular, Akt activation has been shown to correlate with advanced disease and/or poor prognosis in some tumor types. Approximately 40% of breast and ovarian cancers and more than 50% of prostate carcinomas exhibited increased Akt1 kinase activity; and nearly 80% of tumors with activated Akt1 were high grade and stage III//IV carcinomas40. Activation of the Akt2 kinase was observed in 30–40% of pancreatic and ovarian cancers41. Increased Akt3 enzymatic activity was found in estrogen receptor-deficient breast cancer and androgen-insensitive prostate cancer cell line, suggesting that Akt3 may contribute to the aggressiveness of steroid hormone-insensitive cancers42.
Activation of Akt is primarily the result of aberrant upstream molecules of Akt, which include overproduction of growth factors, upregulation and/or mutation of phosphatidylinositol 3-kinase (PI3K), PTEN (Phosphatase and Tensin homologue deleted on chromosome Ten), NF1 and LKB1, and downstream tuberous sclerosis complex 2 (TSC2), Forkhead Box Class O (FOXO) and eukaryotic initiation factor 4E (eIF4E). Several of these proteins (AKT, eIF4E, and both the p110α catalytic and p85α regulatory subunits of PI3K) can behave as oncoproteins when activated or overexpressed, while others (PTEN, FOXO, LKB1, TSC2/TSC1, NF1, and VHL) are tumor suppressors 43.
Activated Akt is a well-established survival factor, exerting antiapoptotic activity in part by preventing the Akt phosphorylates and inactivates the proapoptotic factors BAD and procaspase-9. Moreover, Akt phosphorylates and inactivates the FOXO transcription factors, which mediate the expression of genes critical for apoptosis, such as the Fas ligand gene. Akt also activates IkB kinase (IKK), a positive regulator of NF-kB, which results in the transcription of antiapoptotic genes44. In another mechanism to thwart apoptosis, Akt promotes the phosphorylation and translocation of Mdm2 into the nucleus, where it downregulates p53 and thereby antagonizes p53- mediated cell cycle checkpoints45.
39 Akt activation mediates cell cycle progression by phosphorylation and consequent inhibition of glycogen synthase kinase 3b to avert cyclin D1 degradation46.
Akt activates the downstream mTOR kinase by inhibiting a complex formed by the tumor suppressor proteins TSC1 and TSC2, also known as hamartin and tuberin.
mTOR broadly mediates cell growth and proliferation by regulating ribosomal biogenesis and protein translation47 and can regulate response to nutrients by restricting cell cycle progression in the presence of suboptimal growth conditions.
Each of these tumor suppressors is a negative regulator of the Akt-mTOR pathway, which, when deregulated, results in altered translation of cancer-related mRNAs that regulate cellular processes such as cell cycle progression, autocrine growth stimulation, cell survival, invasion, and communication with the extracellular environment48.
40 1.4. PTEN
PTEN (phosphatase and tensin homolog deleted on chromosome ten), also known as MMAC1 (mutated in multiple advanced cancers-1) or TEP1 (tensin-like Phosphatase-1), was originally identified in 1997 by three independent groups led by Parsons, Steck, and Sun49,50,51.
PTEN is now known to play major roles not only in suppressing cancer but also in embryonic development, cell migration and apoptosis.
Figure 1.22: Overall view of the PTEN structure, including the N-Terminal Phosphatase Domain and a C-Terminal C2 Domain. The dotted line indicates the region deleted in the crystallized protein.
PTEN is a lipid phosphatase for the second messenger PtdIns(3,4,5)P3. By converting PIP3 into PtdIns(4,5)P2, PTEN negatively regulates the PI3 kinase-Akt signaling pathway.
Figure 1.23: The tumor suppressor PTEN opposes the action of PI3K by dephosphorylating the signaling lipid phosphatidylinositol (3,4,5) trisphosphate.
41 1.4.1 Structure
PTEN encodes a 403–amino acid protein that contains multiple domain structures. PTEN protein comprises a phosphatase domain, a C2 domain that binds phospholipid membranes, and a C-terminal tail that contains 2 PEST sequences with Ser/Thr phosphorylation sites and a PDZ (PSD95/Dlg/ZO1) binding motif.
Figure 1.24: The domain structure of PTEN. The numbers denote the amino acid positions of individual domains or motifs of human PTEN.
The phosphatase domain is the catalytic center of PTEN; predictably, some cancer- derived missense mutations are clustered in this region52.Phosphatase domain has a structure resembling previously characterized protein tyrosine phosphatases but containing an enlarged active site that can account for its ability to bind PtdIns(3,4,5)P3. Such enlargement of the active site, however, would be expected to entail diminished specific phosphotyrosine binding and protein phosphatase activity.
In the center of the protein, PTEN has a C2 domain that is required for its interaction with the membrane53. This C2 domain might orient the catalytic domain appropriately for interactions with PtdIns(3,4,5)P3 and other potential substrates.
Several studies have identified intriguing regulatory features in the C-terminal tail of PTEN. This region has several recognizable motifs, including a cluster of putative phosphorylation sites, a PDZ binding motif, and two PEST motifs. All these motifs are implicated in the physiological regulation of PTEN function54.
42 1.4.2. Mechanism of action of PTEN
PTEN phosphatase activity has been observed against both lipid and protein substrates. Overall, the primary physiological substrate of PTEN appears to be the signaling lipid PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 is a major product of PI3K, which is activated by cell receptors including various tyrosine kinase growth factor receptors and integrins.
Figure 1.25: PTEN Negatively Regulates PI3K Signals
PTEN cleaves the 3' phosphate from PtdIns(3,4,5)P3 to generate PtdIns(4,5)P2, which lacks the activities of PtdIns(3,4,5)P3 but has its own actions on cytoskeletal function. By antagonizing the action of PI3K, PTEN affects a number of cell biological processes. In addition, it can dephosphorylate the signaling molecule inositol (1,3,4,5)-tetrakisphosphate, although the biological importance of this activity is not yet clear55.
In vitro, PTEN can also remove phosphate residues from phosphotyrosine-containing peptides and proteins although the relative importance of this enzymatic function in vivo compared with its lipid phosphatase activity has been controversial56.
43 1.4.3. Physiological role of PTEN
1.4.3.1. Role in Cell Cycle regulation
PTEN is a key regulator of progression through the mammalian cell cycle.
Though typically thought of as a survival factor, multiple lines of evidence suggests that Akt may be the major downstream target for PTEN mediated G1 arrest. Among the Akt substrates thought to play an important role in cell cycle control are the Forkhead transcription factors FKHR, AFX and FKHRL1, GSK3 and substrates, such as TSC2, that play a role in regulating mTOR signaling57.
1.4.3.2. Regulation of Cell Migration
PTEN suppresses migration of a variety of cell types, including primary human fibroblasts and tumor cells primarily due to its ability to dephosphorylate proteins rather than lipids. The regulation of cell migration and adhesion can be divided into two components:
1. a directionally persistent migratory component promoted by the FAK signaling pathway, which involves enhanced orientation of the actin cytoskeleton, increased focal contacts and directional migration; and
2. a random-motility component promoted by association of SHC with GRB2 (Growth Factor Receptor-bound Protein-2) and subsequent activation of the Ras/Raf/MEK1/ERK pathway, which involves a modest increase in F-actin levels, random orientation of the actin cytoskeleton and more random cell movement58.
The FAK signaling pathway is activated by Integrin receptors and is linked to cell motility and migration and other cellular activities like cell survival, proliferation and differentiation. The SHC-GRB2 pathway is activated by receptors that include various tyrosine kinase receptors and Integrins59.
PTEN's protein phosphatase activity is able to down-regulate both the pathways by direct dephosphorylation of FAK and SHC which leads to the inactivation of the Ras/MAPK pathway. PTEN-regulated FAK and SHC activity further appears to impinge on cell adhesion, cell migration, and cell invasion. It therefore emerges that the loss of PTEN activity may confer increased survival ability, proliferative
44 potential, and invasive capacity on cells, and thereby may promote progression towards a more malignant phenotype60.
1.4.3.3. Apoptosis
The role of PTEN in apoptosis is clearer. A particularly important role of PTEN is in the form of apoptosis termed anoikis. Anoikis is the induction of apoptosis in cells after loss of contact with the extracellular matrix. This property may be a central feature of normal epithelial cell function (and perhaps certain other cell types) that prevents growth at abnormal sites, especially in suspension. This anchorage dependence of survival is defective in many transformed and malignant cells.
PTEN modulates apoptosis by reducing levels of PtdIns(3,4,5)P3. This signaling lipid regulates activation of Akt through the kinases PDK-1 and PDK-2. Akt is well known as a central regulator of apoptosis. Re-expression of PTEN in various tumor cell lines decreases PtdIns(3,4,5)P3 levels and reduces Akt activation61.
Figure 1.26: Reported sites of action of PTEN.
45 1.4.4. Alteration of PTEN
1.4.4.1. Cancer
Although the PTEN gene was originally identified through its mutation in several types of sporadic tumour, substantial evidence now indicates that loss of expression of the protein, through as yet largely unidentified mechanisms, may be of far greater significance in terms of patient numbers. Using conventional mutation detection techniques that have been highly successful with other tumour suppressors and with PTEN in tumours such as glioblastoma and endometrial carcinoma, mutation of the PTEN gene has not been detected at high frequency in common tumour types such as lung, breast, prostate or colon cancer62.
Germ-line mutations in PTEN cause three rare autosomal dominant inherited cancer syndromes with overlapping clinical features: Cowden disease, Lhermitte–Duclos disease, and Bannayan–Zonana syndrome. These syndromes are notable for hamartomas, benign tumors in which differentiation is normal, but cells are highly disorganized. Whether the type of mutation in PTEN contributes to the distinct features of these three hamartomatous syndromes remains unclear, but other (i.e., modifying) loci probably play the primary role in determining the spectrum of abnormalities evoked by a given mutation.
PTEN function is required for normal development and that loss of PTEN function contributes to carcinogenesis. PTEN appears to suppress cell growth by distinct mechanisms in different types of tumors, producing G1 cell cycle arrest in glioblastoma cells, but inducing apoptosis in carcinomas.
PTEN negatively regulates intracellular levels of PIP3 in cells, which is critical for chemo-attractant gradient sensing. PTEN accumulates at a contralateral subcellular side of the migration leading edge and is required for proper chemotaxis. Although PTEN controlscell migration, the exact role of PTEN in tumor invasion and metastasis is still elusive63.
Emerging data suggests that PTEN suppresses the hypoxia-mediated stabilization of hypoxia inducible transcription factor 1 (HIF1). HIF1, when stabilized, upregulates the expression of vascular endothelial growth factor a potent stimulator of new blood vessel formation. Thus, loss of PTEN or activation of PI3K/Akt may also endow tumors with angiogenic properties64.
46 1.5. mTOR
The mammalian target of rapamycin (mTOR) also known as mechanistic target of rapamycin or FK506 binding protein 12-rapamycin associated protein 1 (FRAP1) is a protein which in humans is encoded by the FRAP1 gene. mTOR is a 289 kDa protein that belongs to the phosphatidylinositol 3-kinase-related kinase family.
Figure1.27: Overall view of the mTOR structure
The TOR protein is conserved in yeast, flies, worms, plants and mammals. Although there is a single mTOR gene in mammals, the mTOR product functions as a component of two complexes, mTOR Complex 1(mTORC1) and mTOR Complex 2 (mTORC2).
1.5.1 Structure
mTOR complexes 1 and 2 (mTORC1/2) differ in their composition, regulation and functions. Each complex contains mTOR and the proteins mammalian lethal with Sec13 protein 8 (mLST8) and DEP domain-containing mTOR- interacting protein (DEPTOR) whereas the other components define each complex.
Specifically, the proteins regulatory-associated protein of mTOR (raptor) and proline-rich Akt substrate of 40 kDa (PRAS40) are specific components of mTORC1 whereas rapamycin insensitive companion of mTOR (rictor), mammalian stressactivated map kinase-interacting protein1 (mSin1) and protein observed with rictor (protor)1/protor2 specifically define mTORC2 (Fig. 1.28). A detailed
47 description of mTORC1 structure that can reveal the organisation of the components is still missing.
Nevertheless several lines of evidence now have suggested that mTORC1 exists as a dimer.
Figure 1.28: Schematic representation of mTORC1 and mTORC2.
Both mTOR complexes function predominantly in the cytoplasm. However, experiments using a nuclear export receptor inhibitor indicate that mTOR may actually be a cytoplasmic-nuclear shuttling protein. This nuclear shuttling appears to play a role in the phosphorylation of mTORC1 substrates induced by mitogenic stimulation and in the consequent upregulation of translation65.
1.5.2 Regulation of mTOR signaling networks
Diverse environmental cues, including growth factors (e.g. insulin, insulin- like growth factor 1, epidermal growth factor (EGF)) and nutrients (e.g. amino acids, energy), promotemTORC1 signaling. Insulin signals via its receptor to activate the PI3K. The lipid products of PI3K, PIP3, recruit both PDK1 and Akt to the plasma membrane. Akt is subsequently phosphorylated on T308 by PDK1 and by mTORC2 (consisting of mTOR, mLST8 and Rictor) on S473, leading to full activation. Akt then phosphorylates numerous targets to promote growth and survival, including the TSC1/2 complex (the tumor suppressor TSC, composed of TSC1 (hamartin) and
48 TSC2 (tuberin)). By phosphorylating TSC2, Akt inactivates TSC2s GAP activity for the small G protein Rheb. GTP-bound Rheb is a potent activator of the rapamycin- sensitive complex mTORC1 (consisting of mTOR, mLST8 and Raptor). mTORC1 phosphorylates several targets, including the translation control proteins S6K and 4EBP1.
Figure 1.29: A model illustrating how mTOR fits within the insulin/Akt signaling network.
Under basal conditions, S6K and 4EBP1 are bound to a scaffolding protein, the eukaryotic initiation factor 3 (eIF3) translation initiation complex; but upon stimulation, mTOR phosphorylates and activates S6K and 4EBP1, causing their dissociation from eIF366. After activation by mTOR, S6K phosphorylates the ribosomal protein S6, leading to an increase in translation of a subset of mRNAs.
mTOR leads to enhancement of translation through S6K, 4EBP1 and other molecules, since it has been postulated that mTOR may serve as a nexus that controls up to 15% of the bulk translation of the cell (Jefferies et al., 1994). Moreover, translation regulation – especially through eIF4E – directly increases the size (and hence the growth) of cells via regulation of key growth promoting proteins such as c- Myc and cyclin D167.
49 1.5.3. Physiological role of mTOR
1.5.3.1. Role in Cell Cycle regulation
mTOR is one of the factors that coordinate the cell cycle transition. mTOR positively regulates the G1/S transition by suppression of cyclin D1 turnover, and by enhanced degradation of the cyclin dependent kinase (cdk) inhibitor, p27.
Moreover, mTOR, in particular mTORC1, contributes to the early development and differentiation/growth of lung acinar epithelium, chondrocytes, myocytes and adipocytes. In these tissues, mTOR mediates activation of the transcription factor, hypoxia-inducible factor-1α (HIF-1α) and regulates the glycolytic enzymes, leading to increased glucose uptake and glycolysis68.
1.5.3.2. Lipid metabolism
Recent studies show a role for mTORC1 signaling in the control of lipid metabolism and adipogenesis. Sterol response elementbinding protein 1c (SREBP1c) is a transcription factor that regulates the expression of proteins involved in lipid metabolism. The activation of SREBP1c by insulin is controlled by mTORC1, which promotes the cleavage and subsequent nuclear accumulation of SREBP1c and the expression of genes that it regulates69.
1.5.3.3. Transcription and ribosome biogenesis
The protein synthetic capacity of a cell depends on the amounts of ribosomes and transfer RNAs (tRNAs) present. Transcription of ribosomal RNAs (rRNAs) and tRNAs by RNA polymerases I and III account for as much as 80% of nuclear transcriptional activity and is tightly regulated by the mTOR pathway70.
The activity of several other transcription factors, particularly those involved in metabolic and biosynthetic pathways, including signal transducer and activator of transcription -1 and -3 (Stat-1 and Stat-3) 71 and the nuclear receptor peroxisome proliferator-activated receptor-γ72, are also regulated by mTORC1-mediated phosphorylation in a rapamycin-sensitive manner.
50 1.5.4. Alteration of mTOR
1.5.4.1. Cancer
The signaling pathways that activate mammalian target of rapamycin (mTOR) are altered in many human cancers.
Cancer related changes in mTOR kinase substrates and their associated proteins are reported. For instance, S6K1 is overexpressed or constitutively active in several tumor cell lines, and in the early stages of transformation in ovarian surface epithelium with BRCA1 mutations73. S6K1 is also amplified in some breast carcinomas74. Generally, for tumors that have an amplification of S6K1, there is a corresponding increase in the level of S6K1 protein74.
The gene coding for eukaryotic initiation factor 4E (eIF4E) that is positively regulated by mTOR, is altered in a number of tumors. Progressive amplification of the eIF4E gene is associated with late-stage head and neck carcinoma, ductal cell breast carcinoma and thyroid carcinoma75-77. Levels of eIF4E are elevated in some colon carcinomas in comparison to normal colon cells78,79. The levels of eIF4E are also increased in some bladder and breast cancers that have a poor outcome80. In these cancers, a corresponding increase in vascular endothelial growth factor (VEGF) was also observed81.
Overexpression of eIF4E also results in increased frequency of late-onset cancers82. These data point to the possibility that at least under some conditions eIF4E may act as an oncogene.
In certain types of cancer the relationship between the levels of the mTOR substrate 4EBP1, and the protein whose function it inhibits, eIF4E, might be a predictor of metastasis. In colon carcinoma both eIF4E and 4EBP1 are frequently overexpressed, but the 4EBP1 levels are the most elevated in patients that have little or no metastatic disease83.
Therefore, therapeutic approaches targeting mTOR pathway using small molecules or other clinically applicable pharmacological tools have great potential for treatment of numerous disorders.
51
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