Phosphoinositide 3-Kinases, Protein Kinase B, and Signalling through Insulin Receptor
and involves insulin receptors that are recovered from lipid rafts and which then associate with the adaptor protein APS.52
The role of PI 3-kinase in activation of protein synthesis
Insulin is a growth factor. Again, operating through the PI 3-kinase pathway, it increases protein synthesis by a mechanism that involves the GTPase Rheb and that acts upon the initiator factor-4E (eIF-4E) and the ribosomal p70 S6- kinase-1 (S6K1). We return to the role of Rheb below.
eIF-4E is the limiting ribosomal initiation factor in most cells and it plays a principal role in determining global translation rates. It is regulated by phosphorylation, for instance through the ERK pathway (see page 341), but more importantly by binding to the translational repressor 4E-BP. eIF4E binds to the 7-methylguanine (m7G) cap at the 5 -end of mRNA and
enables the recruitment of other initiation factors. These, together with the 40S ribosomal subunit, form the pre-initiation complex which promotes the search for the start codon (AUG) on the mRNA (see Figure 18.12). Progression is facilitated by the helicase complex eIF4A/eIF4B which breaks
the intramolecular base-pairing in the mRNA. Recognition of the AUG codon requires tRNAMet, which contains the anticodon 3 -UAC-5 and comes to the ribosomal pre-initiation complex bound to eIF-2 .GTP. As the AUG codon is recognized, GTP is hydrolysed and the ribosomal 60S particle, carrying the peptidyl transferase activity, joins the initiation complex, so initiating the elongation phase of protein synthesis. Binding of 4E-BP to eIF-4E prevents all this.
In response to glucagon and to numerous growth factors, the p70 S6-kinase1 associated with eIF-3 (a large complex of small initiation factors), acts to phosphorylate rbS6, a component of the 40S ribosomal subunit.53,54 Cells lacking rbS6 are smaller in size, but how it regulates cell size remains an enigma. Cellular localization of rbS6 is not restricted to the 40S ribosomal particle, so it may control cell size independently of protein synthesis at the ribosomal level55. p70 S6-kinase1 also phosphorylates the regulatory domain of eIF-4 A (helicase).
Rheb and TSC
TSC1 and TSC2, are downstream components of the PI 3-kinase pathway, discovered as loss-of-function mutants in patients suffering from tuberous sclerosis.56,57
The TSC1/TSC2 complex constitutes a GAP that holds Rheb in its GDP-bound inactive state.62 Phosphorylation of TSC2 by PKB inhibits this activity, so allowing accumulation of Rheb.GTP which activates mTOR (Figure 18.11).63,64 The kinase mTOR when complexed with Raptor and G L binds to eIF-3
A missense point mutation present in the kinase domain of PKB is present in members of a family showing autosomal dominant inheritance of severe insulin resistance.
This finding provides additional evidence for the important role for PKB in insulin signal transduction.51
The S6 protein kinases form a subfamily of serine/threonine protein kinases with members KS6A1-A6, KS6B1, and KS6B2. The S6 kinase discussed here is KS6B1. It phosphorylates the ribosomal S6 protein (rbS6) at six serine residues. Two of these are also phosphorylated by RSK, a protein kinase downstream of the Ras/ MAPK pathway.
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Tuberous sclerosis (TSC). First described by Désiré-Magloire Bourneville in 1880 (and called Bourneville’s disease in France),58,59 TSC is a rare (30–100 per million, worldwide), multisystem genetic disease characterized by the growth of benign tumours in the brain and on other vital organs including the kidneys, heart, eyes, lungs, and skin. It is caused by mutations on either of two TSC genes, 1 or 2, which encode for the proteins hamartin and tuberin respectively. These act as tumour suppressors, regulating
cell proliferation and differentiation. PTEN, which acts in the same signal transduction pathway, is also a tumour suppressor but loss of its function is linked to Cowden disease in which patients have a predisposition to
malignancies such as prostate cancer, glioblastoma, endometrial tumours, and small-cell lung carcinoma.60,61
FIG 18.11 Regulation of mTOR by PKB-mediated phosphorylation of TSC2.
The complex TSC1/TSC2 acts as a GAP, maintaining Rheb in its (GDP) inactive state and holding protein synthesis in check (1). Insulin augments the rate of protein synthesis through activation of Rheb. This occurs through phosphorylation and inactivation of TSC2 by PKB (2). Rheb then accumulates in its GTP bound state (probably involving an as yet unidentified GEF) and interacts with the kinase mTOR (3), complexed to Raptor, FKBP38 (and G L, not shown). Rheb bound to GTP sequesters the inhibitor FKBP38 and this unveils the kinase activity of mTOR and causes the complex to associate with eIF3 (4).
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and coordinates the assembly of the initiation complex through a series of ordered phosphorylation events. First, mTOR/Raptor phosphorylates the eIF-4E binding protein, 4E-BP, at multiple sites, causing its detachment65 and allowing the association of eIF-4G with the initiation complex (Figure 18.12). Importantly, binding of eIF-4G allows interaction of the 5 -mRNA initiation complex with the 3 -mRNA poly A binding proteins (PAPB) (Figure 18.12). This drives translation and ensures that only intact mRNA is translated.
mTOR/Raptor also phosphorylates S6K1,66 which then dissociates from eIF-3 and associates with PDK1, which phosphorylates the activation segment of S6K1 causing full activation. (This mechanism resembles the phosphorylation/ activation of PKB by PDK1: see Figure 18.07.) It phosphorylates the S6 ribosomal
When first discovered, the G-protein -like subunit, G L, was thought to
be a novel subunit, the sixth in the human genome. Although it has
WD repeats, characteristic of subunits, it is otherwise distinct and has now been classified as member of a tiny WD repeat subfamily, WD repeat Lst8.
FIG 18.12 A series of ordered phosphorylations facilitates assembly of the translation initiation complex on mRNA.
(a) Activation and association of mTOR with the pre-initiation complex causes phosphorylation of 4E-BP (BP) (1) and of S6K1. Phosphorylated 4E-BP detaches from eIF-4E and phosphorylated S6K1 detaches from eIF-3. (eIF-3 and the 40S ribosomal particle are represented as a single entities). (b) Dissociation of 4E-BP (2) and S6K1 permits the association of the large initiation factor eIF-4G (3) that interacts with eIF-4E, eIF-3, and eIF-4. S6K1, phosphorylated in its regulatory domain (including the hydrophobic motif) encounters PDK1 (4) causing phosphorylation of its activation segment. S6K1 now phosphorylates the ribosomal proteins S6 and eIF-4B, the regulatory subunit of the DNA helicase eIF-4 A (5). (c) The conditions now favour binding of poly-A binding protein (PABP) which is attached to the 3 -poly A tail of the mRNA (6). The pre-initiation complex moves towards the start AUG codon (7) where it is joined with the 60S ribosomal particle.
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protein and also eIF-4B, a regulatory subunit of the eIF-4 A helicase.67 As eIF-2 .GTP and tRNAMet combine with the pre-initiation complex, the small ribosomal subunit starts its progression towards the 3 end. It halts when it attains the AUG codon, binding to the anticodon of tRNAMet. Protein translation commences. The domain architectures of the components of the TSC/mTOR pathway are shown in Figure 18.13.
Although insulin-mediated activation of ERK is without significant effect on glucose transport or glycogen synthesis, it does have a significant effect on
FIG 18.13 Domain architecture of the components of the mTOR signalling pathway.
Many of the components of the mTOR pathway are large proteins, some having still poorly defined domains. TSC2 carries a GAP domain responsible for activation of hydrolysis of GTP by Rheb. The numerous phosphorylation sites have either stimulatory effects (sites phosphorylated by AMPK1), or inhibitory effects (red). mTOR bears the signature sequence of a PI 3-kinase but operates as a protein kinase. S6K1 has an autoinhibitory and an AGC kinase C-terminal domain with hydrophobic motif, both of which have to be phosphorylated before phosphorylation and activation of the activation segment. 4E-BP has
to be phosphorylated at four residues before it effectively detaches from eIF-4E. mTOR binds directly at the C-terminus or indirectly via Raptor. mTOR phosphorylates at least four residues, one of which is not sensitive to rapamycin and can thus be phosphorylated by another kinase. eIF-4G has a number of domains and regions that interact with other components of the initiation complex. Note the interaction with Mnk, a member of MAPkinase family (see Figure 12.22, page 351 and 12.15, page 340).
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protein synthesis.68,69 This occurs through ERK1/2-mediated phosphorylation of TSC2 at sites distinct from those targeted by PKB and it also causes inhibition of the GAP activity.70 This occurs through RSK1, one of the kinases downstream of ERK that phosphorylates TSC2.71
Control of translation and transcription
Both the Ras/MAPkinase and PKB pathways have profound and selective effects on the pattern of expression of individual proteins that occurs quite independently of their effects on gene transcription. Thus, after ectopic expression of constitutively activated forms of K-Ras or PKB in mouse glialprogenitor cells, polyribosome complexes engaged in the translation of mRNAs that encode proteins involved in the regulation of growth, transcription, and cell–cell interactions, become enriched. How the mRNAs are selected remains to be determined.72 It seems that protein translation, in addition to gene transcription, is also an important target of transforming proteins.
In order to give a further boost to protein synthesis, mTOR/Raptor also controls the transcription of the different forms of RNA required for ribosomal biogenesis and formation of the translation machinery. It stimulates RNA polymerase-I leading to the transcription of ribosomal RNA, RNA polymeraseII leading to the transcription of mRNA coding for ribosomal proteins (approximately 82), and RNA polymerase-III, which transcribes the 32 different transfer RNAs.73
Integration of growth factor and nutrient signalling
A lack of amino acids or the presence of 5 -AMP (see page 243 and Figure 9.1, page 244) must intervene with the growth factor action of insulin in order
to prevent the production of unwanted or unfinished proteins.74 Although the mechanism remains elusive, amino acids regulate mTOR through the intervention of Rheb, but independently of TSC1/2.75 Regulation by 5 -AMP occurs at the level of AMP-activated kinase (AMPK) (Figure 18.14) leading to phosphorylation of the TSC1/2 complex on residues distinct from those phosphorylated by PKB (compare Figure 18.14 with Figure 18.11, also see Figure 18.13). This prevents its inactivation by PKB. Consequently, the Rheb-GAP activity of TSC1/2 remains elevated and protein synthesis is inhibited through failure to stimulate mTOR.
5 -AMP is not a direct activator of AMPK in the way that cyclic AMP activates PKA. Instead, similar to the activation of PKB by PI(3,4)P2, it acts as a recruiting agent. By binding to the -regulatory subunit of AMPK, 5 -AMP induces a conformational change that allows the interaction of the enzyme with LKB1, a constitutively activated kinase. This results in phosphorylation and activation of AMPK. The importance of this pathway in the regulation of protein synthesis is illustrated by the finding that lack of LKB1 activity predisposes to cancer
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FIG 18.14 Signalling through AMP kinase.
Hints of ATP depletion are manifested by the leakage of 5’-AMP from mitochondria which binds the regulatory subunit of AMPK. Subsequent phosphorylation by LKB1 leads to its activation. AMPK phosphorylates TSC2 thereby preventing the inhibitory control by PKB. The active TSC1/2 complex maintains Rheb in its GDP-bound state thus preventing activation of mTOR. Protein synthesis is suppressed.
(Peutz–Jeghers syndrome). LKB1 thus qualifies as a tumour suppressor. It prevents excessive stimulation of the mTOR pathway and in this way it
maintains strict control over protein synthesis.76 Further evidence for a tumour suppressor role of LKB1 comes from the finding that its activation, through the induction of its pseudokinase partner STRAD causes repolarization of transformed epithelial cells with the reformation of tight junctions. LKB1 thus appears to classify as both a polarity and a tumour suppressor gene.77
PI 3-kinase: regulator of cell size, proliferation, and transformation
The (p110) catalytic subunit of PI 3-K class I A is the only component of this family that has been implicated in cancer. Activating mutations or mutations that cause its over-expression are particularly frequent in colorectal cancers and in glioblastomas.78 Indeed, after K-Ras, it is the most mutated gene implicated in cancer. On the other hand, activating mutations in PKB are not associated with human cancers though over-expression has been detected (particularly PKB ), again predominantly in colorectal cancers.
The first indication that PI 3-kinase might act as a regulator of cell size and proliferation came from studies of Drosophila. Over-expression of Dp110
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FIG 18.15 Overexpression of Drosophila type I PI 3-kinase (Dp110) yields flies with giant eyes (and wings). From Leevers et al.85
(catalytic subunit of PI 3-kinase) results in enlarged wings or eyes. Conversely, kinase-defective mutants have very small organs79 (Figure 18.15). The PI 3- phosphatase, PTEN, correspondingly reduces both cell size and number.80 PKB stimulates those pathways that specifically regulate cell and compartment size independently of cell number.81
Why are PTEN inactivation mutants so often linked to malignancy, but TSC1/2 mutants only rarely so? PTEN has many roles. Not only does it regulate
cell size, but it is also a determinant of cell survival and it regulates the cell cycle through expression of cyclin D (acting in the G1 phase). Indeed, this may explain why its loss of function gives rise to a ‘higher level’ of cell transformation. A second explanation has emerged from a recent study in which mice lacking TSC2 manifest deficient signalling of the PKB pathway,
which may limit the transforming capacity. However, loss of a single copy of the PTEN gene, so-called haplodeficiency, restores the activation level of PKB and with it, its transforming consequences.82
Over-expression of eIF-4E can induce full tumorigenic transformation.83 It also leads to enlarged cells and increased proliferation rates and it can
rescue protein synthesis even when mTOR is inhibited by rapamycin.84 Not surprisingly, the PI 3-kinase/mTOR pathway is regarded as a promising target for therapeutic intervention and many derivatives of rapamycin are now being tested both in animal models and in clinical trials. Oncogenes and tumour suppressors linked to the PI 3-kinase/mTOR pathway are listed in Table 18.1.
Rapamycin is a triene macrolide antibiotic obtained from Streptomyces
hygroscopius, first dug up out of the earth of Rapa Nui (Easter Island). It binds to a cellular receptor FKBP12 and, together, they form a complex with mTOR and Raptor (mTORC1). This prevents interaction with substrates. Such inhibitory complex
formation does not occur when mTOR is associated with Rictor (mTORC2).
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