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FIG 18.7 Activation of PKB.
(a) PI(3,4)P2 serves as a membrane recruitment signal for both PKB and PDK1 (1). A first phosphorylation of PKB (by PDK2) occurs in the C-terminal hydrophobic motif (2). Close apposition of PKD1 causes binding of the phosphorylated HM to the C-helix of PDK1 (3), which, now fully competent, phosphorylates the activation segment of PKB so that full kinase activity is achieved (4). Detachment of activated PKB from the membrane may occur after dephosphorylation of PI(3,4)P2 by PTEN (5 and 6). (b) Domain architecture of the AGC kinases. The activities are controlled by paired phosphorylation sites, one in a hydrophobic motif, sometimes part of a larger autoregulatory domain, the other in the activation segment. For these kinases, PDK1 acts as the master switch by phosphorylation of the activation segment. AGC signifies AGC kinase C-terminal domain; other domains as in Figure 18.2.
PDK1, an AGC kinase, has an activation segment that is stabilized by autophosphorylation. It therefore only requires stabilization of theC-helix to express
full activity. This occurs through interaction with substrates that possess a phosphorylated hydrophobic motif such as other members of the AGC family of protein kinases. PDK1 may therefore be considered as a constitutively active protein kinase.
Activated PKB phosphorylates its substrates either at the membrane, or, following its detachment, in the cytosol. The viral oncogene product, v-Akt, has a lipid anchor (myristoyl group) and its attachment to the membrane may facilitate its activation or prevent its deactivation by phosphatases. It may also
prevent phosphorylations that ensure negative feedback, thus perpetuating the signal.
Insulin: the role of IRS, PI 3-kinase, and PKB in the regulation of glycogen synthesis
From the insulin receptor to PKB
Insulin signalling pathways all commence at the insulin receptor, INSR. The product of a single gene, this is post-translationally cleaved into an extracellular -subunit (containing a ligand binding site) and a transmembrane -subunit (which has a tyrosine protein kinase catalytic domain) (Figure 18.8). The mature functional receptor comprises four components, two and two , held together by disulfide bonds.
In common with the related IGF-1R and INSRR (an insulin-related orphan receptor), the insulin receptor is dimeric in its inactive state. In this respect, it differs from all other tyrosine kinase-containing receptors. Binding of the
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Phosphoinositide 3-Kinases, Protein Kinase B, and Signalling through Insulin Receptor
FIG 18.8 Insulin receptor structure.
(a) The insulin receptor is a homodimer. In the mature form, each component is present as two chains, and . Each monomer possesses seven domains, L1 (leucine rich region-1), CR (cysteine rich region), L2, FnIII-1 (fibronectin-III-like domain 1), FnIII-2, FNIII-3, and the intracellular tyrosine kinase domain (structure not shown). Both the and chains contribute to the FnIII-2 domain. The long insert region (shown as a dotted line) separating FnIII-2a from - 2b contains three disulfide bridges (2) that link the monomers. FNIII-1 provides a fourth disulfide bridge (1). b) A possible arrangement of the two peptides is shown. The insulin-binding space is between the central -sheet of L1 and the bottom loops of the FnIII-1 domain. (c) Domain architecture showing locations of the positions numbered in (a).
From McKern et al.42 (2dtg42).
ligand, insulin or IGF-1, must alter the receptor conformation or the relative position of the receptor subunits in order to allow the intracellular catalytic domains to approach each other (or to remove an inhibitory constraint). Three transphosphorylations in each activation segment ensue in orderly sequence. All are required for maximal activation.43 Further phosphorylations then occur in positions beyond the catalytic site (see Figure 18.9).
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FIG 18.9 Activation of PI 3-kinase by the insulin receptor.
Insulin binding to the receptor dimer (1) induces a conformational change (2) that causes transphosphorylation of the activation segments at three tyrosine residues (3). Further phosphorylation follows on both sides of the catalytic domain. The IRS-1 binds pY960 with its PTB domain (4). Phosphorylation on further tyrosine residues follows (5) generating a docking site for the SH2 domains of the p85 regulatory subunit and activation of PI 3-kinase (6) leading to the phosphorylation, at the 3-position, of inositide lipids (7).
A second peculiarity of the insulin receptor is that it signals through the intervention of large docking proteins. These insulin receptor substrates (IRS- 1–4) all possess PH and PTB domains. The PTB domains bind directly to the tyrosine phosphorylated region of the receptor, which corresponds to pY960 as illustrated in Figure 18.9, and results in phosphorylation of the docking protein. Of the four docking proteins, IRS-1 and -2 are essential for insulin signalling; lack of IRS-1 is linked to insulin resistance in muscle and adipose tissue. In mice, absence of IRS-2 causes insulin resistance in the liver (together with many other developmental defects).
The p85 regulatory subunit of PI 3-kinase (type IA) binds phosphorylated IRS-1/2 through its SH2 domain44 (Figure 18.9), so negating the inhibitory constraint imposed on the catalytic subunit and bringing the kinase into the vicinity of its substrate. Prolonged activation of PI 3-kinase and the production of 3-phosphorylated polyphosphoinositides encourage a number of serine/ threonine kinases to associate with the plasma membrane. Important among these is PKB.
The intervention of PI 3-kinase in the activation of PKB is well supported.45 It is quiescent in serum-starved fibroblasts but becomes active shortly after the addition of insulin or platelet-derived growth factor (PDGF). This fails in cells
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Phosphoinositide 3-Kinases, Protein Kinase B, and Signalling through Insulin Receptor
that contain a mutant form of PKB lacking the PH domain and depends on the presence of phosphotyrosines on IRS-1 or on the PDGF receptor. Note that PI(3,4)P2 rather than PI(3,4,5)P3 appears to activate PKB and that this correlates with the ability of both the intact kinase and its isolated PH domain to bind to this particular phospholipid.46 Of the three members of the PKB family, PKB plays the major role. PKB is not involved in insulin signalling.47
Similar to insulin, PDGF is able to activate PKB but is without immediate effect on glucose metabolism. Other factors must be in place to give direction
to the signal transduction pathways downstream of these receptors. For instance, the proximity of phosphorylated IRS-1 to the insulin receptor relays the activation of PKB in the sense of glucose metabolism. This connection is absent in the case of the PDGF receptor.
Lastly, note that the insulin receptor does not signal exclusively through the IRS, because it can also bind the adaptor APS. This regulates membrane expression of the glucose transporter GLUT-4.48 Moreover, p85a (regulatory subunit of group IA PI 3-kinases) can bind directly to the C-terminal end
of INSR.
From PKB to glycogen synthase
The minimum sequence motif required for efficient phosphorylation
of small peptides by PKB is RxRxx(S/T)(F/L) and there are several substrates in which this is present. With respect to insulin signalling, one such is glycogen synthase kinase-3, GSK3 . This has two unusual features. First, the activation segment is fully structured when dephosphorylated so that it needs no further modification to become active. Second, it recognizes only those substrates that offer an array of serine phosphorylation sites,
of which the most C-terminal is already phosphorylated (‘primed substrate’). (We considered a similar situation in relation to the phosphorylation of - catenin – see page 432). Phosphorylation of the most N-terminal serine
by PKB then exposes a 10-residue pseudo-substrate sequence that neatly fits the catalytic cleft of GSK3 (see Figure 18.10b). Having a proline, not serine, four residues upstream, it blocks rather than activates kinase activity.32,49 Importantly, inactivation of GSK3 necessarily reduces phosphorylation at regulatory serine residues on glycogen synthase, so that it becomes activated.
This alone, however, is insufficient to allow an abrupt onset of glycogen synthesis. In order for this to occur, it is also necessary to activate the protein phosphatase-1G (PP1G) which removes phosphate groups from glycogen synthase (Figure 18.10a). This is mediated through the action of the insulinstimulated protein kinase ISPK (not shown). The insulin-stimulated cell is now fully engaged in the synthesis of glycogen.
Diabetes mellitus affects 1–2% of people in Western societies, being especially prevalent among Hispanic and native American peoples. Type 1 (or early onset) diabetes is due to autoimmune destruction of the insulin-producingcells in the islets of Langerhans in the pancreas, and is the more severe form of
the disease. It accounts for most instances of blindness in Western populations. Multiple genes are involved that determine a
susceptibility, but not an inevitability, to express the condition that might be precipitated by a viral infection.
The more common type II (or late onset) form
of diabetes appears to be a correlate of good living. It is associated with obesity, though genetic factors undoubtedly play a part. It is characterized by resistance to insulin and indeed, the early stages are characterized by very high levels of
the hormone which are, however, insufficient to overcome the resistance.
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FIG 18.10 Insulin-mediated activation of glycogen synthase.
(a)Binding of insulin to its receptors leads to the activation of PKB. One of the substrates of PKB is glycogen synthase kinase-3 , which becomes inactivated and so unable to phosphorylate and inactivate glycogen synthase. Also, the phosphatase PP1G ensures rapid dephosphorylation and activation of glycogen synthase allowing glycogen synthesis to recommence. In addition, phosphorylase a is inactivated by dephosphorylation. Thus glycogenolysis is arrested.
(b)Detail of the inhibition of GSK3 by PKB. The N-terminal (pseudosubstrate) segment of GSK3 is most likely disordered, a wagging tail projecting out
of the kinase. When phosphorylated (at S9) it takes on the characteristics of a primed substrate and binds at the catalytic cleft. However, lacking a second serine at a position four amino acids upstream, the catalytic action is hindered and the peptide remains firmly attached. Access of other substrates is prevented. (c) Comparison of the N-terminal sequence of GSK3 with the C-terminal sequence of glycogen synthase. The lack of a second serine renders the phosphorylated N-terminal sequence a pseudo-substrate.
Insulin induces translocation of the GLUT4 transporter
Insulin is instrumental in the control of glucose uptake by the tissues, in particular muscle, fat, and those areas of the brain that regulate metabolic homeostasis.50 It does this by the recruitment of GLUT4 transporters to the plasma membrane from intracellular vesicular stores.
The mechanism by which this occurs is not entirely clear. The insulin acts at two levels, activating the Rap GTPase on the vesicles and then orchestrating the assembly of the exocyst complex at the plasma membrane. Activation of Rap occurs through the PI 3-kinase/PKB pathway and it involves phosphorylation and deactivation of RapGAP, so promoting the GTP-bound state. In this form it interacts with the docking proteins of the exocyst. Which of the many Raps initiates the docking process is not known. Insulin is also
involved in exocyst assemblage in a process that is independent of PI 3-kinase
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