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12.2: Receptors, Second Messengers and Kinases

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  • ACG Kinase


    put in iCn3D or morph pymol of inactive-active PrK ACG - find model


    The figure below shows the generic structure of simple ACG kinases in the active site, in which the C-helix in the N-lobe has been repositioned to form a salt-bridge with K72 to position it so it can stabilize the bond substrate by ion-dipole and H-bond interactions. T197 has been phosphorylated which  allows the active loop, which changes its conformation to a more local organized structure, allowing repositioning of active site side chains in the catalytic loop and allow substrate access.  The normal substrate would be a protein with a Ser or Thr presented for phosphorylation by bound ATP.



    Protein Kinase A



    Protein Kinase C



    Protein Kinase B also know as preferably as AKT

    There are three human AKT isozymes.  Each is a Ser/Thr AGC kinase, and are involved in cell growth, division, survival and metabolism.  As such it is activity/regulation is often aberrant in cancers, so it is a drug target. As with other members of the AGC Kinase family, it has a N-lobe, C-lobe, and catalytic and activation loops (described above, TBA).  What makes AKT so interesting its activation requires its recruitment to the inner leaflet of membranes by its binding to phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3 or more simply PIP3).  This is generated through through membrane receptors for insulin and growth factors, which on binding to their receptors activate phosphoinositide 3-kinase (PI3K), causing phosphorylation of phosphatidy inositols. Binding of PKB to inner leaflet PIP3 causes a conformational change activates AKT for phosphorylation by phosphoinositide-dependent kinase 1 (PDK1).  One activated the now active AKT dissociates from the membrane and acts enzymatically in the cytosol and nucleus (??). Hence, AKT has an extra domain, the pleckstrin homology (PH) domain, that binds PIP3.     

    The structure of AKT bound to a novel allosteric inhibitor is shown below (3o96).

    Key parts of the enzyme are shown as described below:


    • PH (at N-terminus, bind to PIP3) - Salmon main chain trace
    • N Lobe: light blue
    • C Lobe: light gray


    • Activation Loop:  dark blue
    • Catalytic Loop: red

    The blue activation loop is shown in two parts as the interior part, which contains T305 (equivalent to T197 in AGC kinases without a PH domain) is too disordered to resolve.  Table \(\PageIndex{1}\) shows some the numbering of key amino acid and features of generic AGC kinase and the corresponding numbers in AKT.  They are different since AKT has an N-terminal PH domain.

    Table \(\PageIndex{1}\)



    Akt (+108)

    N lobe



    N lobe



    C lobe, cat loop



    C lobe, cat loop



    C lobe, act loop



    C lobe, act loop



    missing in this structure)

    Approx Cat Loop




    Approx Act loop

    Start DFG (292-294) to APE



    308Tmiss toAPE end 319

    The allosteric inhibitor shown in the structure above especially interesting in that it requires the both the PH domain as well as the kinase domains for its effect.  

    The model below shows the structural overlap between the inactive form (shown above, 3o96 containing a bound allosteric inhibitor) with an active form of AKT(3cqw), which has a bound substrate (from glycogen synthase kinase-3 beta, yellow spacefill), and the inactive form (3o96) with the allosteric.  The bound decapeptide substrate (GRPRTTSFAE) in the active form becomes phosphorylated on the Ser chain by active AKT.

    The N-lobe is shown in cyan, while the catalytic loop is in red and the activation loop is in blue.  By pressing the "a" key you can toggle between the inactive 3o96 form and the active 3cqw from.  Note the large change in the blue activation loop.  


    The figure below shows a series of coupled equilibria reactions which regulate the activity of AKT1.  


    Citation: Wu W-I, Voegtli WC, Sturgis HL, Dizon FP, Vigers GPA, Brandhuber BJ (2010) Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition. PLoS ONE 5(9): e12913.  This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

    The top part of the figure shows the cytoplasmic, non-membrane bound form of the enyzme.  The PH domain is shown in orange.  The top-right figure shows the inactive N-and C-lobes of the kinase loosely interacting with an "out" (or away) conformation of the PH domain with respect to the kinase domains. In the presence of the allosteric inhibitor (green hexagon, green bound ligand),  the kinase domains tightly interacti with the PH domain in the "in" conformation. 

    The bottom three structures shows AKT bound to the membrane through the interaction of the PH domain with PIP3 (purple).  The bottom right kinase domains are identical in representation to the two in the top part of the figure, showing that they are inactive.  Only when bound to the membrane is AKT phosphorylated on Thr 308 (red in bottom middle figure), which activates the enzyme.  The bottom left and middle structures show the yellow kinase domains in a different conformation (3cqw),both of which are phsophorylated (red).The active form can bind ATP and protein substrates for phosphorylation.  It can also bound ATP analogs which would competitively inhibit the active form of the enzyme by occupying the ATP binding site. 

    The activation loop in the inhibited form is missing part of its sequence which reflects its disorder. In this state is partially occludes substrate interactions.  On phosphorylation of Ser 308 in the activation loop, the loop adopts a different conformation which allows less restricted access to the active.  The loop itself on phosphorylation has more local ordering as it shifts away from the active site as seen in the iCn3D model above. Another change decreases inhibitory noncovalent interactions of activation loop amino acids with catalytic residues, which increases catalytic efficiency.   In summary, these two types of changes in the activation loop leads to more access by substrates and enhanced catalysis of bound substrates.

    Additional regulation of the kinase occurs through the PH domain which adds additional conditions on Akt conformational changes and subsequent activity.  The PH domain "appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain".  

    Now let's look at an isolated PH domain (1unq) binding to the inner surface of a bilayer through its interaction with just the head group of PIP3, which is named inositol-(1,3,4,5)-tetrakisphosphate (ITKP).  Waters that are H bonded to the ligand are not shown in the model.

    Here is a close up of the interaction of a separate PH domain bound to INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE) without the PI  (1unq).  Waters H bonded to the ligands are not shown.


    Compared to all other structures of bound isolated PH domains, ITKP causes a large conformational change in the PH domain. These, d enable PKB to be activated by PDK1."

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