28.3: The Next step - The Kinome and Activation of Kinases at the Cell Membrane

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Learning Goals 

 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

The Kinome: Structure and Mechanism of Protein Kinases

• Describe the conserved structural features of active AGC kinase domains—including the activation loop, catalytic loop, αC-helix, and key residues (K72, E91, D166, D184, R165, pT197)—and explain how phosphorylation of the activation loop by an upstream kinase converts the enzyme from its inactive to its active conformation.
• Explain why most protein kinases are classified as RD kinases, and describe how the catalytic mechanism uses Asp as a general base, Mg²⁺ to stabilize ATP phosphates, and a conserved Lys-Glu salt bridge to position ATP for phosphoryl transfer.

cAMP-Dependent PKA and DAG/IP3-Dependent PKC

• Describe the allosteric activation of Protein Kinase A: explain how cAMP binding to the regulatory (R) subunits of the R₂C₂ holoenzyme causes dissociation of the catalytic (C) subunits, and identify the major cellular substrates phosphorylated by active PKA (including glycogen synthase, phosphorylase kinase, hormone-sensitive lipase, and CREB).
• Explain how phospholipase C-generated second messengers DAG and IP3 cooperate to activate Protein Kinase C: describe the roles of the C1 (DAG-binding), C2 (Ca²⁺-binding), C3 (ATP-binding), and C4 (substrate-binding) domains, and explain how an internal pseudosubstrate sequence maintains PKC in its autoinhibited state until membrane recruitment relieves inhibition.
• Identify PDK1 as a master upstream Ser/Thr kinase that phosphorylates the activation loops of PKA, PKC, and AKT, and explain why this shared regulatory step creates a point of convergence across multiple signaling pathways.

Receptor Tyrosine Kinases: Activation Mechanisms and Cancer Relevance

• Explain the cis-autoinhibition of RTK kinase domains by the activation loop, juxtamembrane region, and C-terminal sequences, and describe how ligand-induced dimerization leads to asymmetric trans-phosphorylation that activates the kinase domains and creates docking sites for SH2 domain-containing downstream effectors.
• Using EGFR/ErbB receptors as a case study, compare the dimerization and rotation/twist models of ligand-induced RTK activation, and explain how HER2 overexpression in breast cancer drives excessive signaling and how trastuzumab (Herceptin) and antibody-drug conjugates (T-DM1/Kadcyla) exploit HER2 overexpression therapeutically.

AKT (Protein Kinase B): Linking RTK Signaling to Phosphoinositide Metabolism

• Trace the activation pathway of AKT from RTK autophosphorylation → PI3K recruitment via SH2 domains → conversion of membrane PIP2 to PIP3 → AKT recruitment via its PH domain → PDK1-mediated phosphorylation of the activation loop, and explain how the phosphatases PTEN, PP2A, and PHLPP terminate AKT signaling at different points in this pathway.

In the previous section, we examined how cell surface GPCRs indirectly activate membrane-bound enzymes, adenylyl cyclase and phospholipase C, and how RTKs become active enzymes (kinases) upon ligand-induced dimerization. We will now explore what happens next in signaling by examining the products of these activated enzymes and how they continue the signaling process. The products of adenylyl cyclase (cAMP) and phospholipase C (diacylglycerol - DAG - and IP₃) are second messengers. These activate key protein kinases in the cell. The products of the activated RTKs are phosphoproteins (including themselves) that are phosphorylated on tyrosines by the RTK. Target proteins bind to the autophosphorylated RTKs through SH2 domains on the target protein. First, we will explore protein kinases and their general mechanisms.

The Kinome

There are 518 different protein kinases (about 1.7% of the human genome), and as a group, they are the key players in most eukaryotic signaling pathways. Collectively, they are referred to as the kinome. They help regulate every aspect of cellular life, including metabolism, cell growth, differentiation, cell division, and programmed cell death. In humans, most (388) are Ser/Thr kinases. There are 90 tyrosine kinases, and 40 are classified as atypical. In this section, we will focus on the AGC kinases (60 in total), which include Protein Kinase A (PKA), Protein Kinase C (PKC), and Protein Kinase B (PKB, also known as AKT). We'll wait for another section to explore Protein Kinase G (PKG), which is activated by cGMP, not AMP. We'll also study the receptor tyrosine kinases (RTKs)

Luckily, kinases can be grouped into families based on their structures and mechanisms. The ones directly activated by the second messengers cAMP (Protein Kinase A) and DAG (Protein Kinase C) are members of one family of kinases, the AGC Protein Kinase family. The clustered families of protein kinases are shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): The family of protein kinases. https://peerj.com/articles/126/. Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com)

Luckily, most kinases, which catalyze the phosphorylation of OH-containing amino acid side chains (Tyr, Ser, and Thr) on proteins, have similar catalytic sites. Given that phosphorylation of proteins often acts as an on/off switch for protein activity and function, it makes great sense that the catalytic site of protein kinases is not available for substrate binding and/or catalysis in the off state. Protein kinases have catalytic and activation loops, and conformational changes upon activation typically involve movement of the activation loop and rearrangement of the catalytic residues. These conformational changes are often initiated by phosphorylation of key groups in the activation loop by an upstream active protein kinase! Figure \(\PageIndex{2}\) illustrates another representation of the various families of protein kinases and their shared structural features.

Figure \(\PageIndex{2}\): Family organization and common structural features of protein kinases. Trends in Pharmacological Sciences, November 2019, Vol. 40, No. 11 https://doi.org/10.1016/j.tips.2019.09.002"https://doi.org/10.1016/j.tips.2019.09.002. Creative Commons Attribution (CC BY 4.0)

Pane (A) displays the nine eukaryotic and one atypical protein kinase groups, along with their respective numbers of kinases, structures, and inhibitors (indicated in black), as well as mutations (in red) and single-nucleotide polymorphisms (SNPs) (in green). Here, the atypical protein kinases are defined as all non-eukaryotic protein kinases, including non-protein kinase-like kinases. The middle of Panel (B) illustrates the activation and catalytic loops, as well as other common features of protein kinase active sites. The left and right parts of Panel (B) show two different kinases. The protein kinase domain of epidermal growth factor receptor (EGFR) is shown on the left, and the Ser/Thr protein kinase domain of mTOR (mammalian or mechanistic Target Of Rapamycin), a protein we will explore in a separate section, is shown on the right.

AGC Kinases

These serine/threonine kinases are activated by the second messengers cAMP (A Kinase), cGMP (G kinase, which we will see later), and by diacylglycerol (DAG) formed by activation of phospholipase C. Figure \(\PageIndex{3}\) shows the generic structure of active kinase domains (including tyrosine kinases)

Figure \(\PageIndex{3}\): Generic structure of the active form of ACG kinases (Adapted from the superlative text, Cell Signaling by Lim, Mayer, and Pawson)

A generic kinase has an N-terminal and C-terminal lobe between which the substrates ATP and the target protein containing the key serine, threonine, or tyrosine side chain bind. In the active conformation, Glu 91 (E91) in the αC-helix of the N-lobe forms a salt bridge (ion-ion interaction) with Lys72, positioning it to stabilize ATP binding through ion-dipole and Hydrogen-Bond interactions. In the active state, Thr 197 (T197) in the activation loop is phosphorylated (pT197) by an upstream kinase, resulting in a less flexible loop conformation. This facilitates optimal repositioning of active site side chains in the catalytic loop, enabling substrate access, and catalysis. Asp 166 (D166) in the catalytic loop acts as a general base in abstracting a proton from the target Ser, Thr, or Tyr. Arginine 165 (R165) in the catalytic loop forms a salt bridge with phosphotyrosine 197 (pT197), stabilizing the catalytically competent conformation of the activation loop. Aspartic acid 184 (D184) plays a key role in stabilizing Mg²⁺, which in turn stabilizes the negative charges on the phosphates of ATP and in the developing transition state.

Most kinases are also classified as RD kinases since they have a key arginine (R)- aspartic acid (D) sequence in the catalytic loop. The normal protein substrate for an AGC kinase would be a protein with a Ser or Thr projecting into the active site for phosphorylation by bound ATP. The normal sequence for phosphorylation in protein targets of PKA is Arg-Arg-X-Ser/Thr-X (or more fully as XR(R/K)X(S/T)B, where B is a hydrophobic amino acid). Again, we will focus on three AGC kinases here: PKA, PKC, and AKT (also known as PKB), and defer our discussion of PKG to another section.

cAMP-dependent Protein Kinase A - PKA

Before studying PKA, the formation of the second messenger cAMP is reviewed in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Synthesis of cAMP on activation of GPCRs and adenylyl cyclase

PKA contains a kinase domain with short N- and C-terminal extensions, making it a prototypical and simple model for study. In the absence of the second messenger cAMP, this prototypic AGC kinase exists as a holo-heterotetramer (or dimer of a heterodimer). It contains two catalytic kinase subunits (C) and two regulatory subunits (R). cAMP produced upon GPCRs activation of adenylyl cyclase binds to the regulatory subunits, causing them to dissociate from the heterotetramer, freeing the catalytic subunits for activity. This is illustrated in Figure \(\PageIndex{5}\).

Figure \(\PageIndex{5}\): Activation of Protein Kinase A by the second messenger cAMP

The cAMP serves as an allosteric activator of protein kinase A.

Figure \(\PageIndex{6}\) shows the actual structures of the protein kinase A RIIb tetrameric holoenzyme (3TNP)

Figure \(\PageIndex{6}\): Protein Kinase A RIIb tetrameric holoenzyme (3TNP)

The inhibited catalytic subunits are shown in red, and the two regulatory subunits are shown in cyan.

Figure \(\PageIndex{7}\) shows only one catalytic subunit of protein kinase A as it goes from the more closed inactive holoenzyme (tetramer, 3tnp) containing the regulatory subunits (not shown), to the more open apo-form (monomer, 1J3HA), without the regulatory subunit. The conformational change opens the free catalytic subunit to substrate binding, including protein and ATP.

Figure \(\PageIndex{7}\): Conformational change on the conversion of the catalytic subunit of protein kinase A from the more closed inactive holoenzyme (tetramer, 3tnp) containing the regulatory subunits to the more open apo-form (monomer, 1J3HA) without the regulatory subunit

Side chains required for MgATP binding and phosphoryl transfer are pre-formed in the apo form, but some changes still occur on the binding of substrate.

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of the mouse catalytic subunit of cAMP-dependent protein kinase complexed with MnATP and a peptide inhibitor (1ATP)

Figure \(\PageIndex{8}\): Catalytic subunit of cAMP-dependent protein kinase complexed with MnATP and a peptide inhibitor (1ATP) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...B8Xyks9kMdbZN9

Zoom in on the model to view each labeled residue. All of the features shown in Figure \(\PageIndex{3}\) are displayed in this model. The activation loop is shown in dark blue, and the catalytic loop is shown in red. The peptide inhibitor is shown in cyan. The normal target motif for phosphorylation by Protein Kinase A (Arg-Arg-X-Ser/Thr-X) has been replaced in this model by Arg-Arg-X-Ala-X. Why does this make the peptide an inhibitor instead of a substrate?

Many primary signals activate adenylate cyclase through G protein-coupled receptors (GPCRs), producing cAMP as a secondary messenger. These include corticotropin, dopamine, epinephrine (β-adrenergic), follicle-stimulating hormone, glucagon, many odorants, prostaglandins E1 and E2, and many tastants. All of these would activate protein kinase A. Some enzymes regulated by cAMP-dependent phosphorylation by PKA are shown in Table \(\PageIndex{1}\) below

Enzyme	Pathway
Glycogen Synthase	Glycogen synthesis
Phosphorylase Kinase	Glycogen breakdown
Pyruvate Kinase	Glycolysis
Pyruvate Dehydrogenase	Pyruvate to acetyl-CoA
Hormone-sensitive Lipase	Triacylglycerol breakdown
Tyrosine Hydroxylase	Synthesis of DOPA, dopamine, and norepinephrine
Histone H1	Nucleosome formation with DNA
Histone H2B	Nucleosome formation with DNA
Protein Phosphatase 1 Inhibitor 1	Regulation of protein dephosphorylation
CREB	cAMP regulation of gene expression
PKA consensus sequence	XR(R/K)X(S/T)B (B = hydrophobic amino acid)

Table \(\PageIndex{1}\): Proteins phosphorylated by Protein Kinase A.

An example of how epinephrine (a flight or fight hormone) can lead to the breakdown of glycogen (your main carbohydrate reserves in muscle and liver) is shown in Figure \(\PageIndex{9}\).

Figure \(\PageIndex{9}\): Cascade activation of glycogen phosphorylase through GPCR and cAMP signaling.

A cascade of events begins with the hormone binding to its receptor, followed by the activation of adenylate cyclase and the formation of the second messenger cAMP. This activates PKA, which phosphorylates and activates the enzyme phosphorylase kinase. Following the naming convention we discussed earlier, phosphorylase kinase is a protein kinase whose target enzyme is glycogen phosphorylase, which is NOT a kinase. When active, glycogen phosphorylase uses inorganic phosphate (P_i) as a nucleophile in a phosphorolysis reaction to cleave glucose from the end of glycogen polymers, forming glucose-1-phosphate. This is the first step in mobilizing glycogen as an energy reserve. It is no wonder that a complex, highly regulated signaling pathway governs this process.  

The second messengers DAG and IP_3, and their activation of Protein Kinase C

The activation of protein kinase C (PKC, a Ser/Thr kinase member of the AGC protein kinase family) is very similar to that of protein kinase A. To start, an extracellular signal molecule binds to a GPCR receptor (again, with no intrinsic enzyme activity), triggering a conformational change in the receptor that propagates across the membrane to its intracellular domain. That then triggers the exchange of GDP with GTP on the alpha subunit of the bound heterotrimeric G protein, which contains the specific G_αq subunit. The G_αq subunit dissociates and binds to the membrane protein enzyme phospholipase C (not adenylyl cyclase). Once activated, it cleaves the phospho-head group from the membrane phosphatidyl inositol-4,5-bisphosphate (PIP2) into two second messengers - diacylglycerol and inositol trisphosphate (IP3). These products are shown in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): Cleavage of phosphatidyl inositol-4,5-bisphosphate (PIP2) into two second messengers - diacylglycerol and inositol trisphosphate (IP₃)

The formation of the second messengers IP₃ and DAG is presented in Figure \(\PageIndex{11}\). Note that phospholipase C is a peripheral membrane protein bound to the inner leaflet of the cell membrane.

Figure \(\PageIndex{11}\): Synthesis of DAG and IP₃ from PIP₂ by phospholipase C

Diacylglycerol binds to and activates protein kinase C (PKC). The IP₃ binds to ligand-gated receptors/Ca²⁺ channels on intracellular membranes, leading to an influx of calcium ions into the cytoplasm. The released calcium ions also activate protein kinase C (PKC). These steps are illustrated in Figure \(\PageIndex{12}\) in more detail below. The activation of the peripheral membrane phospholipase C (PLC) is very similar to that of the integral membrane protein adenylyl cyclase, both of which produce second messengers.

Figure \(\PageIndex{12}\): GPCR activation of phospholipase C, generation of second messengers IP₃, DAG, and Ca²⁺ ions, and downstream activation of Protein Kinase C

You might also expect the regulation of the activation of protein kinase C (PKC) activity to be similar to the regulation of the activation of protein kinase A (PKA). It is, but with a major difference. In contrast to the structure of PKA, which cycles between an inactive R₂C₂ (R is the regulatory and C the catalytic subunit) and an active separated form, PKC is a single chain that has a regulatory domain and a catalytic domain, as shown in Figure \(\PageIndex{13}\). Variants of PKC are also shown.

Figure \(\PageIndex{13}\): Domain structure of conventional, novel, and atypical protein kinase Cs. Kaleli et al. Cells 2020, 9(3), 553; https://doi.org/10.3390/cells9030553https://www.mdpi.com/651098Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Protein kinase C was named "C" because of its activation by Ca²⁺ ions, but you could also remember it because it requires the upstream activation of phospholipase C.

There are more than 500 PKCs.  They are divided into 15 subgroups, whose downstream functions ultimately regulate gene expression. They all have four common domains, C1-C4. C1 and C2 are the functional regulatory domains, and C3 and C4 are in the overall catalytic domain. They have the following activities.

• C1 binds diacylglycerol (DAG) and phorbol esters (commonly known activators of PKC)
• C2 binds Ca²⁺, another second messenger, which activates the protein; Novel PKCs use DAG and phosphatidyl ethanolamine but not Ca²⁺ to activate the protein, while atypical ones use only DAG.
• C3 binds ATP
• C4 binds target proteins for phosphorylation.

The structure of the phorbol 12-myristate 13-acetate, which activates PKC, is shown in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\): Phorbol 12-myristate 13-acetate

In a novel approach to keep the protein inactive, it contains a small peptide sequence (a pseudosubstrate) that mimics the amino acids surrounding the target phosphorylation site (at the target Ser/Thr). This binds in the active site of the inactive form of PKC, acting as a competitive inhibitor and preventing PKC activity.

PKCα is found in the cytoplasm, cell membrane, nucleus, and mitochondria. Its activation by DAG suggests it becomes localized to membrane surfaces. The protein is conformationally very flexible (note the hinge domain in Figure \(\PageIndex{13}\)), making it challenging to obtain detailed structures.

Figure \(\PageIndex{15}\) shows an interactive iCn3D model of the PKC (alpha)-C2 domain complexed with Ca²⁺ and PtdIns(4,5)P2 (IP3) (PDBID 3GPE). The hydrogen bonds and ion-ion interaction between the C2 domain and IP3 are detailed and labeled.

Figure \(\PageIndex{15}\): PKC (alpha)-C2 domain complexed with Ca²⁺ and PtdIns(4,5)P2 (IP3) (3GPE) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...gX24dYC1NhSXi7

Protein kinase C is targeted to the membrane (as shown in Figure \(\PageIndex{12}\)), but it lacks the Pleckstrin Homology (PH), which most phospholipase Cs use to interact with PIP₂ in the membrane.

Before PKC activation by DAG and Ca²⁺, it must be phosphorylated sequentially. Before these phosphorylations, PKC binds loosely to the membrane with the activation loop open, allowing it to be phosphorylated by kinases. These are the subsequent steps:

1. phosphorylation of Thr500 (PKC βII) in the activation loop of PKC by an upstream kinase, PDK1 (a kinase which also phosphorylates other AGC kinases such as AKT discussed below). This kinase can bind to the exposed activation loop
2. autophosphorylation at the C-terminus of PKC, which leads to conformational positioning of side chains needed for catalysis and substrate binding, and access to the substrate binding site

PKC engages in a very dynamic cycle. It begins as the inactive cytoplasmic form, which is autoinhibited by its pseudosubstrate sequence. It then moves to the membrane, where the autoinhibition is relieved. All of this requires flexibility, which makes it difficult to determine its structure. Figure \(\PageIndex{16}\) shows how the inactive cytoplasmic form of PKC becomes activated at the cell membrane.

Figure \(\PageIndex{16}\): Conversion of the cytoplasmic inactive PKC to its membrane-bound active form (after Tatyana I. Igumenova. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979571/)

The regulatory domain, which contains the C1 (DAG-binding) and C2 (Ca²⁺) domains, binds to the membrane, freeing and activating the kinase domain upon release of the bound internal pseudosubstrate.

Figure \(\PageIndex{17}\) shows the optimal consensus sequence flanking both sides of the phosphorylation site in target proteins (based on model peptides) and the sequence of the internal pseudosubstrate motif for several PKCs.

Figure \(\PageIndex{17}\): Optimal consensus substrate and actual internal pseudosubstrate sequences for PKC. Nishikawa et al. JBC. 272, 952–960, 1997. https://creativecommons.org/licenses/by/4.0/

Boxed amino acids show structural similarity between the target and internal pseudosubstrate. Position 0 indicates the serine that is phosphorylated in the target. Note that it is replaced with alanine in the pseudosubstrate motif. Also, note the abundance of positively charged side chains.

Figure \(\PageIndex{16}\) also shows that phosphorylation of key side chains facilitates the activation of the enzyme. The upstream kinase 3-phosphoinositide-dependent protein kinase 1 (PDPK1 or PDK1) phosphorylates the key Ser/Thr in the activation loop, as we discussed above. PDK1 is a "master" Ser/Thr kinase that phosphorylates and activates many proteins, including PKA, PKC, and protein kinase B (which we will explore below).

Figure \(\PageIndex{18}\) shows an interactive iCn3D model of the Protein Kinase C beta II (3PFQ)

Figure \(\PageIndex{18}\): Protein Kinase C beta II (3PFQ) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ym5qJhtdWsdN96

The C2 domain (magenta)has two bound Ca²⁺ ions (gray spheres) and interacts with the bottom leaflet of the cell membrane. The C1 domain (purple) has two bound Zn²⁺ ions. The N lobe of the kinase domain is shown in green, and the C lobe is shown in brown. A nonhydrolyzable ATP analog, AMPPNP (ANP), is shown in spacefill between the N and C lobes. There is an additional NFD helix preceding the C-terminal tail, which can adopt two positions, one of which confers low activity and the other high PKC activity. The Phe629 in this region is outside the active site in the low-activity form and interacts with the adenine of ATP in the high-activity form.

Some signals that activate phospholipase C and produce IP3 and diacylglycerol include acetylcholine (a different class from the type located at the neuromuscular junction discussed in the last chapter), angiotensin II, glutamate, histamine, oxytocin, platelet-derived growth factor, vasopressin, gonadotropin-releasing hormone, and thyrotropin-releasing hormone.

In the inactive form of PKC, the arginine-rich basic autoinhibitory pseudosubstrate interacts with acidic side chains in the substrate binding site.An acidic patch in the substrate-binding site (Figure 6.2). When PKC is activated by phosphorylation of the regulatory domain, it phosphorylates arginine-rich sites in protein substrates. This also releases pseudosubstrates from some inactive target protein kinases, allowing them to become active kinases in turn. PKC physiological substrates include receptors, cytoskeleton proteins, protein kinases, proteases, and nuclear proteins

The Ca²⁺ ions also act as second messengers. The calcium ions bind to the calcium-modulatory protein calmodulin, which, in turn, binds to and activates the calmodulin-dependent kinase (CAMK), a topic we will discuss later. Some kinases regulated by calcium and calmodulin include myosin light chain kinase, PI-3 kinase, and CAM-dependent kinases. Ca/CAM also regulates other proteins, which include: adenylate cyclase (brain), Ca-dependent Na channel, cAMP phosphodiesterase, calcineurin (phosphoprotein phosphatase 2B), cAMP-gated olfactory channels, NO synthase, and plasma membrane Ca/ATPase.

Protein Phosphorylation by Activated Receptor Tyrosine Kinases (RTKs)

Figure \(\PageIndex{19}\) shows the dimeric structure of RTKs driven by extracellular signal binding.

Figure \(\PageIndex{19}\): Dimeric structure of selected RTKs driven by extracellular signal binding. Sareshma Sudhesh Dev et al. Front. Pharmacol., 15 November 2021 | https://doi.org/10.3389/fphar.2021.772510. Creative Commons Attribution License (CC BY).

Table \(\PageIndex{2}\) below shows the classification of RTKs into classes and families.

Table \(\PageIndex{2}\):EGFR: epidermal growth factor receptor; InsR: insulin receptor; PDGFR: platelet-derived growth factor receptor; VEGFR: vascular endothelial growth factor receptor; FGFR:fibroblast growth factor receptor; CCK: colon carcinoma kinase; NGFR, nerve growth factor receptor; HGFR: hepatocyte growth factor receptor; EphR: ephrin receptor; Axl: from the Greek word anex-elekto, or uncontrolled, a Tyro3 protein tyrosine kinase; TIE:tyrosine kinase receptor in endothelial cells; RYK: receptor related to tyrosine kinases; DDR: discoidin domain receptor; Ret: rearranged during transfection; ROS: RPTK, expressed in some epithelial cell types; LTK: leukocyte tyrosine kinase; ROR: receptor orphan; MuSK: muscle-specific kinase; LMR: Lemur. Sareshma Sudhesh Dev et al. Front. Pharmacol., 15 November 2021 | https://doi.org/10.3389/fphar.2021.772510. Creative Commons Attribution License (CC BY).

We introduced RTK activation in the previous section and noted that ligand-induced dimerization led to their activation. Let's expand on that now. As in the case of PKC, the intracellular kinase domains of RTKs are inhibited by specific structures within their chains, known as cis-autoinhibition. These include the activation loop, the C-terminal sequences, and the region linking the C-terminal domain to the transmembrane domain. This is called the juxtamembrane region. All of these must be phosphorylated to activate kinase activity. Autoinhibition is relieved upon ligand binding and dimerization. The kinase domain of one dimer can allosterically activate the kinase domains of the other dimer. Each domain phosphorylates the cytoplasmic domain of the other, so it's called trans-phosphorylation (i.e., a kinase domain on one monomer does not autophosphorylate itself, which would be called cis-phosphorylation). The now-active kinase domains recruit target proteins containing SH2 domains, which bind to p-Tyr peptides or proteins, thereby propagating signaling. These include proteins involved in other signaling pathways, such as the MAPK and phosphoinositide-3-kinase (PI3K)/Akt pathways, which we will discuss later. An RTK also activates a particular phospholipase Cγ (PLC-γ).

Cancers can arise when the RTK signaling pathways, which regulate cell growth and division, become overactive. Figure \(\PageIndex{20}\) shows four different mechanisms for the expression and/or activation of RTKs that could lead to cancer.

Figure \(\PageIndex{20}\): Possible mechanisms for expression and/or activation of RTKs in cancer cells. Dev et al. Front. Pharmacol., 15 November 2021 | https://doi.org/10.3389/fphar.2021.772510. Creative Commons Attribution License (CC BY).

We will focus on the epidermal growth factor receptor (EGFR) for the remainder of our discussion. One of the most interesting questions is how ligand binding in the extracellular domain of the biotopic integral membrane proteins leads to an intracellular signal in the cytoplasmic domain. It isn't easy to imagine such an activation propagating through a single transmembrane helix. We will discuss how ligand-promoted dimerization of the receptor appears to be the primary mechanism of activation for RTKs.

Figure \(\PageIndex{21}\) shows the domain structure of three different RTKs, including the insulin receptor (IR) family. The insulin receptor is also synthesized as a single chain but undergoes proteolysis and interchain disulfide bond formation to give the "dimeric" structure shown below.

Figure \(\PageIndex{21}\): L,leucine-rich; CR, cysteine-rich; Ig, immunoglobulin-like; FnIII, fibronectin type III; ID, insert domain. The L1, CR1, L2, and CR2 domains of the ErbB family are alternatively termed Domains I–IV. Ichiro N. MaruyamaCells. Cells. 2014 Jun; 3(2): 304-330. doi: 10.3390/cells3020304. (http://creativecommons.org/licenses/by/3.0/). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092861/

The EGFR is a member of the ErbB family, which comprises four members: EGFR (also known as Erb1 or HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). The name ErbB arises from the avian ERythroBlastosis oncogene B. They are also called HER after the Human Epidermal Growth Factor Receptor). The HER2 gene is often dysregulated in breast cancer. Members of the ErbB family have unique numbers and positions of tyrosine in the C-terminal kinase domains. EGFR has 20 tyrosine residues, of which 12 are phosphorylated. The EGFR is also unique in that it has only one tyrosine in the activation loop that is phosphorylated; however, the tyrosine itself is not required for kinase activity. Although we suggested earlier that RTKs are activated upon dimerization, studies show that RTKs. However, an increasing number of studies demonstrate that RTKs exist as inactive dimers in the absence of ligand.

As shown in Figure \(\PageIndex{21}\), ErbB receptors have four extracellular domains, a transmembrane domain, the juxtamembrane region (about 40 residues), the cytoplasmic kinase domain, and a C-terminal extension that gets autophosphorylated and binds downstream target protein through its SH2 domains. Extracellular domains I ( L1) and III/L2 have β-helix solenoid secondary motifs that bind the ligand. Domains II/CR1 and IV/CR2 are cysteine-rich disulfide bonds. Some fraction (>80% by cross-linking studies) of ErbB receptors exists as dimers at the cell surface in the absence of ligand, in contrast to the older view that ligand binding is required for dimerization.

The structure of the extracellular domains of the free ErbB and ligand-bound EGFR receptors shows conformational changes that are required for dimer formation. In the absence of the ligand, a "tether" arm, denoted by an open triangle in domain IV in Figure \(\PageIndex{22}\), is close to a buried "dimerization" arm (asterisk *) in domain II of the extracellular regions of EGFR, ErbB3, and ErbB4, effectively inhibiting dimerization. When the ligand is bound, domains I and III interact, freeing the dimerization domains to interact (** in EGFR). These features are illustrated in Figure \(\PageIndex{22}\).

Figure \(\PageIndex{12}\): Schematic representations of the structures of the extracellular regions of the ErbB family. EGFR, ErbB3, and ErbB4 adopt the tethered conformation in the absence of ligand. In contrast, ErbB2 adopts an extended, or untethered, conformation that resembles the ligand-activated, dimerization-competent EGFR protomer in the ligand-bound form of the EGFR dimer, shown at the right. The ‘dimerization arm’ and ‘tethering arm’ are shown by an asterisk * and an open triangle, respectively. Ligands are shown in red. Domains I–IV correspond to the domains shown in Figure \(\PageIndex{21}\). Not drawn to scale. Ichiro N. MaruyamaCells. Cells. 2014 Jun; 3(2): 304–330. doi: 10.3390/cells3020304' (http://creativecommons.org/licenses/by/3.0/). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092861/

Additionally, the tether arms on both monomers now interact with each other in the dimer. Note that the ligand binding region is not involved in dimer formation and is not in the dimer interface, as it is with other RTKs, where the ligand is involved in direct contact in the dimer interface. Mutations in the II/IV domains that inhibit their interactions do not lead to receptor activation, so ligand binding is still required. ErbB2 exists in an extended conformation (no ligand is known for it), so it is free to interact with another ErbB chain in a heterodimer.

Structural studies now suggest that the EGFR kinase dimer has a symmetrical inactive conformation in which the activation loop is packed and occludes the active site. Additionally, it has an asymmetrical active one in which the activation loop is unpacked and the active site is open. How is this transmitted across the subunits? It appears that the C-lobe of the "activator/donor" kinase interacts with the N-lobe of the adjacent "receiver/acceptor" kinase, which activates it through a conformational change. When the ligand binds, the inactive dimer eventually leads to the asymmetric active dimer.

Two models have been proposed for ligand-gated activation of the Erb dimers: the dimerization and rotation/twist models, as shown in Figure \(\PageIndex{23}\).

Figure \(\PageIndex{23}\): Models for ligand-induced activation of the ErbB family. (A) ‘Dimerization’ model. (B) ‘Rotation/twist’ model. Not drawn to scale. Ichiro N. MaruyamaCells. Cells. 2014 Jun; 3(2): 304–330. doi: 10.3390/cells3020304' (http://creativecommons.org/licenses/by/3.0/). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092861/

• A. Dimerization model: Ligand binding to the I and III extracellular domains of a monomer leads to dimerization by releasing the tether arm between I and III, allowing the dimerization arms to become free and interact with each other. This causes the kinase domains to adopt the active state
• B. Rotation/Twist model: The receptor is already a dimer in the unliganded state, with the extracellular region untethered and the intracellular kinase domains in an inactive symmetric state. Ligand binding causes the two dimerization arms to extend, causing a twist in the transmembrane segment. This causes the kinase domains to adopt an active, asymmetric state, with the activator kinase domain of one monomer activating the receiver kinase domain of the second monomer in the dimer, resulting in each forming an extended conformation.

Figure \(\PageIndex{24}\) shows an interactive iCn3D model of the intracellular dimeric EGFR kinase domains in complex with an ATP analog-peptide conjugate (2GS6)

Figure \(\PageIndex{24}\): Dimeric intracellular EGFR kinase domains in complex with an ATP analog-peptide conjugate (2GS6) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...iw1V4T8rsfxDZ7

The two EGFR kinase domains are shown in cyan and magenta. The ATP analogs in each domain (spacefill) are thiophosphoric acid O-((adenosyl-phospho)phospho)-S-acetamidyldiester. The peptide substrates (green sticks) are 13-mers with a tyrosine (sticks, labeled Y, minus the OHs) connected to the ATP analog.

Figure \(\PageIndex{25}\) shows an interactive iCn3D model of a single EGFR kinase domain showing N and C terminal lobes in complex with an ATP analog-peptide conjugate (2GS6)

Figure \(\PageIndex{25}\): A single EGFR kinase domain showing N and C terminal lobes in complex with an ATP analog-peptide conjugate (2GS6) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...VLu1jPJYJNoih9

The ATP part of the ATP-peptide conjugate is sandwiched between the N-lobe (green) and the C-lobe (brown), just as in PKA. The ATP in the peptide conjugate is shown in spacefill, while the peptide is shown in gray. The tyrosine (Y) linked to the peptide is labeled.

Figure \(\PageIndex{26}\) shows an interactive iCn3D model showing the similarity in structure between the PKA kinase domain (1J3H A chain) and the EGFR kinase domain (2GS6)

Figure \(\PageIndex{26}\): Alignment of the PKA kinase domain (1J3H A chain) and the EGFR kinase domain (2GS6) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?mVfpwk3Y7EDcM5mq9

The gray structure by itself is the second kinase domain of the EGFR dimer. The superimposed chains are on the other side. Red indicates identical residues, and blue nonconserved residues in the structural alignment. Zoom in on the aligned sequences in blue and red to show how similar the kinase domains are.

In the next chapter section, we will explore the downstream effects of signaling mediated by the second messengers cAMP, DAG, and IP3, as well as the substrates phosphorylated by ligand-activated receptor tyrosine kinases.

EGFR and HER2 in breast cancer

The HER2 receptor can form homo or heterodimers with single-chain ErbB1-ErbB4 (same as HER1-HER4). HER receptors exist as both monomers and dimers, either homo- or heterodimers. Rapid dimerization of HER1, HER3, or HER4 occurs if they form heterodimers with HER2. In addition, any ErbB dimer with HER2 leads to strong intracellular signaling compared to other HER heterodimers. Ligand binding to HERI, HER3, or HER4 induces rapid receptor dimerization, with a marked preference for HER2 as a dimer partner. Since noncancer cells have very little HER2, correspondingly few HER2 heterodimers are found. If HER2 is overexpressed, as in HER2+ breast cancer cells, more heterodimers form, and signaling levels become anomalously high. This resulted in a poorer prognosis for HER2+ breast cancers in the past.

A revolution in breast cancer therapy has changed that situation. Humanized antibodies, which are human antibodies produced in mouse cells, targeting HER2 are now used in treatment. These antibodies are known as trastuzumab (Herceptin). Since the antibodies are derived from human genes, they are not targeted as foreign by the immune system. In early-stage HER2+ breast cancers, the antibody trastuzumab (Herceptin) is administered intravenously periodically over a 1-year period. The antibody selectively binds to HER2 on breast cancer cells. Once bound, the Fc portion of the antibody signals the immune system, recruiting immune cells and modulators to the tumor cell, ultimately resulting in its destruction.

If the cancers are at a later stage or if tumor cells are found in lymph nodes, a variant of the anti-HER2 antibody can be administered with a chemotherapeutic drug covalently attached to it. The structure of the drug Ado-trastuzumab emtansine (T-DM1), also known as Kadcyla, is shown in Figure \(\PageIndex{27}\).

Figure \(\PageIndex{27}\): Ado-trastuzumab emtansine (T-DM1) and its interaction with HER2 positive tumor cells. Sadeghi et al. Pharmacogenomics and Personalized Medicine. 2014 Oct 15;7:329-38. doi: 10.2147/PGPM.S47524. Creative Commons Attribution – Non Commercial (unported, v3.0) License. http://creativecommons.org/licenses/by-nc/3.0/.

When the antibody-drug complex binds to receptors on cancer cells, it is taken up by the cells. The antibody is degraded, releasing the toxic drug into the cell, sparing all other cell types (only those with the receptor are targeted). The released drug binds to microtubules, which are part of the cell's internal cytoskeleton, and prevents the changes necessary for cell division. HER2 is expressed at very low levels in noncancer cells, which can lead to side effects. However, these are much less severe than traditional chemotherapy, which targets all dividing cells. The antibody acts as a homing device, bringing the attached chemotherapy predominantly to tumor cells. It can be likened to a smart bomb or a cruise missile guided to just one target.

A better image of DMI and the linker connecting to the antibody is shown in Figure \(\PageIndex{28}\). The toxic chemotherapy drug is chemically linked to the antibody via a non-reducible thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

Figure \(\PageIndex{28}\): Ado-trastuzumab emtansine (T-DM1) conjugate used in HER2+ cancers

Figure \(\PageIndex{29}\) shows a mechanism for the actual cross-linking reaction. The reaction of the maytansine derivative through its free thiol and the human antibody (trastuzumab) through a free amine should be readily understandable based on the reactions present in Chapter 2.

Figure \(\PageIndex{29}\): Cross linking reactions to connect emtansine and trastuzumab

The final linker after the leaving of the N-hydroxysuccinimide is designated MCC.

The C-MET receptor, illustrated in Figure 19, is a receptor tyrosine kinase (RTK) involved in cell proliferation and survival; as such, alterations in its expression can lead to cancer development. Its mature form results from selective proteolysis by furin. Its physiological ligand is hepatocyte growth factor (HGF), a multisubunit protein in its mature form. It also binds a naturally occurring smaller splicing variant of HGF called NK1. The structure of C-MET bound to both HGF and NK1 has been solved. Binding of either leads to dimerization of the C-MET receptor, as illustrated in the cartoon representation of Figure \(\PageIndex{30}\).

Figure \(\PageIndex{30}\): Cartoon representation of a working model for HGF and NK1-induced c-MET activation. HGF induces an asymmetric c-MET signaling complex, whereas NK1 induces a symmetric c-MET signaling complex. The bindings of a second HGF and heparin strengthen the 2:1 c-MET/HGF active complex. Uchikawa et al. Nature Communications (2021) 12:4074 | https://doi.org/10.1038/s41467-021-24367-3 . Creative Commons Attribution 4.0 International License, http://creativecommons.org/ licenses/by/4.0/.

The glycosaminoglycan heparin enhances c-MET activation by HGF. This is illustrated in Figure 30 above. Heparin binds between the N domain of HGF I and the IPT1 domain of c-MET II, facilitating their interaction. In addition, a sufficiently long heparin chain could bridge both HGF I and II, further strengthening the entire complex. Structural representations of the c-MET:HGF asymmetric dimer are shown in cartoon form in Figure \(\PageIndex{31}\).

Figure \(\PageIndex{31}\): Overall structure of the c-MET/HGF holo-complex.

Panel (a) shows the domain structures of human c-MET and HGF. The proteolytic cleavage site of c-MET is located between Arg307 and Ser308. The proteolytic cleavage site of HGF is located between Arg494 and Val495. C-MET927-LZ and full-length HGF were used for structural determination in this study. The dashed boxes indicate the domains that were unsolved in cryo-EM maps. Panel (b) shows the 3D reconstruction of the 2:2 c-MET/HGF holo-complex and the corresponding ribbon representation of this complex fitted into the cryo-EM map at 4.8 Å resolution, shown in two orthogonal views. Panel (c) shows the ribbon representation of the c-MET/HGF holo-complex shown in two orthogonal views.

Figure \(\PageIndex{32}\): Shows the structure of the c-MET/NK1 symmetrical dimer.

Figure \(\PageIndex{32}\): Overall structure of the c-MET/NK1 complex. A 3D reconstruction of the 2:2 c-MET/NK1 complex, and the corresponding ribbon representation of this complex fitted into the cryo-EM map at 5 Å resolution, shown in two orthogonal views. b The ribbon representation of the 2:2 c-MET/NK1 complex is shown in two orthogonal views.

Nuclear RTKs

What makes signal transduction so complicated yet interesting is its unexpected nature. It turns out that some RTKs (EGFR, VEGFR, FGFR, IR, and NGFR have been found in the nucleus. It's experimentally easy to localize proteins in cells using immunofluorescence microscopy. It's hard to determine their functions. ErbB-2 is also found in the nucleus. Kinase inhibitors blocked the expression of ErbB-2 in the nucleus, suggesting that its kinase activity is required for it to translocate to the nucleus. The structure of the membrane forms of ErbB-2, EGFR, and ErbB-3 appears to be the same as the structure of the nuclear forms.

The carboxy-terminal ends of EGFR and ErbB-4 can activate gene transcription as measured by the expression of luciferase (a fluorescent protein) reporter proteins. Hence, they appear to act as transcription factors that bind DNA to promote gene expression. For example, nuclear EGFR increases cyclin D1 expression, the progression through the cell cycle. ErbB-2 appears to activate transcription from the promoter of the gene for cyclooxygenase 2 (COX-2). The protein COX-2 leads to the synthesis of inflammatory prostaglandins. It also increases blood vessel growth and is dysregulated in tumors.

Since proteomic analysis of these growth factor receptors shows no DNA binding motifs or domains, they must promote gene transcription through binding to other protein transcription factors in the nucleus.

Back to AGC Kinase - AKT (Protein Kinase B)

We've just explored the:

• Activation of Protein Kinase A (an AGC Kinase) through GPCR signaling, activation of the integral membrane protein adenylyl cyclase, production of the second messenger cAMP, which binds to inactive PKA and leads to its activation
• Activation of Protein Kinase C (an AGC Kinase) again through GPCR signaling, which leads to activation of the peripheral membrane protein phospholipase C, production of the second messengers DAG and IP3 from PIP₂, and activation of PKC at the membrane.
• Activation of receptor tyrosine kinases (RTKs) leading to autophosphorylation of the cytoplasmic kinase domain, followed by recruitment of downstream signaling proteins through binding to the phosphorylated RTKs through the downstream protein's SH2 domain

Now let's explore another AGC kinase called AKT or protein kinase B (PKB) that links signaling through RTKs to phosphoinositol-related signaling in a fashion similar to the link between phospholipase C and Protein Kinase C activities. 

Since Protein Kinase B is often referred to as AKT, we will use that abbreviation. As with other AGC kinases, AKT is a Ser/Thr protein kinase. There are three variants, AKT1, AKT2, and AKT3. These are involved in metabolism, growth, and proliferation, making them key players in signaling. It is a key player in the uptake of glucose into cells as it regulates the translocation of the glucose transporter SLC2A4/GLUT4 to the cell surface in response to insulin.

You would expect that aberrant expression of these would lead to cancer. The abbreviation AKT appears to derive from "a serine/threonine protein kinase encoded by the oncogene in the transforming retrovirus isolated from the thymoma cell line AKT-8, which is derived from the Stock A Strain k AKR".

As with Protein Kinase C and phospholipase C, AKT is recruited to the inner leaflet of membranes. Recruitment is mediated by its binding through its pleckstrin homology (PH) domain to phosphatidylinositol (3,4,5)-trisphosphate, a modified form of PIP₂, abbreviated either as PtdIns(3,4,5)P₃ or more simply as PIP₃. Note that PIP₂ has three phosphate groups while PIP₃ has four.) The mechanism of membrane recruitment is similar to that of phospholipase C, which also binds membrane PIP₂ through its pleckstrin homology (PH). (This is in contrast to PKC, which is recruited through its C1 and C2 domains.)

PIP₃ is generated in the membrane from PIP₂ by the enzyme phosphoinositide 3-kinase (P13K), a lipid kinase, whose own activation occurs through stimulation of insulin and growth factor receptor tyrosine kinases (RTKs). Class 1 PI3K has a regulatory/adapter subunit (p85) and a 110 kDa catalytic subunit (p110). The regulatory subunit contains SH2 domains, which enable it to bind to autophosphorylated RTKs.

Figure \(\PageIndex{33}\) shows how membrane PIP₂ can be converted to the second messengers DAG and IP₃ by phospholipase C, or to PIP₃ by the enzyme phosphoinositide 3-kinase (P13K), which is a lipid kinase.

Figure \(\PageIndex{33}\): Conversion of membrane PIP₂ to the second messengers DAG and IP₃ by phospholipase C, or to PIP₃ by phosphoinositide 3-kinase (P13K)

The binding of AKT (PKB) to inner leaflet PIP₃ through its PH domain causes a conformational change that activates AKT for phosphorylation by phosphoinositide-dependent kinase 1 (PDK1), a membrane protein kinase.  When activated, AKT dissociates from the membrane and acts enzymatically in the cytosol and nucleus. The overall activation of AKT is shown in Figure \(\PageIndex{34}\).

Figure \(\PageIndex{34}\): Activation of AKT through RTK signaling and PI3K activation https://commons.wikimedia.org/wiki/F...activation.png

.

The blunt arrows in the figure above indicate inhibition by the indicated enzymes. These (PTEN, PP2A, and PHLPP 1/2) are phosphatases.

• PTEN is a lipid phosphatase (phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase), which removes a phosphate from the lipid PIP₃.
• PP2A and PHLPP 1/2 are protein phosphatases that remove phosphates added by the kinases PDK1 in the kinase domain and mTORC2 in the C-terminal domain.

Figure \(\PageIndex{35}\) shows an interactive iCn3D model of AKT bound to a novel allosteric inhibitor (3o96).

Figure \(\PageIndex{35}\): AKT bound to a novel allosteric inhibitor is shown below (3o96) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...wTWYdpU1Y5JnP9

The PH domain (at the N-terminus, binds to PIP3) is shown in magenta, the N-lobe in green, and the C-lobe in brown. The activation loop is in dark blue, and the catalytic loop is in 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 the numbering of key amino acids and features of generic AGC kinase and the corresponding numbers in AKT. They differ because AKT has an N-terminal PH domain.

Table \(\PageIndex{1}\)

SITE	Generic	Akt (+108)
N lobe	K72	K180
N lobe	E91	E199
C lobe, cat loop	R165	R273
C lobe, cat loop	D166	D274
C lobe, act loop	D184	D292
C lobe, act loop	T197	T308* missing in this structure)
Approx Cat Loop	163-179	271V—287H
Approx Act loop Start DFG (292-294) to APE	184-200	292-308 308Tmiss toAPE end 319

The allosteric inhibitor shown in the structure above is particularly interesting because it requires both the PH and kinase domains for its effect.

Figure \(\PageIndex{36}\) shows an interactive iCn3D model of 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).

Figure \(\PageIndex{36}\): Alignment of inactive AKT (3o96 with bound allosteric inhibitor) and active AKT(3cqw with bound substrate) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...V2LbSdXLdTA9NA

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, 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 form. Note the significant change in the blue activation loop.

Figure \(\PageIndex{37}\): below shows a series of coupled equilibria reactions that regulate the activity of AKT1.

Figure \(\PageIndex{37}\): 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. https://doi.org/10.1371/journal.pone.0012913. 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, nonmembrane-bound form of the enzyme. 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 interact with the PH domain in the "in" conformation.

The bottom three structures show AKT bound to the membrane via the PH domain's interaction with PIP3 (purple). The bottom-right kinase domains are identical in representation to those in the top part of the figure, indicating they are inactive. Only when bound to the membrane is AKT phosphorylated on Thr 308 (red in the bottom middle figure), thereby activating the enzyme. The bottom left and middle structures display the yellow kinase domains in different conformations (3cqw), both of which are phosphorylated (red). The active form can bind ATP and protein substrates for phosphorylation. It can also bind ATP analogs, which would competitively inhibit the enzyme's active form 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, the loops partially occlude substrate interactions. Upon phosphorylation of Ser 308 in the activation loop, the loop adopts a different conformation, allowing less restricted access to the active site. The loop itself undergoes more local ordering upon phosphorylation as it shifts away from the active site, as seen in the iCn3D model above. Another change reduces the inhibitory noncovalent interactions between activation loop amino acids and catalytic residues, thereby increasing catalytic efficiency. In summary, these two types of changes in the activation loop lead to greater substrate access 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".

Figure \(\PageIndex{38}\) shows an interactive iCn3D model of the separate AKT Pleckstrin Homology (PH) domain bound to the inner member through just the head group of PIP3 (inositol (1,3,4,5)-tetrakisphosphate) (1unq). Waters H bonded to the ligands is not shown.

Figure \(\PageIndex{38}\): AKT Pleckstrin Homology (PH) domain bound to inositol (1,3,4,5)-tetrakisphosphate (1unq) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...h9LwZyV9WrJsN6

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter traces the intracellular signaling events that follow receptor activation at the cell membrane, focusing on the protein kinases that serve as the primary effectors of second-messenger signaling. The 518-member human kinome is organized into structurally related families, most of which share a conserved bilobal kinase domain with a catalytic loop, an activation loop, and an αC-helix. Activation almost universally involves phosphorylation of a key threonine in the activation loop by an upstream kinase, a conformational change that repositions catalytic residues and opens the active site to substrate access.

Three AGC-family Ser/Thr kinases take center stage. Protein Kinase A (PKA) is held inactive as an R₂C₂ heterotetramer; cAMP produced by adenylyl cyclase binds the regulatory subunits and releases the active catalytic subunits, which then phosphorylate a broad array of metabolic and transcriptional targets, including glycogen synthase, phosphorylase kinase, hormone-sensitive lipase, and the transcription factor CREB. Protein Kinase C (PKC) is activated downstream of phospholipase C by the joint action of DAG (binding to the C1 domain) and Ca²⁺ (binding to the C2 domain); an internal pseudosubstrate sequence keeps PKC autoinhibited in the cytoplasm until membrane recruitment and phosphorylation by PDK1 relieve inhibition. AKT (PKB) bridges RTK signaling to phosphoinositide metabolism: RTK autophosphorylation recruits the regulatory subunit of PI3K via its SH2 domain, PI3K converts membrane PIP2 to PIP3, and AKT is recruited to PIP3 through its pleckstrin homology (PH) domain, where PDK1 phosphorylates and activates it. The phosphatases PTEN, PP2A, and PHLPP terminate AKT signaling by removing phosphate groups from PIP3 or from the kinase itself.

RTK activation involves cis-autoinhibition of the kinase domain that is relieved by ligand-induced dimerization and asymmetric trans-phosphorylation between monomers. The ErbB/HER family illustrates the medical stakes: HER2 overexpression in breast cancer drives excessive proliferative signaling, and this has been countered therapeutically by the humanized antibody trastuzumab (Herceptin), which recruits immune destruction of HER2⁺ cells, and by the antibody-drug conjugate T-DM1 (Kadcyla), which delivers a microtubule-disrupting chemotherapeutic agent selectively to HER2-overexpressing tumor cells. Across all these pathways, the master upstream kinase PDK1 emerges as a unifying regulatory node, phosphorylating and activating PKA, PKC, and AKT alike.