Skip to main content
Biology LibreTexts

12.4: The next step - Downsteam intracellular signaling

  • Page ID
    75919
  • \( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \) \( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\)

    Intracellular signaling from activated PKA and PKC

    We discussed the basics of the activation of the protein kinase A holoenzyme (R4C4) by cAMP binding to the regulatory subunit, which frees the catalytic subunit C for activity. Likewise we discussed activation of PKC at the cell membrane by DAG, Ca2+ ions and phosphorylation of key Ser/Thr in the protein. Where in the cell do activated PKA and PKC phosphorylate target proteins to continue downstream signaling? This is a much simpler question for RTKs since downstream signaling proteins come to them through interaction of their SH2 domains with the autophosphoryated RTKs. For activated PKA and PKC, it turns out that there location is controlled by scaffolding proteins, which bind them either before their activation or after.

    Let's discuss a particularly important scaffolding protein, the A-kinase-anchoring proteins (AKAP). There are 13 classes of these containing 50 different members. These proteins bind PKA through its regulatory subunit, where cAMP can mediate the activation of the holoenyzme (R4C4). In addition, AKAPs can bind other signaling proteins including PKC and phosphatases, the latter which in turn counter-regulate signaling by phosphoproteins. For example, the bound phosphatases can dephosphorylate PKC to deactivate it as well as other downstream phosphoproteins. AKAPs can also bind phosphodiesterase, the enzyme that converts cAMP to AMP, returning signaling to baseline levels. AKAPs localize key signaling enzyme to sites where biologically appropriate protein targets are localized. In addition, they decrease indiscriminate phosphorylation of other off-target proteins elsewhere in the cell. They may also allosterically regulate the activity of bound signaling proteins.

    There are at least 50 A-kinase anchoring proteins or A-kinase anchor proteins (AKAPs) which bind the regulatory subunit of protein kinase A (PKA) and localize PKA to specific sites in the cell. By binding multiple signaling enzymes at specific sites, they integrate signaling pathways mediated by cAMP (for example) with others such mediated by PKC (again for example).

    Here are some examples of AKAPs in humans (from Uniprot). Note that one (12) also binds PKC

    • 1, mitochondrial: Binds to type I and II regulatory subunits of protein kinase A and anchors them to the cytoplasmic face of the mitochondrial outer membrane;
    • 6: Binds to type II regulatory subunits of protein kinase A and anchors/targets them to the nuclear membrane or sarcoplasmic reticulum;
    • 7 isoforms alpha and beta: Targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel;
    • 7 isoform gamma: targets cAMP-dependent protein kinase (PKA) to the cellular membrane or cytoskeletal structures;
    • 8: Acts as an anchor for a PKA-signaling complex onto mitotic chromosomes, which is required for maintenance of chromosomes in a condensed form throughout mitosis;
    • 8-like: Required for cell cycle G2/M transition and histone deacetylation during mitosis and recruitment of signaling enzymes into the nucleus;
    • 9: assembles several protein kinases and phosphatases on the centrosome and Golgi apparatus;
    • 12: Anchoring protein that mediates the subcellular compartmentation of protein kinase A (PKA) and protein kinase C (PKC)
    • 17A: Splice factor regulating alternative splice site selection for certain mRNA precursors. Mediates regulation of pre-mRNA splicing in a PKA-dependent manner.

    Figure \(\PageIndex{1}\) below illustrates localization/binding of signaling enzyme (PKA, PKA substrates, PDE, other kinases) to AKAPs.

    targetingprotprotinteractionsAKAPs_Fig2.svg
    Figure \(\PageIndex{1}\): Localization/binding of signaling enzyme (PKA, PKA substrates, PDE, other kinases). Calejo et al. Frontiers in Pharmacology 6:192 (2015) DOI: 10.3389/fphar.2015.00192. License CC BY 4.0

    Note that some AKAPs can also bind PKA substrates, facilitating their phosphorylation and minimizing phosphorylation of the wrong targets.

    AKAPs use an amphilic helix to interact with the R2 regulatory dimer of the PKA. Some AKAPs bind to just one of the regulatory subunits. Note that some AKAPs can also bind PKA substrates, facilitating their phosphorylation and minimizing phosphorylation of the wrong targets.

    Figure \(\PageIndex{2}\) below shows specific AKAP complexes in the heart that could be targets for drug therapies.

    targetingprotprotinteractionsAKAPsFig3.svg
    Figure \(\PageIndex{2}\): AKAP complexes in the heart.

    Panel (A): Disruption of the AKAP18γ/δ-PLB (another phospholipase involved in signaling) interaction prevents PLB phosphorylation on Ser16 and dislocation from SERCA2 (Sarcoplasmic/endoplasmic reticulum calcium ATPase 2). This inhibits SERCA2 activation and consequently Ca2+ uptake into the sarcoplasmic reticulum

    Panel (B): Disruption of the nesprin-1α /mAKAP interaction promotes AKAP/PKA complex dissociation from the perinuclear membrane and might be a strategy to reduce hypertrophy. Nesprin 1 is a protein which forms a linking network between organelles and the actin cytoskeleton to maintain the subcellular spatial organization.

    Panel (C): Disruption of the connexin 43-ezrin interaction could prevent PKA-mediated phosphorylation increasing inter-cardiomyocyte conductivity which could be cardioprotective following myocardial infarction damage. Connexin is a gap junction protein. Ezrin is involved in connections of major cytoskeletal structures to the plasma membrane.

    To add to the complexity of PKA activation and signaling, there are different forms of the regulatory subunits of the holoenyzme PKA. These include RIalpha (RIA), RIbeta (RIB), RIIalpha (RIIA) and RIIbeta (RIIB). They have different affinities for cAMP, the catalytic subunits Cs, and for different AKAPs.

    Figure \(\PageIndex{3}\) below shows an interactive iCn3D model of a the amphiphilic anchoring peptide AKAP-IS from an AKAP binding to the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA (2IZX)

    Amphiphilic anchoring peptide AKAP-IS _ RIIalpha Rsubunit_PKA  (2IZX).png
    Figure \(\PageIndex{3}\): Amphiphilic anchoring peptide AKAP-IS from AKAP binding to the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA (2IZX) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...pWKiYgQeGA32e6

    The brown represents the RII dimer D/D domains of the regulatory subunit. The anchoring peptide AKAP-IS shown in gray. In both, the side chains involved in binding of the peptide to the regulatory subunit domains are shown as sticks and colored coded based on hydrophobicity of the side chains. Green indicates most hydrophobic. Rotate the model carefully to differentiate the side chains and note that the hydrophobic face of the peptide is interacting with hydrophobic side chains projecting into a groove made by the two RII dimer D/D domains. Polar side chains in AKAP help target to the correct isoform of the R subunit.

    In addition to binding to some AKAP scaffold, PKC also binds to Receptors for Activated C-Kinases (RACKs), heat shock proteins (HSP), importins and annexins (AnxA1, A2, A5 and A6). The interactions of activated PKC with RACK1 and downstream events is shown in Figure \(\PageIndex{4}\) below.

    nuclearimportPKCbyRACKTxFig9.svg
    Figure \(\PageIndex{4}\): Interactions of PKC with RACK1 and downstream signaling. Meng et al. PLoS ONE · (2014) DOI: 10.1371/journal.pone.0109111. Creative Commons Attribution License - https://creativecommons.org/licenses/by/4.0/

    The insect protein BR-C (Broad Complex) has a DNA binding domain (two zinc fingers) domains for the activation of gene transcription and a BTB) domain that allows binding to RACK1. On binding the PKC:RACK1 complex, BR-C is phosphorylated at Ser373 and Thr406, after which it is translocated into the nucleus where it binds DNA and activates gene transcription

    The binding of PKC to RACK1 stabilizes PKC for phosphorylation of targets. PKC binds to RACK through its C2 regulatory domains. Binding may be to specific forms of PKC include unphosphorylated, and inactive and activated phosphorylated form and to specific isozymes of PKC. RACK1 may also recruit PKC to the ribosome and it inhibits the activity of SRC kinases which we will discuss later. PKC activity occur in many cellular locations, including the cell membrane, nucleus, Golgi apparatus, mitochondria, and cytosol. RACKS also bind and recruit other signaling proteins including PLCγ, Src, and integrins. In addition to interactions of PKC with RACK through the C2 domain, PKC can localize through its C1 domain.

    Structure of RACK1

    RACK1 (317 amino acids) has a very interesting structure. It is a member of a family of over a 100 proteins that have tryptophan-aspartate (WD) repeats that are 44-60 amino acids longs and ending in WD or a variant. It is homologous to the beta subunit of the heterotrimeri Gαβγ signaling protein. RACK 1 interacts with proteins through a 7-bladed propeller structure that allow binding of proteins with SH2 domains, plextrin homology (PH) domains and C2 domains (PKCs). Figure \(\PageIndex{5}\) below shows the WD repeats in human RACK1. Note that the N-terminal end of the WD repeat often is glycine-histidine (GH).

    RACK1 - A multifaceted scaffolding proteinStructFnFig1.svg
    Figure \(\PageIndex{5}\): Sequence of RACK1 depicting WD-repeats. RAdams, D.R., Ron, D. & Kiely, P.A. RACK1, A multifaceted scaffolding protein: Structure and function. Cell Commun Signal 9, 22 (2011). https://doi.org/10.1186/1478-811X-9-22; https://creativecommons.org/licenses/by/2.0 )

    Figure \(\PageIndex{6}\) below a model for the interaction of RACK1 and PKC-βII.

    RACK1 - A multifaceted scaffolding proteinStructFn-3A.svg RACK1 - A multifaceted scaffolding proteinStructFn-3B.svg

    Figure \(\PageIndex{6}\): Model for PKC-βII and RACK1 interaction. Adams et al. Cell Communication and Signaling 2011, 9:22 http://www.biosignaling.com/content/9/1/22. Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)

    Panel (A) shows the resting state with no interaction between RACK1 and inactive PKCbII. Panel (B) shows how activation of PKC-βII leads to its interaction with RACK1. RACK1 followed by substrate binding and phosphorylation. R is a receptor and L its ligand.

    Figure \(\PageIndex{7}\) below shows how RACK1 can translate into the nucleus after ligand (L) gated activation of GPCRs (R) through adenylyl cyclase production of cAM and activatio of Protein Kinase A.

    RACK1 - A multifaceted scaffolding proteinStructFn-4A.svg RACK1 - A multifaceted scaffolding proteinStructFn-4B.svg

    Figure \(\PageIndex{7}\)Model for cAMP/PKA-mediated nuclear translocation of RACK1. Adams et al. Cell Communication and Signaling 2011, 9:22 http://www.biosignaling.com/content/9/1/22. Creative Commons Attribution License. (http://creativecommons.org/licenses/by/2.0)

    Panel (A) shows the resting state of RACK, which forms homdimers and heterodimers with the homologous Gβ subunit of the Gαβγ complex. Panel (B) shows how activation of PKA leads to dissociation if RACK which then can translate into the nucleus, where it leads to increase transcription of brain-derived neurotrophic factor (BDNF).

    Figure \(\PageIndex{8}\) below shows an interactive iCn3D model of a the human Rack1 (4AOW) color coded as in Figure \(\PageIndex{5}\).

    Human Rack1 Protein4AOW .png
    Figure \(\PageIndex{7}\): human Rack1 (4AOW) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...7WDaKw2dxGK87A

    Downstream signaling from activated receptor tyrosine kinases

    To review once again, when receptor tyrosine kinases get activated by binding a primary messenger such as a growth factor, the receptors dimerize, activating their cytoplasmic kinase domains. The activated RTK then autophosphorylates itself. The phosphorylated intracellular domain provides a docking site for other cellular "adaptor" proteins that contain an SH2 domain. The bound adaptor protein binds other signaling molecules within the cell leading to downstream propagation of the signal. Figure \(\PageIndex{8}\) below shows some RTKs and downstream signaling events. We have already discussed activation of the lipid kinase phosphoinositide 3-kinase (P13K) which leads to activation of Akt (PKB). In this rest of this section we will focus on the the next step after activation of RTKs. We focus on the epidermal growth factor receptor (EGFR, ErbB1) again.

    RTKsandSignaling PathwaysFig2Signaling.svg
    Figure \(\PageIndex{8}\): Some RTKs and downstream signaling events Dev et al. Frontiers in Pharmacology, 12 (2021). https://www.frontiersin.org/article/...ar.2021.772510. DOI=10.3389/fphar.2021.772510. Creative Commons Attribution License (CC BY).

    1. Downstream signaling from the epidermal growth factor receptor.

    Figure \(\PageIndex{9}\) below shows events associated with the activation of EGFR.

    RASnucleotidecyclingGRB_SOS_RASimageFig2.svg
    Figure \(\PageIndex{9}\): Near downstream signaling molecules after activation of EGFR

    Once bound and activated by binding of growth factors protein signals, the intracellular domains of the now dimeric EGFR autophosphorylates itself on selected tyrosine side chains. This then recruits a protein called Growth factor receptor-bound protein 2 (GRB2), which has an SH2 (Sarc Homology 2) domain that binds phosphotyrosine motifs in proteins. GRB2 acts as an adaptor protein in that in addition to the SH2 domain, it has two SH3 (Sarc Homology 3) domains which bind proline-rich domains on other signaling protein, including the protein Son of sevenless homolog (SOS). GRB2 does not have enzymatic activity.

    The adaptor protein GRB2 binds through its SH3 domain to the protein SOS, which then interactions with the protein Ras. This last member in the recruited complex is named because it causes Rat Sarcomas. There are many variants of these protein but 3 are key in humans, HRas, KRas and NRas. Ras is perhaps the key member of the family of intracellular small G proteins that bind GDP/GTP and are activated on the exchange of GTP for GDP. The proteins also have intrinsic GTPase activity as is found in the Gα protein of the eterotrimeric Gαβγ protein.

    So what does SOS do? The SOS bound to RAS facilitates the exchange of GTP for GDP on Ras, maintaining it in an active state. SOS is a member of a another fascinating class of small proteins that catalyze the exchange of GTP for GDP. The family of GTP/GDP exchange proteins are called Guanine nucleotide Exchange Factors (GEFs). We will explore in the next section.

    The EGFR -GRB2-SOS-Ras complex in the figure above looks somewhat similar to the structure of a GPCR-heterotrimeric G protein GαGβGγ complex, where Gα is also a GTP/GDP exchange protein with intrinsic GTPase activity. When the ligand binds to the GPCR, a conformational changes ensues which facilitates the exchange of GTP for GDP on the Gα protein, activating intracellular signaling.

    Once RAS is activated (bound to GTP), it binds and activate key kinases in the cell, including the lipid kinase PI3K and RAF, a kinase shown in Figure \(\PageIndex{8}\) above. Control of RAS activity is critical in signaling. It is one of mostly commonly mutated proteins in cancer cells. Mutations the inhibit the intrinsic GTPase activity keep the protein active, leading to unregulated cell growth, proliferation and differentiation, hallmarks of cancer cells.

    The domain structures of GRB2 and SOS are shown in Figure \(\PageIndex{10}\) below. The proline-rich domain (motif) is not shown in the figure.

    GRB2 domain structureGRB2domain.png (green SH3 domains)
    SOS1 domain structureSOS1domain.png

    Figure \(\PageIndex{10}\): Domain structure of GRB2 and SOS1 (proline rich domain not shown in SOS)

    Note

    As we discussed in Chapter xx (domains), new genes encoding proteins with new functionalities can be created by duplicating and adding gene segments for different domains in a preexisting gene. As we have seen with the SH2 and SH3 domains, signaling proteins often share common domains. Table \(\PageIndex{1}\) below, adapted from the excellent book Cell Signaling by Lim, Mayer and Pawson, shows some common domains found in signaling proteins.

    Domains in Signaling Molecules
    Domain Binding Target Cellular Process Example protein

    Pdb file (examples)

    Find your own in the PDB

    Bromo Acetyl-Lys Chromatin reg. BRD4 2YYN
    C1 diacylglycerol Plasma memb recruitment Raf-1 3OMV
    C2 Phospholipid (Ca dependent) Membrane targeting, vesicle trafficking PRKCA 3IW4
    CARD Homotypic interactins apoptosis CRADD 3CRD
    Chromo Methyl-Lys Chromo reg, gene txn CBX1 3F2U
    Death (DD) Homotypic inter. Apoptosis Fas 3EZQ
    DED Homotypic inter. Apoptosis Caspase 8 1F9E
    DEP Memb, GPCRs Sig trans, prot trafficking

    Dsh

    human dishevelled 2

    2REY
    GRIP Arf/Art G prot Golgi traffic Golgin-97 (Golga5) 1R4A
    PDZ C-term peptide motifs Diverse, scaffolding

    PSD-95

    Or discs large homolog 4

    1L6O
    PH Phospholipids Membrane recuirt Akt

    1O6L

    3CQW

    PTB Phospho-Y Y kinase signaling

    Shc 1

    SHC-transforming protein 1

    1UEF

    1irs

    RGS GTP binding pocket of Galpha Sig trans RGS4 1EZT
    SH2 Phospho-Y pY-signaling Src 4U5W
    SH3 Pro-rich sequence Diverse, cytoskelet Src 2PTK
    TIR Homo/Heterotypic Cytokine and immune TLR4 3VQ2
    TRAF TNF signaling Cell survival TRAF-1 3ZJB
    VHL hydroxyPro ubiquitinylation VHL 1VCB

    Figure \(\PageIndex{11}\) below shows an interactive iCn3D model of the GRB2-SH2 domain in complex with a high affinity phosphopeptide KPFpYVNVEF (1BMB)

    GRB2-SH2 domain in complex with a high affinity phosphopeptide.png
    Figure \(\PageIndex{11}\): GRB2-SH2 domain in complex with a high affinity phosphopeptide KPFpYVNVEF (1BMB) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...gBEpnsN6D8yrR6

    Grb2 exists in both a monomeric state which can bind SOS, and a dimeric state which can't. You would expect the equilbrium between the monomer and dimeric form wold be highly regulated. When a phospho-tyrosine ligand is bound to Grb2 through its SH2 domain, the dimer dissociated. The occurs also on phosphorylation of tyrosine 160 (Y160) on Grb2, a post-translational modification found in human prostate, colon and breast cancers.

    Figure \(\PageIndex{12}\) below shows an interactive iCn3D model of the GRB2 N-terminal SH3 domain complexed with a ten-residue proline-rich peptide (1135 Ac-VPPPVPPRRR-NH2) derived from SOS (1GBQ)

    Grb2 N-terminal SH3 domain _ proline-rich peptide SOS (1GBQ).png
    Figure \(\PageIndex{12}\): GRB2 N-terminal SH3 domain complexed with a ten-residue proline-rich peptide (derived from SOS (1GBQ). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...k2jNGaHexaUmT8

    We will explore interactive structures of the small G protein Ras with the guanine nucleotide exchange factor SOS in the next chapter section.

    After activation of Ras through GTP exchange for GDP, the GTP-Ras protein binds to and activate the kinase Raf. We will continue our exploration of that later in this section.

    2. Downstream signaling from the insulin receptor.

    When insulin binds to the receptor tyrosine kinase (RTK), it phosphorylates itself, which then leads to the binding of other proteins to the activated receptor and their phosphorylation. These lead to more intracellular signaling and ultimately changes in gene transcription. We'll focus on a specific adaptor protein, the Insulin Receptor Substrate 1, IRS1, a "scaffolding protein", which leads to the movement of the glucose transport protein GLUT4 to the cell surface, allowing glucose uptake. These activities are shown schematically in Figure \(\PageIndex{13}\) below.

    MetabolomcsTypeIandIIDiabetesFig1.svg
    Figure \(\PageIndex{13}\): Binding to insulin stimulates the association of the receptor with downstream mediators, including insulin receptor substrate-1 (IRS-1) and PI3K. The insulin receptor can activate PI3K either directly, by binding to the p85 regulatory subunit, which produces PIP3, or indirectly, which leads to phosphorylation and the activation of AKT. Afterward, AKT phosphorylates the Ser9 site of GSK-3β and inhibits its activity. The PI3K/AKT/GSK-3β signaling pathway is involved in the insulin signaling transduction, and GSK- 3β is regulated and controlled by insulin in this signaling pathway. Arneth et al. Int. J. Mol. Sci. 2019, 20, 2467; doi:10.3390/ijms20102467. Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

    Human IRS1 has two domains, a PH domain for binding to membranes through phosphorylated IP2 derivartives and a IRS/PTB domain which binds phosphotyrosines on proteins. The PTB and the SH2 domains are the most common domains for binding phosphotyrosines on proteins. PTB binds the NPXY, where X is a pTyr.

    We show a more detailed view in Figure \(\PageIndex{14}\) below, in part, to review many of the signaling proteins we have seen before, including PI3K and PDK1.

    insulin signaling
    Figure \(\PageIndex{14}\): A more detailed view of the insulin receptor-mediated translocation of GLUT4 to the membrane.

    Likewise, another review of PIP2 derivatives is warranted. After phosphorylation by the activated insulin receptor protein tyrosine kinase, IRS-1 binds phosphatidylinositol 3-kinase (PI3K) that causes phosphorylation of the 3'OH on phosphatidyl inositol (PI) in the inner leaflet of the membrane to form PI(3)P. PI3K is a member of a family of kinases that phosphorylates PIP2. The metabolic pathway centered on pI3K is one of the most mutated in human cancers. PI(3)P in turn recruits to the membrane other inactive kinases, phosphoinositide-dependent kinase 1, PDK1 and Akt, also known as PKB. Figure \(\PageIndex{15}\) below shows phosphorylated phosphatidylinositol derivatives.

    phosphorylated phosphatidylinositol derivatives
    Figure \(\PageIndex{15}\): Phosphorylated phosphatidylinositol derivatives

    On binding of PI(3)P, PDK1 becomes an active kinase, which phosphorylates and activates Akt. The family of three Akt kinases are major Ser/Thr protein kinase that phosphorylates proteins involved in a host of cell activities, including regulation of glucose transport, cell proliferation and death. In the insulin signaling pathway, active (phosphorylated) Akt leads to movement of the GLUT4 protein from intracellular endosomal vesicles to the cell surface, which offers a quicker way to import glucose into the cell that if Akt activated GLUT 4 gene expression. PDK1 phosphorylation of Akt2-T309 is required for insulin-stimulated Glut4 translocation. If T309 is mutated to A309 or if PDK1 is inhibited, GLUT 4 is not translocated to the cell membrane.

    Figure \(\PageIndex{16}\) below shows an interactive iCn3D model of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog (1IR3))

    Activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog (1IR3).png
    Figure \(\PageIndex{16}\): Activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog (1IR3). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...j11LhWKCUnreV9

    The dimeric form of the kinase is shown. ANP is shown in spacefill. The peptide substrate containing the interior tyrosine (stick) for phosphorylation is shown in blue. Three key tyrosine in the activation loop that are autophosphorylated (Tyr1158, Tyr1162 and Tyr1163) are shown and labeled in the right hand monomer.

    As with a protein kinase, the activation loop of the insulin receptor kinase domain is phosphorylated and the resulting conformational change allows ATP and target protein access. The activation loop get phosphorylated on Tyr1158, Tyr1162 and Tyr1163 with Y1163 being key.

    Downstream signaling from Cytokine activated Receptors- The JAK/STAT pathway.

    Now we explore two signaling pathways mediated by the adaptor protein JAK and STAT. These are abbreviations for the Janus Kinase (JAK) and the Signal Transducer and Activator of Transcription (STAT). These plays key roles in embryonic development, stem cell maintenance, hematopoiesis (formation of blood cells) and immune cells signaling. We all know how important that from our experience with the COVID-19 pandemic. This pathway is stimulated by cytokines and interleukins, protein modulators released by immune cells, as well as growth factors.

    In general there are five grops of cytokines:

    • tumore necrosis factor alpha (TNF-alpha)
    • Interleukin-1 family members (IL-1_
    • Transforming growth factor-beta (TGF-bet)
    • those that signal through RTKs (such as macrophage colony stimulating factor (M-CSF)
    • Chemokines
    • cytokines that signal through JAK/STAT

    In contrast to RTKs, which have kinase domains activated on receptor dimerization, cytokine receptors that work through JAK/STAT do NOT have kinase domains. On cytokine binding to their target cytokine receptor, the now activated receptors activates the prebound inactive Janus kinase through conformational changes. The kinaes domains autophosphorylate themselves in a trans fashion. The active kinase then phosphorylates the cytoplasmic tails of the cytokine receptors. This trigger further signal transduction reactions mediated by the binding of target signaling proteins to the cytoplasmic region of the phosphorylated cytokine receptor. Just to reiterate, the cytokine receptor is NOT a RTK with latent kinase activity. Instead, it becomes phosphorylated by the bound and cytokine-activated JAK. A portion of the pathway is illustrated in Figure \(\PageIndex{17}\) below.

    JAKSTATSignalingBestFig1.png
    Figure \(\PageIndex{17}\): An overview of the JAK/STAT signalling pathway (A) and the domain structures of JAK (B) and STAT (C). Thomas et al. TBr J Cancer 113, 365–371 (2015). https://doi.org/10.1038/bjc.2015.233. Creative Commons Attribution-Non-Commercial-Share Alike 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/

    Panel (A1) shows that the kinase JAK is bound constitutively in an inactive state to the cytokine receptor cytoplasmic region, not through its SH2 domain, but through its FERM domain (Panel B). The figure implies the cytokine receptor is dimeric in the absence of ligand. On cytokine binding, conformational changes and repositioning of the bound JAK activates it kinase domain, which phosphorylated the C-terminal tails of the cytokine receptor. STAT monomers, through their SH2 domains, binds to the phosphorylated cytokine receptor where they get phosphorylated by the active JAK. The phospho-STAT monomers form a dimer, dissociate from the complex, and translocate to the nucleus where they act as transcription factors. the Janus kinase is named after Janus, the two-faced Roman god of beginnings, endings and duality, since JAK has two nearly identical JH kinase domains. One has kinase activity while the other inhibits the first.

    Panels (B) shows the domain structure of JAK. The FERM domain faciliates JAK:cytokine receptor binding. The JH2 pseudokinase domain regulates kinase activity of the JH1 kinase domain. Ps show site on JAK necessary for activation

    Panel (C) shows the domain structure of STAT. The SH2 domain binds phosphorylated tyrosines. The carboxy terminus transactivation domain is required for full transcriptional activation. P marks the conserved tyrosine residue whose phosphorylation is essential for STAT activation.

    The pseudokinase domain of JAK interacts with the kinase domain on the same chain and prevents its activity in the inactive monomer and dimer. Cytokine binding to the cytokine receptor induces a conformational change that promotes the interactions of the pseudokinase domain on one JAK monomer with that the same domain on another, promoting dimerization and freeing the kinase domains for activity. Figure \(\PageIndex{18}\) shows the pseudokinase domain (orange) interactions in the active JAK dimer.

    pseudokinaseinteractions_JanusKinaseDimer7T6F_icn3d_loadable (3).png

    Figure \(\PageIndex{18}\): The pseudokinase domain (orange) interactions in the active JAK dimer.

    The kinase domain of each dimer is shown in red and the pseudokinase domain is shown in orange. The activation loop (cyan) and ADP (spacefill, CPK colors) are shown in the red kinase domains. The orange pseudokinase domain has a bound adenosine (not ADP) that is shown spacefill and CPK color. However it lacks the the
    DFG motif required for catalysis. The phenylalanine cluster (F635, 657, 574) are labeled. The structure is actually for a mutant that has the a V657F mutation that promotes dimerization and JAK activity. Hence the V657F mutation is oncogenic.

    JAK activity can be inhibited by the protein suppressor of cytokine signaling (SOCS). Transcription of the protein is activated by STAT, and the SOCS protein in a feedback inhibition loops binds to JAKs and and also to IFNGR1, which inhibit JAK activity.

    Figure \(\PageIndex{19}\) below shows an interactive iCn3D model of the structure of the inhibitory protein SOCS1 in complex with JAK1 kinase domain (6C7Y)

    SOCS1 in complex with JAK1 kinase domain (6C7Y).png
    Figure \(\PageIndex{19}\): Structure of the inhibitory protein SOCS1 in complex with JAK1 kinase domain (6C7Y). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...tqtfo2SQ5TxF58

    The N-lobe of the JAK1 kinase domain is shown in cyan, and the C-lobe in magenta. ADP (sticks) and Mg2+ (green) are shown in the interface between the lobes. SOCS1 is shown in gray except for the kinase inhibitor region which is shown as spheres and CPK colors. It binds in the substrate binding grove and prevents substrate access.

    Activated JAK activity and signaling does not stop simply with the activation of STAT. In addition to stimulating signaling through phosphorylated dimers of STAT, cytokines also activate other signaling pathways through the same receptors. Examples include the PI3K pathway described in an earlier section and also the MAPK pathways, described in detail later in this section. Both the PI3K and MAPK pathways are activated by binding of the cytokine IL6 to its cytokine receptor. The mechanism for PI3K activation is a bit unclear. The MAPK pathways is activated by a phosphatase called SHP2 for Src homology region 2 domain‐containing phosphatase 1. This protein binds to pTyr759 on the IL-6 receptor. As binding of the cytokine activates the prebound JAK, it also activates SHP2, ultimate activating signaling through Ras (a small G protein), which activates RAF, a kinase. Figure \(\PageIndex{20}\) below shows these three combined signaling pathways for a cytokine receptor: STAT, Ras and PI3K activations.

    Cytokine_Signaling_in_Tumor_Progression_JakStatFig2.svg
    Figure \(\PageIndex{20}\): Schematic representation of the JAK-STAT pathway. Lee et al. Immune Netw. 2017 Aug;17(4):214-227. https://doi.org/10.4110/in.2017.17.4.214. pISSN 1598-2629·eISSN 2092-6685. Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/)

    The cytokine receptor induces activation of JAKs after cytokine stimulation following the phosphorylation of STATs. Furthermore, phosphorylated STATs undergo dimerization and translocate to the nucleus to activate target gene transcription. SOCS, suppressors of cytokine signaling; PI3K, phosphatidyl inositol 3 kinase; Akt, protein kinase B; FOXO, Forkhead box protein O; mTOR, mammalian target of rapamycin.

    Figure \(\PageIndex{21}\) below shows an interactive iCn3D model of the Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA (1BF5)

    Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA (1BF5).png
    Figure \(\PageIndex{21}\): Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA (1BF5). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...VEjHZ5t3baKC37

    The dimer chains are shown in brown and gold except for the those colored coded by secondary structure (helix red, sheet yellow). The backbone of the nucleotides are shown in spacefill cyan and gray. Zoom in to see noncovalent interactions between the bound DNA and protein monomers. Two phospho-tyrosines are labeled pTR701 are shown as well. The DNA binding domain of the STAT dimer has an immunoglobulin fold and forms a "C-shaped clamp" around the DNA. The domains colored by secondary structure are SH2 domains, with each recognizing and binding to the phosphorylated Tyr 701 (labeled pTR701), a very interesting use of the SH2 domain.

    Figure \(\PageIndex{22}\) below shows an interactive iCn3D model of the active Janus Kinase (JAK) dimer complexed with the intracellular domains (spacefill) of the interferon lambda(a cytokine) receptor (7T6F).

    active Janus Kinase (JAK) dimer complexed with cytokine receptor intracellular domainV2 (7T6F).png

    Figure \(\PageIndex{22}\): Active Janus Kinase (JAK) dimer complexed with intracellular domain of the interferon lambda(a cytokine) receptor (7T6F). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...9s3VBNMxsk7WM7

    Domain/proteiin Coloring

    • Red: PTKc-JAK-rpt2 kinase catalytic domain
    • Orange: PTK-JAK1-rpt1 pseudokinase domain
    • Yellow: FERM F2
    • Magenta: FERM C-JAK1
    • Cyan: SH2
    • Brown: FERM F1
    • Navy Blue and Blue spacefill: cytoplasmic domains of the cytokine receptor interferon lambda receptor 1 dimer
    • The gold large sphere represents interferon bound to the extracelluar domain of the interferon lambda receptor 1 dimer

    Figure \(\PageIndex{23}\) below shows a model combining the active Janus kinase (JAK) containing the intracellular domain of the interferon alpha receptor 1 dimer (navy and blue spacefill) with AlphaFold models of the extracellular and transmembrane domain of the interferon alpha receptor ((Q8IU57))

    JanusKinase_inferonalphareceptormodel.svg

    Figure \(\PageIndex{23}\): AlphaFold model of Interferon lambda receptor 1 Extracellular Domain and transmembrane domain (Q8IU57) with Active Janus Kinase (JAK) dimer complexed with cytokine receptor intracellular domain (7T6F)

    Again, to reiterate as we did above, the cytokine receptor (in the figure above the interferon lambda receptor) is NOT a RTK with latent kinase activity. Instead, it bcomes phosphorylated by the bound and cytokine-activated JAK which is resident in the cytoplasm.

    Here is a lin to a second iCn3D model which show the phenylalaine cluster that promotes pseudokinase domain interactions in the active JAK dimer.

    Downstream Intracellular signaling though Src Family Kinases - Membrane associated non-receptor tyrosine protein kinases

    Another family of intracellular protein kinases - the Src family - are often activated on upstream activation of many different types of receptors including GPCRs, RTKs, cytokine receptors, as well as integrins and adhesion receptors that we explore in a later chapter section. We have already touched on them when we discussed proteins containing the src homology domains SH2 and SH3. Src, an intracellular Tyr kinase (MW 60,000), is the founding member of this family of protein kinases. Src is activated when it binds through its own SH2 domain to a phosphorylated membrane receptor.

    Src has many names including Proto-oncogene tyrosine-protein kinase Src, proto-oncogene c-Src, pp60c-src and p60-Sr. These membrane-associated, non-receptor tyrosine kinases regulate cell proliferation, differentiation, apoptosis, migration, metabolism and cytoskeleton organization. They are one of major class of intracellular kinases which are activated after upstream activation of the membrane receptors mentioned above (GPCRs, RTKs, cytokine receptors, as well as integrins and adhesion receptors). They in turn activate further downstream protein kinases by phosphorylation. They even phosphorylate the upstream membranes which led to their activation.

    There are 10 members in the Src family: Src, Frk, Lck, Lyn, Blk, Hck, Fyn, Yrk, Fgr, and Yes. They all share the same expanded domain structure shown in Figure \(\PageIndex{24}\) below.

    The_Crosstalk_between_Src_and_HippoYAP_Signaling_PFig1.svg
    Figure \(\PageIndex{24}\): The domain structure of Src (panel A) and its activation (panel B). Hsu et al, Cancers 12(6):1361. DOI: 10.3390/cancers12061361. CC BY 4.0

    They have a SH4 N-terminal region which can by post-translationally modified with fatty acids (myristoylation and palmitoylation), which can anchor it to the membrane. The spacer SH2-SH3 spacer is intrinsically disordered and differ in sequence among members of the Src family. Two key phosphoryation sites (Tyr416 and Tyr527) are important. In inactive Src, Try 527 is phosphorylated, allowing it to bind the SH2 domain. The SH2-kinase linker also binds to the SH3 domain. This occludes the active site region and prevents the phosphorylation of Try 416 in the "activation loop" of the SH1 kinase domain. When Try 527 is dephosphorylated, a conformation change ensues the opens the binding site allowing autophosphorylation of Tyr 416 and its activation. Hsu et al, Cancers 12(6):1361. DOI: 10.3390/cancers12061361. CC BY 4.0

    Figure \(\PageIndex{25}\) below shows an interactive iCn3D model of a the Human tyrosine-protein kinase C-Src in complex with AMP-PNP (2SRC)

    Human tyrosine-protein kinase C-Src in complex with AMP-PNP (2SRC).png
    Figure \(\PageIndex{25}\): Human tyrosine-protein kinase C-Src in complex with AMP-PNP (2SRC). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...L45honKx6rrEu7

    pY527 is shown in stick and labeled. It binds to the SH2 domain (151-248) shown in cyan. The SH3 domain is in magenta and the kinase domain is shown in gray. The activation loop in the kinase domain is shown in red with the pY416 needed for activation shown in stick and labeled. ANP is shown in spacefill.

    We have now seen the structure of many kinase domains. Figure \(\PageIndex{26}\) below shows an interactive iCn3D model of the alignment of human c-Src (452 amino acid) (2SRC) and Erk2 (362 amino acids) also called MAPK1 (2Y9Q), a protein kinase will be explore at the end of this section

    Aligned structures of human c-Src (2SRC) and Erk2 (2Y9Q).png
    Figure \(\PageIndex{26}\): Aligned structures of human c-Src (2SRC) and Erk2 (2Y9Q). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ETS1puViPtrQu7

    Red shown conserved sequences, blue aligned (but not conserved) and gray nonaligned. Use the "a" key to toggle between the states.

    After much discuss of the binding of p-Tyrosine side chains to SH2 domain, we now present Figure \(\PageIndex{27}\) below, which shows an interactive iCn3D model of a phosphotyrosine peptide bound to the SH2 domain of Fer tyrosine kinase (6KC4)

    Phosphotyrosine peptide bound to the SH2 domain of Fer tyrosine kinase (6KC4).png
    Figure \(\PageIndex{27}\): Phosphotyrosine peptide bound to the SH2 domain of Fer tyrosine kinase (6KC4). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...3eqrcT7NwcHpr8

    The SH2 domain is shown in gray. The phosphopeptide (DEpYENVD) is shown in cyan with the labeled pY in stick. The side chains on the SH2 domains interacting with the pY are shown in stick and labeled.

    We are about to explore the last but incredibly important downstream kinases activated in signal transduction cascades, the mitogen activate protein kinase (MAPK) cascade. It could also be called the Erk cascade. Before we do that, we present parts of three pathway mediated by activated RTKs to refresh your minds and also because the more you see key players in the pathway, the more you start to remember the names, structural features and function of the signaling molecules.

    Figure \(\PageIndex{28}\) offers a quick and abbreviated looks at signaling through activated RTKs that proceed through the adaptor protein and the GEF SOS, leading to activation of Ras, a key small G protein. Ras in turn activates a protein (MAPKKK) in the MAPK cascade.

    Signaling pathways downstream to receptor tyrosineFig1.svg

    Figure \(\PageIndex{28}\): Simplified cartoon showing the activation of the MAPK cascade protein MAPKKK. Cordover E, Minden A. Signaling pathways downstream to receptor tyrosine kinases: targets for cancer treatment. J Cancer Metastasis Treat 2020;6:45. http://dx.doi.org/10.20517/2394-4722.2020.101. Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/),

    Figure \(\PageIndex{29}\) below shows an abbreviated version of the activation of the lipid kinase PI3K and through the activation of PDK1 and AKT, the activation of two major kinase complex, mTORC1 and mTORC2, which we will explore in its own Chapter sectrion.

    Signaling pathways downstream to receptor tyrosineFig3.svg
    Figure \(\PageIndex{29}\): Activation of mTORC1 and mTORC2, through activation of PI3K, PDK1 and AKT. Cordover E, Minden A. Signaling pathways downstream to receptor tyrosine kinases: targets for cancer treatment. J Cancer Metastasis Treat 2020;6:45. http://dx.doi.org/10.20517/2394-4722.2020.101. Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/)

    Finally, Figure \(\PageIndex{30}\) show the combined activation of both the MAPK (ERK) cascade pathway and the mTOR complex through GPCR signaling using the adaptor protein Grb2.

    GRBIMAGEfIG6.svg
    Figure \(\PageIndex{30}\): Combined activation of both the MAPK (ERK) cascade pathway and the mTOR complex through GPCR signaling using the adaptor protein Grb2Yang et al. Journal of Cellular and Molecular Medicine 22(6). DOI: 10.1111/jcmm.13447. License CC BY 4.0

    Downstream signaling through the Mitogen Activated Protein Kinase Cascade

    Active upstream kinases like PKA, PKC and RTKs phosphorylate target proteins and in doing so change their activities. The usual protein targets are kinases, which become active on phosphorylation. They in turn activate other kinases, resulting in a complex cascade and amplification of the original signal. Often the end product of such cascade is a phosphorylated transcription factor which can alter gene expression. Perhaps the most described of these cascades is the Mitogen Activated Protein Kinase (MAPK) pathway. Mitogen are chemical species lead to mitosis (cell division). The MAPK system has three layers and a confusing nomenclature (until you are used to it). The end (down stream) product of the cascade is the enzyme mitogen activated protein kinase, abbreviated MAPK. It can be phosphorylated several times to produce MAPKP or MAPKPP where the last Ps in the abbreviation signify the number of added phosphate groups.

    The kinase immediately upstream that phosphorylates MAPK is abbreviated MAPKK (for mitogen activated protein kinas kinase) or MAP2K. MAPKK (MAP2K) is activated by yet another upstream kinase called MAPKKK or MAP3K. If these are also targets of another upstream unrelated kinase, they would be abbreviated MAP3KPP for example.

    Now of course, there are families of the MAPK cascade enzyme, each with their own name. Figure \(\PageIndex{31}\) below shows the names of a four different mammalian MAP3Ks leading to the activation of 5 different MAP2Ks which lead to the activation of 4 different MAPKs. Some of these enzymes are so widely discussed in textbooks and journal articles that it is good to remember them specifically with their alternative names. These include the MAP3K enzymes Raf (Rapidly Accelerated Fibrosarcoma) and MEK, and the MAPK enzymes ERK (Extracellular Related Kinase) and JNK (c-Jun N-terminal Kinase)

    MAPK-pathway-mammalian.png
    Figure \(\PageIndex{31}\): Simplified overview of the mammalian MAPK cascades https://commons.wikimedia.org/wiki/F...-mammalian.png. Creative Commons Attribution-Share Alike 3.0 Unported

    Figure \(\PageIndex{32}\) shows another representation of the MAPK cascade with some different enzyme names and added phosphates shown in circles.

    Cardiac_Fibroblast_p38_MAPK_A_Critical_Regulator_o_Fig1.svg

    Figure \(\PageIndex{32}\): Another representation of the MAPK cascade. Journal of Cardiovascular Development and Disease 6(3):27. 2019. 10.3390/jcdd6030027. CC BY 4.0

    One way to organize a seminar on a complex topic is to use these 3 steps: tell your audience what you will tell them, tell them, and then tell them what you told them. Following that advice, we present in Figure \(\PageIndex{33}\) below an integrated view of signaling starting from the membrane and moving inward to three enzymes in the MAPK kinase cascade, RAF (a MAP3K), MEK (a MAP2K) and ERK (a MAPK). Upstream signaling to the MAPK cascade comes RTKs, GPCRs and Ca2+ signaling, which we will discuss later.

    MAPERK_Signaling_in_Developing_Cognitive_and_EmotiFig1.svg
    Figure \(\PageIndex{33}\): Upstream signaling to the MAPK cascade from RTKs, GPCRs and Ca2+ signaling Albert-Gasco et al. International Journal of Molecular Sciences 21(12):4471 (2020). DOI: 10.3390/ijms21124471. License CC BY 4.0

    The sequence of events is:

    • binding of external signal to membrane receptor and activation of receptor kinase
    • phosphorylation of receptor kinase and interaction with an activator GTP binding protein like ras
    • binding of activated G-protein to and activation of a mitogen activated protein kinase kinase kinase (MAPKKK)
    • MKKK phosphorylates and activates another kinase, MAPKK
    • MKK phosphorylates and activates mitogen activated protein kinase, MAPK
    • MAPK phosphorylates inactive transcription factors (or other proteins) and activates them. Unfortunately (from a naming point of view) when the activated proteins are themselves protein kinase, they are called mitogen activated protein kinase activated protein kinases (MAPKAPK)

    There are seven types of MAPKs, four conventional and three atypical. Four typical ones are described in the table below.

    Activator GTP binding protein Ras:GTP      
    MAPKKK or MAPK3 Raf-1A/B
    c-Mos
    MEKK1-4
    DLK
    MLK2
    MEKK1-4
    DLK
    MLK2
    MEKK2/3
    Tpl-2
    MAPKK or MAPK2 MEK1,2 MEK4,7 MEK3,6 MEK5
    MAPK or MAK ERK1,2 JNK1-3 p38 ERK5
    MAPKAPK RSK 1-4
    MNK2
    MSK 1,2
    MK2,3 MSK1,2
    MK2,3
    RSK1-4
    An eventual
    Protein Target
    c-Jun c-Jun    

    iconexternal_link.gifMAP Kinase System from Cell Signaling

    Structural the protein are similar in size and domain structure as we have seen for other kinases. Figure \(\PageIndex{34}\) below shows an interactive iCn3D model of the alignment of MEK 1 (4U7Z) and ERK2 (5NHJ)

    alignment of MEK 1 (4U7Z) and ERK2 (5NHJ).png
    Figure \(\PageIndex{34}\): . (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Vq6RR7BA6o1Qb7

    We now present multiple visual images of the activation of the MAPK cascade. Figure \(\PageIndex{35}\) shows two.

    Details:

    EGFR_signaling_pathway.png

    EGFR_signaling_pathway.png. public domain

    MAPKpathway_diagram.svg
    Figure \(\PageIndex{x}\): https://commons.wikimedia.org/wiki/F...ay_diagram.svg. Creative Commons Attribution-Share Alike 4.0 International

    Figure \(\PageIndex{35}\): Two visual representation of the activation of the MAPK cascade through to activation of gene transcription.

    As with protein kinase A and protein kinase C, signaling efficiency and specificity with minimal phosphorylation of wrong targets occurs when multiple signaling partners are scaffolded. This is also true of the MAPK cascade trio of kinase. Figure \(\PageIndex{36}\) shows the role of scaffolds KSR and Ste5 in MAPK signaling.

    scaffoldsshapeMAPKsignalingFig1.svg
    Figure \(\PageIndex{36}\): The role of scaffolds KSR and Ste5 in MAPK signaling. (A) In quiescent cells an inactive KSR/MEK complex exists in the cytosol. (B) Upon stimulation of the cell, KSR translocates to the cell membrane and forms an active complex with phosphorylated Raf, MEK, ERK. Activated ERK detaches from the scaffold with three outcomes; (1) ERK dimerizes in the cytoplasm where the dimer remains or translocates to the nucleus; (2) ERK translocates to the nucleus; (3) KSR acts as a platform where ERK dimers are assembled and the new complex can interact with substrates in the cytoplasm. (C) Schematic of the yeast mating pheromone response pathway. Witzel et al. Front. Physiol., 21 December 2012 | https://doi.org/10.3389/fphys.2012.00475. Creative Commons Attribution License,

    Systems Biology: MAPK Cascade

    You might think that the interconnected reactions of the MAPK cascade are really complicated. However, as presented in the figures above, it really only consists of 3 enzymes, MAPKKK (MAPK3 or MK3), MAPKK (MAPK2 or MK2) and MAPK. We added complexity by converting the actual enzymes from an inactive state to an active state by phosphorylation. In reality this pathway is simple compared to the complete signal transduction pathways they are part of, and very simple compared to whole catabolic and anabolic pathways that we will see in Part Two of this book.

    We have discussed kinetics in earlier chapters and have shown how it can be used to more fully understand an enzyme and its regulation/control. We need to extend such kinetic analyses to whole pathways as well. We can do that using the VCell. Let's look at two different models of the MAPK cascade using Vcell. One particularly interesting feature is the regulation of the pathway. We will see in the next section on metabolism that pathways are often regulated by the end product of the pathway. This makes sense since if that end product is abundant, it would make biological sense for that product to inhibit the first enzyme in the pathway to avoid making more of the ultimate end product. Of course that inhibition would be relieved as the concentration of the end product falls. Hence there is a temporal sense to the inhibitio.

    Let's look at two models for the MAPK Cascade, one with no end product inhibition and one with inhibition of the first step. Since we dealing with linked kinases, the inhibition of the first enzyme (MAP3K=MKKK) and hence the first reaction (MKKK ↔ MKKK_P) is not mediated by a chemical product of the last enzyme (MAPK_PP) but by phosphorylation of the first enzyme (MKKK) by the last (MAPK_PP).

    VCellLogo.png

    MAPK Cascade - Model 1: No feedback inhibition of the MAPK cascade by inhibition of the first step (MKKK ↔MKKK_P) by the "end product" of the cascade (MAPK__PP)

    Vcell OMEX-SBML file

    Model: 

    NoFeedbackMAPKCascadeVCellModel.png

    Reference: Kholodenko2000___Ultrasensitivity_and_negative_feedback_bring_oscillations_in_MAPK_cascade

    The various concentration vs time curves in the output graph should make "intuitive" sense. There are no surprises!

    Now let's add a twist. What if the last active enzyme in the pathway, MAPK that is doubly phosphorylated (MAPK_PP), the "finaL product" of the cascade, can, in a feedback reaction, inhibit the very first reaction of the cascade, MKKK → MKKK_P by an inhibiting phosphorylation. Run the simulation in Vcell to find out!

    VCellLogo.png

    MAPK Cascade - Model 2: Feedback inhibition of the MAPK cascade by inhibition of the first step (MKKK ↔MKKK_P) by the "end product" of the cascade (MAPK__PP)

    Vcell OMEX-SBML file

    Model: 

    OscillationsMAPKCascadeVCellModel.png

    Reference: Kholodenko2000___Ultrasensitivity_and_negative_feedback_bring_oscillations_in_MAPK_cascade

    Kholodenko2000 - Ultrasensitivity and negative feedback bring oscillations in MAPK cascade. https://www.ebi.ac.uk/biomodels/BIOMD0000000010. Based on Kholodenko BN. Negative feedback and ultrasensitivity can bring about oscillations in the mitogen-activated protein kinase cascades. Eur J Biochem. 2000 Mar;267(6):1583-8. doi: 10.1046/j.1432-1327.2000.01197.x. PMID: 10712587.

    There is utterly no way to have predicted, using intuition or simple mathematical analyses, the oscillatory changes in the concentration of all the enzymes and their phosphorylated variants. Of course, the oscillating concentrations depend on the chosen initial concentrations and on the rate constants.

    When enzymes are linked sequentially in signal transduction pathways, their actions are amplified by the preceding enzyme. If the first kinase (MK3) activates a 1000 molecules of the second kinase (MK2), and each of these activate 1000 of the last kinase (MAPK), the net effect of activating the first kinase is a million-fold amplification of the last! This causes this three kinase pathway to be ultrasensitive to small changes to the first enzyme. Add to this other activating and inhibiting agents and the net activities of the pathway components becomes even more complicated.

    Just the simple feedback inhibition by the last enzyme of the first enzyme in this cascade can bring about the oscillation shown in Vcell model 2. Depending on the concentrations and rate constants used in the model, the oscillations can last from minutes to hours. These oscillations can produces waves of phosphoproteins that propagate through the cytoplasm of the cell.

    Here is a simplified animation of the MAPK cascade that shows changes in MKKK (red dots) and MAPK_PP) blue dots with no feedback inhibition (left, Model 1) and feedback inhibition (right, Model 2) in the cascade. (Animations produced by Shraddha Nayak and Hui Lui.)

    Model 1: No feedback inhibition of the MAPK cascade Model 2: Feedback inhibition of the MAPK cascade
    MAPKCascade_NoOscillations.png MAPKCascade_WithOscillations.png

    You might expect similar oscillatory behavior in proteins (cyclins and cyclin-dependent protein kinases) controlling the movement of cells through the cell cycle. We will see that in a subsequent section.


    12.4: The next step - Downsteam intracellular signaling is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski and Patricia Flatt.