Lecture #12 | Oncogenes in Signal Transduction: GF Receptors, Ras, Raf, MAPK, JNK, PKC

Oncogenes in Signal Transduction

  • Lecture 12 focuses on oncogenes involved in signal transduction, specifically growth factor receptors, Ras, Raf, MAPK, JNK, and PKC.
  • References Weinberg's Cancer Biology textbook, Chapters 5.3, 5.4, 5.6, 5.10 (synopsis) and Chapters 6.5, 6.14, 6.15 (synopsis).
  • Also references the lecture "Cancer Chemoprevention w/ Dietary Phytochemicals" from Lecture 4, Figure 3.

Lecture 12 Overview

  • Ras Identification: How Ras, a human oncogene, was identified.
  • Ras Function: Normal function as a G protein.
  • Ras Mutation: Mutation leading to oncogenic activity.
  • Ras Targeting: Prenylation and targeting of Ras for cancer treatment.
  • Receptor Tyrosine Kinases (RTKs)
  • Kinase Cascade: Raf, MEK, MAPK pathway.
  • Scaffolding Proteins: Their role in signal transduction.
  • Other Pathways:
    • JNK and stress-activated pathway.
    • Protein kinase C and phorbol esters.
  • Kinase Regulation: Regulatory domains of kinases.
  • Phytochemicals: Impact on signal transduction.
  • Abbreviations:
    • PO4 = phosphorylates/phosphate group
    • TF = transcription factor
    • ST = signal transduction
    • Txn = transcription

Learning Objectives

  1. Ras Function: Understand the role of Ras in the signal transduction pathway.
  2. Tumorigenesis: Explain how mutations in Ras contribute to tumorigenesis.
  3. Prenylation: Describe the significance of prenylation in Ras function.
  4. Growth Factor Receptors: Explain how growth factor receptors signal to Ras.
  5. Downstream Targets: Identify the downstream targets of Ras.
  6. Scaffolding: Describe the role of scaffolding proteins in signal transduction.
  7. MAPK vs. SAPK: Differentiate between mitogen-activated (MAPK) and stress-activated (SAPK) signal transduction pathways.

Isolation of Human Ras Oncogene

  • Isolated from human bladder carcinoma using the NIH 3T3 Assay / Foci Assay. Refer to Weinberg 4.1 in supplemental slides.

Viral Oncogenes

  • Several viral oncogenes and their origins:
    • src: Rous sarcoma virus (Chicken)
    • myc: Avian myelocytomatosis virus (Chicken)
    • erb A, erb B: Avian erythroblastosis virus (Chicken)
    • myb: Avian myeloblastosis virus (Chicken)
    • ets: Avian erythroblastosis virus (Chicken)
    • rel: Avian reticuloendotheliosis virus (Turkey)
    • H-ras: Harvey rat sarcoma virus (Rat)
    • K-ras: Kirsten murine sarcoma virus (Mouse)
    • abl: Abelson murine leukemia virus (Mouse)
    • raf: Murine sarcoma virus (Mouse)
    • fos: Mouse osteosarcoma virus (Mouse)
    • fms: Feline sarcoma virus (Cat)
    • fes: Feline sarcoma virus (Cat)
    • sis: Simian sarcoma virus (Monkey)
  • After cloning, the cellular gene isolated from the human tumor was found to be related to known viral oncogenes in rat and mouse.
  • This discovery occurred in the 1980s when biological databases and computer usage were nascent.

RAS Oncogene Cloning

  • Viral Oncogene Identification:
    • Mouse Harvey sarcoma virus -- RasH
    • Mouse Kirsten sarcoma virus -- RasK
  • Human Tumor Isolation (NIH 3T3 foci assay):
    • Bladder carcinoma -- cellular RasH (Cooper & Weinberg, 1981)
    • Lung carcinoma -- cellular RasK
    • Neuroblastoma -- cellular RasN
    • Chemically induced tumor in rats (Weinberg, 1979)

RAS Overview

  • Ras is mutated in approximately 30% of all tumors.
    • 50% of colon cancers.
    • 90% of pancreatic cancers.
    • Less frequent mutations in other cancers.
  • Guanine Nucleotide Binding Protein (G-protein):
    • Binds GDP/GTP.
    • Cycles between inactive (GDP-bound) and active (GTP-bound) states.
    • Transitions are aided by Guanine Nucleotide Exchange Factors (GEF) and GTPase Activating Proteins (GAP).
    • GEF promotes GDP to GTP exchange.
    • GAP stimulates GTP hydrolysis.

Ras Switching Mechanism

  • Two domains of Ras protein (Switch I & II) bind to the γ-PO4 of GTP in the active "on" state.
  • Hydrolysis of the PO4 causes Switch I & II to relax, generating the inactive "off" state.

Ras Mutations and Oncogenesis

  • Activating mutations in Ras:
    • Increase exchange of GDP for GTP.
    • Increase binding to RAF.
    • Block GAP's ability to hydrolyze GTP (e.g., codon 12).
  • Normal cells: ~95% of Ras protein is inactive.
  • Normal cells: ~5% of Ras protein is active.

Point Mutations in Ras

  • A single point mutation (G>T) in codon 12 results in a Gly>Val change, converting the normal Ras gene into an actively transforming oncogene.
  • Normal RasH sequence example: MetThrGluTyrLysLeuValValValGlyAlaGlyGlyMet Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly
  • Activated RasH sequence example: MetThrGluTyrLysLeuValValValGlyAlaValGlyMet Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly
  • Codons 13, 59, and 61 are also hotspots for point mutations that activate Ras.

Guanine Nucleotide Binding Pocket

  • Activating mutations in Ras are often found in the guanine nucleotide binding pocket (codon 12).
  • Mutations in codon 12 (GGC → GTC) inhibit GTP hydrolysis, keeping Ras in the active state.
  • AA substitutions in the effector loop (codons 35, 37, 40) affect interactions with downstream effectors of Ras.
  • Codons 60 and 61 in Switch II are also frequently mutated in cancer.

Ras as a Target of Chemical Carcinogens

  • B[a]P (Benzo[a]pyrene) ==> lung tumor ==> codon 12 mutated
  • AFB1 (Aflatoxin B1) ==> liver tumor ==> codons 12 and 61 mutated
  • Alkylating agent (MNU) ==> breast tumor ==> codon 12 mutated
  • Diethylnitrosamine ==> liver tumor ==> codon 61 mutated
  • In humans, mutations in codon 12 decrease colon cancer survival (causation or correlation?).
  • In animals, mutations in codon 12 are induced by chemical carcinogens.

Ras Protein Family and C-Terminal Domain

  • Members of the Ras family are highly conserved, except for the C-terminal domain (CTD).
  • The CaaM,S motif at the very C-terminus is conserved, suggesting its importance for Ras function.

Prenylation of Ras

  • The C-terminus (at CaaM,S) is prenylated, allowing attachment to the plasma membrane.
    • Prenylation = addition of a prenyl group (like a fatty anchor).
    • Made up of isoprene units.
    • Carried out by farnesyl transferase.
  • Farnesyl transferase inhibitors block prenylation, making them potential anti-cancer drugs.
    • Attachment of RAS to the membrane is required for oncogenicity.
    • If mutated Ras is not properly anchored, it will not be oncogenic.
  • Limonene also blocks prenylation.
    • Found in orange and lemon peels.
    • Used in breast cancer treatment.
  • Ras is anchored to the inner plasma membrane.

Farnesyl Transferase

  • Farnesyl pyrophosphate (FPP) and Limonene are relevant molecules.
  • Farnesyl transferase inhibitors are used to treat leukemias and other diseases.
  • Drug target: Farnesyl Transferase, Methyl Transferase, RAS Protease.

Receptor Tyrosine Kinases (RTKs)

  • Growth factor receptors signal to Ras via adaptor proteins.
  • Dimerization and autophosphorylation are key steps.
  • Four different ways to turn a GF receptor into an oncogene.

Ras Function and Downstream Signaling

  • Ras transduces the signal from membrane-bound GF receptors to a kinase cascade.
  • SH2 and SH3 domains are involved.

Epidermal Growth Factor Receptor (EGFR)

  • EGFR, a receptor tyrosine kinase (RTK), was first cloned as a viral oncogene, v-ErbB.
  • Its kinase domain has homology with the Src kinase domain (SH1).

EGFR Activation

  • EGF binds to dimeric EGFR -> autophosphorylation and transphosphorylation.

Growth Factor Receptors becoming Oncogenic

  • Several ways for a GF receptor to become oncogenic:
    • Ligand-independent dimerization:
      • In certain glioblastomas, the ROS RTK gene is fused with the FIG gene.
      • FIG causes the RTK to dimerize (& autophosphorylate) in the absence of GFs.
    • Ligand-independent activation of a GF receptor.

GF Receptor Signaling to Ras

  • GFs activate GF receptors within seconds.
  • Ras is activated within minutes.
  • 5 minutes after adding EGF to cells, Ras is activated.
  • GF receptors signal to Ras.

MAPK Pathway

  • MAPK = mitogen-activated protein kinase. A mitogen stimulates cell proliferation.
  • Activated Ras is a key initiator of several signaling pathways. The best-characterized is the MAPK pathway.

MAPK Activation

  • Active (GTP-bound) Ras recruits Raf to the membrane.
  • Hydrolysis of Ras-GTP to Ras-GDP releases active Raf.
  • Raf is a Ser/Thr kinase that phosphorylates MEK.
  • MEK is a dual-specificity kinase (Tyr & Ser/thr) that phosphorylates MAP Kinase (MAPK) – a ser/thr kinase.

MAPK Targets

  • Active MAPK phosphorylates targets in the cytosol and nucleus.
  • MAPK phosphorylates other kinases (p90RSK) in the cytosol, which translocate to the nucleus and phosphorylate SRF, a TF.
  • MAPK also translocates to the nucleus & phosphorylates TCF.
  • P-TCF and P-SRF bind the SRE in the promoter of the c-Fos gene (c-Fos is an oncogene).
  • SRF = serum response factor; SRE = serum response element.
  • Serum is full of GFs/mitogens.

Scaffolding Proteins

  • Kinases in the ST pathway are held in place by scaffolding proteins.
  • The scaffold promotes specificity and efficiency of the pathway.
  • Only the appropriate targets are phosphorylated in an efficient manner.
  • The concentration of both the kinases and the scaffolding proteins needs to be just right for a normal ST pathway.
  • Local adaptors anchor the scaffolding protein and hence the kinases to the inner plasma membrane (where Ras resides).

SAPK Pathway

  • Another ST pathway is activated by stress -- SAPK pathway.
  • JNK is the key kinase in this pathway.
  • The stress response results in:
    • Apoptosis.
    • Immune activation.
    • Transformation (c-Jun).
    • Adaptation to stress.
  • Mitogen - proliferative signal, e.g., growth factors
  • Stress - UV, ROS, osmolarity changes, heat shock, protein synthesis inhibitors, inflammatory cytokines
  • ERK = extra-cellular signal regulated kinase (ERK pathway = MAPK pathway)

MAPK Pathway Activation

  • Signals other than growth factors can activate the MAPK pathway:
    • Phorbol esters (tumor promoter) activate Protein kinase C (PKC): ser/thr kinase activated by Ca++ and diacylglycerol (and TPA)
    • Ionizing and non-ionizing (UV) radiation can stimulate the ST pathway by activating growth factor receptors
  • TPA: phosphorylates and activates Raf, a member of a family of PKCs; also phosphorylates c-Jun and other TFs. Overexpression can transform cells.

Kinase Regulation

  • Kinases have regulatory regions that control the activity of the kinase domain.
  • Oncogenic kinases lack the regulatory domains.

Phytochemicals

  • Phytochemicals block PKC, JNK, and other ST and pro-inflammatory pathways.

GFs and Receptors

  • There are many different types of GFs and receptors.
  • GF receptors bind many different adaptor molecules.
  • Ras is central to several different pathways, in addition to MAPK.
  • There are many other G proteins, in addition to Ras.
  • There are many scaffolding proteins.
  • Complexity of ST pathway -> lots of potential drug targets.

Terminology

  • G-protein = switch dependent on GTP/GDP binding (not a kinase)
  • MAPK = mitogen-activated protein kinase
  • Mitogen = any signal that stimulates the growth/proliferation of a cell
  • ERK = extra cellular signal kinase (originally MAPK = ERK. They were identified at the same time by 2 different labs). Now MAPK is used as a general term for a family of related kinases.
  • MAPK/ERK is a specific member of the family, as are p38 and SAPK.
  • MEK = MAPK (ERK) kinase (MAP kinase kinase, MAPKK)
  • Raf is a MAP kinase kinase kinase (MAPKKK)
  • JNK = Jun N-terminal kinase
  • Ligand = small molecule that binds a protein receptor, can be a peptide, a hormone, a vitamin, dioxin, etc.

GF Receptors

  • There are many different types of GF receptors; they all have a kinase domain in the cytoplasmic side.

Adaptor Molecules

  • The cytoplasmic domain of GF receptors binds many different adaptor molecules via p-Tyr.

JNK Pathway Coordination

  • Like the MAPK pathway, factors in the JNK pathway are coordinated by scaffolding proteins.
  • Response to different stimuli/stress is coordinated by different scaffolding proteins.

Mutagenic Outcome Factors

  • Route of exposure.
  • Site of metabolism into a mutagen.
  • Structure of the mutagen.
  • Surrounding sequence in the DNA: hotspots for DNA damage.
  • The rate of DNA repair.
  • Codons 12, 13, 59, and 61 are not the only codons that are mutated. Other mutations may be made in Ras or other genes, but if they do not result in a growth advantage and produce a tumor, they will not be selected for and, hence, not detected.

Chemical Inhibitors of Kinases

  • Many chemical inhibitors of kinases have been developed. They effectively treat cancer and other diseases.