L9 - Kinases

Basics of Kinase Signaling

Phosphorylation

  • Definition: Phosphorylation is a reversible process critical for controlling enzyme activity.

  • It plays a role in coordinated regulation, often necessitating opposing effects (example: glycogen homeostasis).

  • Key Terms:

    • Phosphorylation/Kinase: Addition of a phosphate group to a protein, mediated by kinases.

    • Dephosphorylation/Phosphatase: Removal of a phosphate group, mediated by phosphatases.

  • These are reciprocal reactions critical for cellular function.

Phosphorylation Targets

  • Phosphorylation typically occurs on one of three amino acids:

    • Serine

    • Threonine

    • Tyrosine

  • Types of Kinases:

    • Serine/Threonine Kinases

    • Tyrosine Kinases

  • Phosphorylation can happen at multiple sites on a single protein, facilitated by various kinases.

  • Kinases may exhibit specificity toward a single protein or have broader action across multiple proteins.

  • Consensus Sequences:

    • These are specific amino acid sequences recognized by kinases, often determined via bioinformatics.

  • ATP Role: ATP typically provides the phosphate group during phosphorylation.

Serine/Threonine Kinases

  • Examples include:

    • Protein Kinase A (PKA)

    • Protein Kinase B (PKB)

    • Protein Kinase C (PKC)

    • G-Protein-Coupled Receptors Kinases (GRKs)

    • Mitogen-Activated Protein Kinases (MAPKs)

    • Ca2+-Calmodulin-Dependent Protein Kinase (CAM Kinase)

Mitogen-Activated Protein Kinases (MAPK)

  • Description: A signaling cascade involving serine/threonine kinases (and some tyrosine kinase activity) that modifies transcription and/or enzyme activity.

  • Activation triggers:

    • Mitogens

    • Heat shock

    • Pro-inflammatory cytokines through engagement with G proteins (either trimeric or monomeric) or reactive oxygen species.

  • Functions include regulation of:

    • Gene expression

    • Mitosis

    • Differentiation

    • Proliferation

    • Cell survival and apoptosis.

MAP-Kinase Phosphorylation Pathway

  • Major signaling components include:

    1. Ras

    2. MAP-Kinase-Kinase-Kinase (MAP3K)

    3. Activated Ras Protein

    4. MAP-Kinase-Kinase (MAP2K)

    5. MAP-Kinase (MAPK)

  • Pathway shown:

    • GTP->GDP conversion

    • ATP utilization with kinase cascades to activate different proteins affecting gene expression and enzymatic activity.

Tyrosine Kinases

  • Definition: Phosphorylation of tyrosine residues, which is less common than that of serine or threonine.

  • Categories:

    • Non-Receptor Tyrosine Kinases

    • Receptor Tyrosine Kinases

    • Includes Janus Kinases (JAKs), Epidermal Growth Factor (EGF) receptors, and Insulin receptors.

  • Functionality:

    • JAKs are involved in interferon signaling, promoting phosphorylation of STATS (signal transducers and activators of transcription) which regulate immune functions and angiogenesis.

Janus Kinases (JAKs)

  • Activation Process:

    • Cytokine binding induces a conformational change in the receptor activating JAK kinases.

  • Active JAKs perform reciprocal phosphorylation on each other and on other target proteins.

  • JAKs also phosphorylate STATS, which play a role in gene regulatory processes related to immune response.

Tyrosine Kinase-Linked Receptors

  • General Structure:

    • An extracellular ligand-binding domain

    • A single transmembrane domain

    • A cytoplasmic domain with intrinsic kinase activity

  • Function: These receptors mediate the initial steps in signaling for various hormones including insulin and EGF.

Tyrosine Kinase-Receptor Signaling

  • Mechanism:

    • Ligand binding leads to conformational changes and dimerization of receptors.

    • The cytoplasmic domains phosphorylate each other, activating kinase activity.

    • Kinases then phosphorylate intracellular mediators, initiating common signaling pathways (e.g., MAPK and PI3-Kinase).

EGF Receptor (EGFR)

  • Role: EGF facilitates cell lineage determination, organ development, cell survival, and has roles in oncogenesis.

  • Cancer Association: EGF receptor overexpression is noted in numerous cancers.

  • Mechanism:

    • Binding induces transformation from inactive monomers to active dimers.

    • The intracellular domains undergo phosphorylation on tyrosine residues, enhancing enzymatic activity and catalyzing phosphorylation cascades.

EGFR Pharmacology

  • Monoclonal Antibodies:

    • Examples include Cetuximab (Erbitux) and Panitumumab (Vectibix) which bind EGFR to block EGF availability, reducing signaling.

    • Use: Treatment for metastatic colorectal cancer and head & neck cancer.

  • Small Molecule Inhibitors:

    • Examples: Gefitinib (Iressa) and Erlotinib (Tarceva), classified as TKIs.

    • Mechanism: Prevent phosphorylation on tyrosine residues inhibiting cancer cell proliferation and angiogenesis.

Insulin Receptor

  • Overall Impact: Insulin lowers blood glucose levels by promoting glucose uptake, glycogen production, and stimulating protein and lipid synthesis.

  • Structure:

    • The receptor consists of alpha and beta subunits; alpha binds insulin, while beta transmits intracellular signals.

  • Autophosphorylation: The beta subunit undergoes autophosphorylation on tyrosines post insulin binding, leading to various downstream effects.

IRS-1 and Downstream Pathways

  • Upon activation, IRS-1 associates with the tyrosine-phosphorylated insulin receptor and undergoes phosphorylation on its own tyrosine residues.

  • Two significant pathways from IRS-1:

    1. Ras Pathway:

    • Activated Ras binds GTP, spurring a MAPK cascade affecting gene transcription.

    1. PI3-Kinase Pathway:

    • PI3-Kinase, binding to IRS-1, converts PIP2 to PIP3, activating PKB promoting glycogen synthesis, and various signaling processes.

Protein Kinase B (PKB)

  • Also known as Akt:

    • It's a serine/threonine kinase that interacts with PIP3.

  • Activation involves relocalization from the cytoplasm to the plasma membrane where it is phosphorylated, participating in signaling related to metabolism, proliferation, and apoptosis.

Ras G Protein

  • Part of small GTPases involved in cell signaling processes, specifically in cell growth and differentiation.

  • It operates in a monomeric form and is activated by GTP binding.

Arachidonic Acid Signaling

Arachidonic Acid

  • It is a second messenger generated from DAG via diacylglycerol lipase or derived from phospholipids through phospholipase A2 (PLA2).

  • Serves as a precursor for eicosanoids such as leukotrienes and prostaglandins, which are key mediators in the inflammatory response.

Calcium Signaling

Historical Background

  • Research by Sydney Ringer established the significance of calcium for heart contractions in the 1880s.

  • Direct application experiments in the 1940s confirmed that Ca2+ induces muscle fiber contractions.

Role in Cell Function

  • Recognized as a vital second messenger in numerous processes: secretion, muscle contraction, gene transcription, and cell proliferation.

  • Intracellular calcium concentrations are typically very low:

    • Cytosol (resting cell): 50-100 nM

    • Endoplasmic Reticulum (ER): 30-300 μM

Calcium-Regulated Proteins

  • Include various proteins such as:

    • Ca2+-dependent kinases

    • Phospholipase C

    • Nitric oxide synthase

    • Protein kinase C

    • Other enzymes and pathways modulated by calcium.

Mechanisms for Calcium Homeostasis

  • Maintenance:

    • Pumps and exchangers help keep low intracellular calcium levels.

    • Calcium-binding molecules assist in sequestration.

Increasing Cytosolic Ca2+

  • Mechanisms include:

    • Release through phospholipase C-generated IP3 from internal stores.

    • In excitable cells, voltage-gated Ca2+ channels open upon depolarization.

Calcium Targets

  • Proteins like Protein Kinase C are activated by Ca2+, mediating phosphorylation and signaling pathways.

Ca2+-Calmodulin

  • A calcium-binding protein that can bind up to 4 calcium ions.

  • Structure resembles a dumbbell with two globular regions connected by a flexible alpha helix.

  • When binding Ca2+, conformational changes occur that enhance interaction with target proteins.

Ca2+-Calmodulin-Dependent Kinase (CaM)

  • Concentrated in neuronal tissues including the brain, influencing several cellular processes including memory, ion channel modulation, and regulation of metabolic pathways.

Cyclic GMP & Nitric Oxide

Cyclic GMP Functionality

  • Operates in few cell types (e.g., intestinal mucosa) as a signaling molecule.

  • Intracellular rise in cGMP promotes smooth muscle relaxation via dephosphorylation of myosin light chains, induced through specific ligands that stimulate guanylyl cyclase.

Nitric Oxide (NO)

  • Characteristics:

    • A free radical that can diffuse across membranes, unstable with a short half-life of 5-10 seconds.

    • Reactivity leads to interactions that can change its form (e.g., nitrite and nitrate).

NO and Smooth Muscle

  • Actions include the rapid relaxation of smooth muscle upon NO diffusion and activation of guanylyl cyclase resulting in cGMP production.

Nitric Oxide Synthase (NOS)

  • Different forms:

    • macNOS: Found in macrophages

    • eNOS: Present in endothelial cells

    • nNOS: Found in neuronal tissues

    • iNOS: Inducible form often present in macrophages.

Review & Integration of Second Messenger Systems

Intracellular Signaling Types

  • Direct signaling through channels and metabotropic receptors, and long-term signaling through trophic and endocrine factors which often involve kinase pathways or altered transcription.

Second Messenger Roles

  • Second messengers are essential in mediating signals generated intracellularly from primary signals.

  • Various kinases participate in the signaling pathways, including both second messenger-dependent and independent pathways.

  • Phosphoproteins often execute signal transmission.

Second Messenger Systems

  • Cyclases such as adenylate and guanylate cyclase function to raise the levels of cyclic nucleotides, acting on various cellular mechanisms.

Key Mechanisms of Second Messenger Systems

  • Phospholipase C catalyzes the formation of IP3 and DAG, which affect Ca2+ levels and signaling through kinases like PKC.

  • Phospholipase A2 generates Arachidonic acid, a precursor and signaling molecule within various pathways, especially in inflammatory responses.