Lecture 23 PSC2002

Course Overview

  • Course Title: PSC2002 L23 Other Protein Kinases and Protein Phosphatases

  • Instructor: Dr. Mike Gray

Learning Outcomes

  • Understand and explain the role and regulation of other protein kinases not directly regulated by cAMP.

  • Explain the function and regulation of protein phosphatases.

  • Discuss clinical examples of diseases linked to these protein kinases and phosphatases.

  • Explore examples where there is significant interplay between different intracellular signaling pathways.

Other Protein Kinases (PK)

  • Activation Mechanisms:

    • Some are activated by cyclic GMP, notably Protein Kinase G (PKG).

    • Others activated by increases in Ca2+:

      • PKC: Activated by Ca2+ and/or lipids.

      • Calmodulin-dependent protein kinases:

      • PKB (Akt) and Protein Tyrosine Kinases (discussed in related lectures on Insulin and diabetes).

The cGMP Signaling Pathway

  • Overview:

    • Similar to cAMP pathway; GPCRs are not involved.

    • cGMP synthesized by guanylyl cyclase (GC).

  • Types of GC:

    • Soluble GC (sGC): Activated by nitric oxide (NO).

    • Plasma Membrane Bound GC (pGC): Activated by a small number of peptide agonists.

  • Function of cGMP and PKG:

    • cGMP activates PKG (two isoforms: I and II).

    • PKG phosphorylates serine/threonine residues.

    • cGMP degradation by cGMP-dependent phosphodiesterases (PDEs), e.g., PDE 5.

cGMP in Health and Disease

  • Health Applications:

    • Atrial natriuretic factor (ANP), C-type natriuretic factor (CNP), and Brain natriuretic factor (BNP) activate cGMP signaling.

    • Heat-stable enterotoxin from E. coli activates pGC/cGMP leading to secretory diarrhea via CFTR activation.

    • Nitric oxide (NO) released from endothelial cells activates sGC/cGMP in vascular smooth muscle for vasodilation.

    • Excessive NO production from iNOS can lead to clinical shock.

    • Viagra (sildenafil) inhibits PDE5, raising cGMP levels to relax smooth muscle, treating erectile dysfunction and pulmonary hypertension.

Protein Kinase C (PKC)

  • Structure and Isoforms:

    • Single polypeptide with both regulatory and catalytic domains.

    • 11 different isoforms divided into three groups: conventional, novel, and atypical.

    • Requires phospholipid binding for activity.

  • Activation Process:

    • Ca2+ binds C2 domain, PKC translocates to the plasma membrane, binds diacylglycerol (DAG) via C1 domain.

    • Pseudosubstrate motif disengages from the substrate binding site, allowing substrate binding and phosphorylation.

    • Phorbol esters can chemically activate PKC.

Ca2+/Calmodulin-dependent Protein Kinases

  • Types:

    • Narrow substrate specificity (e.g., phosphorylase kinase).

    • Broad substrate specificity (e.g., Multifunctional CaM Kinase II).

  • Functions:

    • Regulates NMDA receptors, enhances Inositol triphosphate formation, decodes calcium signals, and controls SERCA2 pump activity.

Protein Phosphatases (PP)

  • Function:

    • Remove phosphate groups from Ser/Thr and Tyr residues of phosphorylated proteins.

  • Major Classes:

    • Four classes of Ser/Thr phosphatases (PP1, 2A, 2B, 2C) with varying substrate specificities.

    • One main class for Tyrosine phosphatases: cytosolic and membrane-bound forms.

Chemical Inhibitors of Protein Phosphatases

  • Okadaic acid:

    • Inhibits PP1 and PP2A.

  • Cyclosporin A:

    • Specific inhibitor for calcineurin (PP2B), used clinically for immunosuppression post-transplantation.

Effects of Okadaic Acid

  • Source:

    • Toxin from dinoflagellates, causing diarrhetic shellfish poisoning (DSP) upon ingestion.

  • Symptoms:

    • Severe diarrhea, vomiting, and nausea.

  • Mechanism:

    • Increases paracellular permeability, activates CFTR leading to fluid loss, inhibits PP1 and PP2A activity.

Role of Phosphatase Inhibitory Proteins

  • Function:

    • Regulate protein phosphatase activity; involved in energy metabolism.

  • PKA Relationship:

    • PKA activates PP inhibitory proteins, which inhibit PPs during glycogen breakdown, promoting glycogenolysis through increased cAMP levels.

Interaction of Signaling Pathways

  • Key Interactions:

    • Synergy between cAMP and Ca2+ in skeletal muscle during contraction involving phosphorylase kinase.

  • Phosphorylase Kinase Activity:

    • Requires both PKA phosphorylation and Ca2+ to function effectively.

    • The δ subunit is calmodulin, while γ is the catalytic subunit.

  • Further Signaling Interaction:

    • Interaction between cAMP signaling and tyrosine kinase signaling in regulating insulin release from beta cells.

Conclusion

  • Instructor's Contact: For further inquiries, please email Dr. Mike Gray at m.a.gray@newcastle.ac.uk.

Course Overview

Course Title: PSC2002 L23 Other Protein Kinases and Protein Phosphatases

Instructor: Dr. Mike Gray

Learning Outcomes

  • Understand and explain the role and regulation of other protein kinases that are not directly regulated by cyclic adenosine monophosphate (cAMP), including their mechanisms and significance in various cellular processes.

  • Explain the function and regulation of various protein phosphatases, detailing their interaction with protein kinases and their contributions to cellular signaling dynamics.

  • Discuss clinical examples of diseases linked to dysfunctions in these protein kinases and phosphatases, including cancer, diabetes, and cardiovascular diseases, emphasizing the importance of targeted therapies.

  • Explore specific examples where there is significant interplay between different intracellular signaling pathways, highlighting the complexity and interconnectedness of cellular communication.

Other Protein Kinases (PK)

Activation Mechanisms:

  • Some protein kinases are activated by cyclic guanosine monophosphate (cGMP), notably Protein Kinase G (PKG), which plays a critical role in mediating vascular smooth muscle relaxation and various hormonal responses.

  • Other kinases are activated by increases in intracellular calcium ions (Ca2+):

    • Protein Kinase C (PKC): Activated by Ca2+ and/or lipids; PKC is vital for diverse cellular processes including cell proliferation, differentiation, and apoptosis.

    • Calmodulin-dependent protein kinases: Various subtypes exist, including PKB (Akt) and Protein Tyrosine Kinases which are discussed in detail in related lectures on Insulin and its metabolic implications in diabetes management.

The cGMP Signaling Pathway

Overview:

  • The cGMP signaling pathway is similar to the cAMP pathway; however, unlike cAMP pathways, G protein-coupled receptors (GPCRs) are not directly involved.

  • cGMP is synthesized primarily by guanylyl cyclase (GC), which has two forms:

    • Soluble GC (sGC): Activated by nitric oxide (NO), affecting smooth muscle relaxation and neurotransmission.

    • Plasma Membrane Bound GC (pGC): Activated by a limited number of peptide agonists, influencing various signaling cascades.

Function of cGMP and PKG:

  • cGMP activates PKG, which has two isoforms (I and II) and plays a crucial role in phosphorylation of proteins on serine and threonine residues, thereby modulating several downstream effects.

  • cGMP is degraded by cGMP-dependent phosphodiesterases (PDEs), such as PDE5, which have clinical significance in regulating vascular tone and treating erectile dysfunction.

cGMP in Health and Disease

Health Applications:

  • Atrial natriuretic factor (ANP), C-type natriuretic factor (CNP), and Brain natriuretic factor (BNP) are influential molecules that activate cGMP signaling pathways, playing pivotal roles in cardiovascular health and fluid balance.

  • The heat-stable enterotoxin from Escherichia coli triggers pGC/cGMP activation, leading to secretory diarrhea by stimulating cystic fibrosis transmembrane conductance regulator (CFTR) channels.

  • Nitric oxide (NO) released from endothelial cells activates sGC/cGMP pathways in vascular smooth muscle, facilitating vasodilation and influencing blood pressure regulation.

  • Excess NO production, particularly from inducible nitric oxide synthase (iNOS), can lead to critical conditions such as septic shock.

  • Viagra (sildenafil) functions by inhibiting PDE5, increasing cGMP levels which result in smooth muscle relaxation, thus it is instrumental in treating erectile dysfunction and pulmonary hypertension.

Protein Kinase C (PKC)

Structure and Isoforms:

  • PKC is characterized as a single polypeptide that comprises both regulatory and catalytic domains.

  • There are 11 different isoforms of PKC, categorized into three groups: conventional, novel, and atypical, each with distinct regulatory mechanisms and functional roles.

  • PKC activity requires binding to specific phospholipids, emphasizing its dependency on cellular lipid environments.

Activation Process:

  • Ca2+ ions interact with the C2 domain of PKC, prompting its translocation to the plasma membrane and subsequent binding to diacylglycerol (DAG) via the C1 domain.

  • The pseudosubstrate motif disassociates from the substrate binding site, allowing the kinase to interact with its target substrates for phosphorylation, an essential process in various signaling pathways.

  • Phorbol esters can also chemically activate PKC, providing insight into tumor promotion mechanisms.

Ca2+/Calmodulin-dependent Protein Kinases

Types:

  • These kinases feature varying substrate specificities ranging from narrow (e.g., phosphorylase kinase) to broad (e.g., Multifunctional CaM Kinase II), illustrating their diverse functional applicability.

Functions:

  • They regulate various cellular processes including:

    • NMDA receptor modulation influencing neurotransmission and synaptic plasticity.

    • Enhancing Inositol triphosphate-mediated signaling mechanisms.

    • Decoding calcium signals crucial for cellular responses and functions.

    • Controlling activity of the SERCA2 pump which is pivotal for calcium homeostasis in muscle cells.

Protein Phosphatases (PP)

Function:

  • Protein phosphatases play a critical role in cellular signaling by removing phosphate groups from serine/threonine and tyrosine residues of phosphorylated proteins, influencing their activity and function.

Major Classes:

  • Four primary classes of Ser/Thr phosphatases (PP1, 2A, 2B, 2C) exist, exhibiting diverse substrate specificities and regulatory mechanisms.

  • The primary class of Tyrosine phosphatases encompasses both cytosolic and membrane-bound forms, with implications in cellular growth and differentiation pathways.

Chemical Inhibitors of Protein Phosphatases

  • Okadaic acid: This potent inhibitor specifically targets PP1 and PP2A, affecting cell cycle regulation and apoptosis.

  • Cyclosporin A: A targeted inhibitor for calcineurin (PP2B), utilized clinically to prevent organ transplant rejection via immunosuppression.

Effects of Okadaic Acid

Source:

  • Okadaic acid is derived from dinoflagellates, notably causing diarrhetic shellfish poisoning (DSP) upon ingestion.

Symptoms:

  • Symptoms of DSP include severe diarrhea, vomiting, and nausea, highlighting the toxicological impacts of this compound.

Mechanism:

  • It increases paracellular permeability and activates CFTR channels, leading to fluid loss, while simultaneously inhibiting PP1 and PP2A activity, disrupting normal cellular processes.

Role of Phosphatase Inhibitory Proteins

Function:

  • Phosphatase inhibitory proteins serve to regulate the activity of protein phosphatases, playing critical roles in energy metabolism and maintaining cellular signaling homeostasis.

PKA Relationship:

  • Protein Kinase A (PKA) activates phosphatase inhibitory proteins during glycogen breakdown processes, consequently inhibiting PPs, thereby promoting glycogenolysis through increases in cyclic AMP levels.

Interaction of Signaling Pathways

Key Interactions:

  • There exists synergy between cAMP and Ca2+ pathways, particularly in skeletal muscle where phosphorylation events are critical for contraction mechanisms involving phosphorylase kinase.

Phosphorylase Kinase Activity:

  • Phosphorylase kinase activity is contingent upon phosphorylation by PKA and the presence of Ca2+, where the delta subunit serves as calmodulin while the gamma subunit functions as the catalytic core.

Further Signaling Interaction:

  • There is a noteworthy interaction between cAMP signaling and tyrosine kinase signaling, particularly in the regulation of insulin release from pancreatic beta cells, underscoring the complexity of metabolic regulation.

Conclusion

  • For any further inquiries or clarifications, please reach out to Dr. Mike Gray at m.a.gray@newcastle.ac.uk.