Lecture 5: GPCR Effector Systems
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Questions and Answers

What is the primary factor required for the activation of aPKC?

  • Phorbol esters
  • Phosphatidyl serine (PS) (correct)
  • Diacylglycerol (DAG)
  • Calcium ions

Which of the following components binds to the C1 domain of PKC?

  • Threonine
  • Zinc ions (correct)
  • Phosphatidyl serine
  • Calcium ions

What role does the C2 domain of PKC play after activation?

  • It binds zinc ions to activate the kinase.
  • It acts as a pseudosubstrate for autophosphorylation.
  • It directly phosphorylates phosphatidyl serine.
  • It facilitates translocation to the cell membrane. (correct)

Which phosphorylation is also known as autophosphorylation in the activation process of PKC?

<p>Serine 660 (B)</p> Signup and view all the answers

What is the correct sequence of activation leading to an active C2 domain in PKC?

<p>Phosphorylation by PDK1, autophosphorylation, binding of C2 to Ca2+. (A)</p> Signup and view all the answers

What is the role of the PH domain in PLCβ?

<p>It binds to PI(4,5)P2. (B)</p> Signup and view all the answers

Which of the following statements is true about the PLC isoforms?

<p>PLCδ acts as a prototype. (B)</p> Signup and view all the answers

Which subunit does Gq/11 signaling activate in relation to PLCβ?

<p>Alpha subunit (D)</p> Signup and view all the answers

What does the C2 domain of PLCβ do?

<p>Reinforces attachment to the membrane. (B)</p> Signup and view all the answers

Which of the following G proteins can activate PLCβ?

<p>Gαq (C)</p> Signup and view all the answers

What structural feature enhances the catalytic activity of PLCβ?

<p>X-Y catalytic domains (B)</p> Signup and view all the answers

Which substance does PLCβ utilize as a substrate?

<p>PI(4,5)P2 (C)</p> Signup and view all the answers

Which PLC isoform is most commonly recognized as prototype?

<p>PLCδ (A)</p> Signup and view all the answers

What role do anchoring proteins play for PKC?

<p>They localize PKC to specific areas in the cell. (D)</p> Signup and view all the answers

Which of the following is NOT a mechanism of PKC activation?

<p>Dephosphorylation (A)</p> Signup and view all the answers

What happens to PKC when there is prolonged stimulation with phorbol esters?

<p>PKC undergoes degradation. (B)</p> Signup and view all the answers

Which proteins are involved in directing atypical C-kinases like PKC?

<p>PICKs (B)</p> Signup and view all the answers

The pseudosubstrate domain of PKC must be released to allow what process?

<p>Phosphorylation of substrates (D)</p> Signup and view all the answers

What is the primary consequence of DAG being phosphorylated by DAG kinases?

<p>Inhibition of PKC binding to membranes. (C)</p> Signup and view all the answers

What role do RACKs play in relation to PKC?

<p>They act as scaffolds bringing PKC isoforms close to substrates. (D)</p> Signup and view all the answers

Calcium signaling in the context of PKC activation primarily affects which aspect?

<p>It enhances membrane association of PKC. (B)</p> Signup and view all the answers

What initiates catalysis in PLCβ?

<p>Interaction with Gβγ or Gαq (A)</p> Signup and view all the answers

Which domains are present in the specific isoform of PLCβ mentioned?

<p>One SH3 domain and two SH2 domains (A)</p> Signup and view all the answers

Which substance is known to compete with DAG for its binding site?

<p>Phorbol esters (B)</p> Signup and view all the answers

How many isozymes are present in the human PKC family?

<p>15 isozymes (B)</p> Signup and view all the answers

Which regulatory domain of PKC binds calcium and a phosphoinositide?

<p>C2 domain (D)</p> Signup and view all the answers

Which PKC subfamily is characterized by conventional isoforms?

<p>cPKC (A)</p> Signup and view all the answers

What must happen to the C-lobe in PKC for catalysis to be enabled?

<p>It must be phosphorylated on a Thr (B)</p> Signup and view all the answers

What type of kinase is PKC described as?

<p>Cyclic nucleotide-independent protein kinase (B)</p> Signup and view all the answers

Flashcards

PKC

A family of lipid-regulated Ser/Thr kinases involved in cell signaling.

C1 Domain

The domain in PKC that binds to DAG (diacylglycerol), phosphatidyl serine, and phorbol esters like PMA (phorbol myristate acetate).

aPKC

A type of PKC that lacks the C2 domain, so it does not bind to calcium. It only needs phosphatidyl serine (PS) for activation.

PKC Activation Steps

PKC is activated through a series of phosphorylation events: 1) Phosphorylation by PDK1 at Thr500, 2) Autophosphorylation at Thr641, 3) Autophosphorylation at Ser660, 4) Ca2+ binding to the C2 domain, 5) Binding to phosphatidyl serine on the cell membrane.

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What is the role of the C2 domain in PKC activation?

The C2 domain binds calcium, which helps PKC translocate to the cell membrane. This allows PKC to interact with its targets and regulate cell processes.

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What activates PKC?

PKC is activated by binding of DAG, Ca2+, or phorbol esters, phosphorylation, and autophosphorylation.

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What inactivates PKC?

PKC is down-regulated by prolonged stimulation with phorbol esters, which leads to dephosphorylation and degradation of PKC. DAG kinases also phosphorylate DAG to phosphatidic acid, depleting DAG stores and terminating membrane localization of PKC.

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How does PKC become active?

The pseudosubstrate domain at the N-terminal of PKC is released, making the catalytic region C4 available to phosphorylate its substrate at Ser/Thr residues.

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PKC's Membrane Association

Binding of PKC to DAG and Ca2+ enhances its membrane association, increasing the chances of phosphorylation by PDK1 and subsequent activation of its catalytic domain.

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Anchoring Proteins and PKC

Some PKCs require anchoring proteins, like PDZ- or non-PDZ-containing proteins, to translocate to the plasma membrane.

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RICKS/AKAPs

Receptors for inactivated C-kinases (RICKS/AKAPs) differentially localize different PKC isoforms to specific areas within the cell.

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STICKs

Substrates that interact with inactive C-kinases (STICKs) link the actin cytoskeleton to the plasma membrane.

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PLC-IP3-DAG Pathway

A signaling pathway that involves the activation of phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 triggers calcium release from the endoplasmic reticulum, while DAG activates protein kinase C (PKC).

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Phospholipase C (PLC)

An enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG), leading to downstream signaling events.

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What are the products of PLC activity?

The products of PLC activity are inositol triphosphate (IP3) and diacylglycerol (DAG).

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What are the roles of IP3 and DAG?

IP3 triggers the release of calcium from the endoplasmic reticulum, while DAG activates protein kinase C (PKC).

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What are the four families of PLC isoforms?

The four families of PLC isoforms are PLCβ, PLCγ, PLCδ, and PLCε.

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What is the simplest PLC isoform?

The simplest PLC isoform is PLCδ, which serves as a prototype for understanding the structure and function of other PLC isoforms.

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What are the domains of PLCδ?

PLCδ has several domains: PH, EF-hand, C2, and X-Y catalytic domains.

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How does PLCβ bind to the membrane?

PLCβ binds to the membrane via its PH domain, which interacts with phosphatidylinositol 4,5-bisphosphate (PIP2) present in the membrane.

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PLCβ Activation

PLCβ is activated by G protein subunits Gβγ or Gαq. This initiates catalysis, leading to the production of second messengers.

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PLCβ and RTKs

PLCβ interacts with receptor tyrosine kinases (RTKs) like EGFR through its SH2 domains, which bind to phosphotyrosine residues on the RTKs. This interaction activates PLCβ.

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PLCβ Second Messengers

PLCβ catalyzes the breakdown of PIP2 into two important second messengers: diacylglycerol (DAG) and inositol trisphosphate (IP3).

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PKC Activation

Protein kinase C (PKC) is activated by diacylglycerol (DAG) or phorbol esters like PMA. These molecules bind to the C1 domain of PKC.

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What are Phorbol Esters?

Phorbol esters are compounds like PMA (phorbol 12-myristate 13-acetate) that mimic the action of DAG and activate PKC. They were originally discovered in plants and have tumor-promoting effects.

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PKC's Domains

PKC has several key domains: C1 (binds DAG), C2 (binds Ca2+ and PS), and a catalytic domain (C3, C4). It also has a pseudo-substrate domain and a regulatory domain.

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PKC Subfamilies

The PKC family is divided into three subfamilies based on their structure and activation requirements: conventional (cPKC), novel (nPKC), and atypical (aPKC).

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PKC Activation Mechanism

PKC is activated when its catalytic domain is phosphorylated on a threonine residue. This phosphorylation is enabled by the binding of DAG and/or Ca2+ to the C1 and C2 domains, respectively.

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Study Notes

Lecture 5: Effector Systems and Second Messengers Downstream of GPCRs (Part 2)

  • PLC-(IP3 & DAG)-PKC pathway is a key downstream pathway of GPCRs
  • Main pathways downstream of GPCRs include AC-cAMP-PKA and PLC-(IP3 & DAG)-PKC pathways
  • Phospholipase C (PLC) cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3)
  • DAG activates protein kinase C (PKC)
  • IP3 triggers release of calcium from the endoplasmic reticulum
  • Calcium and DAG are activators of PKC
  • Mammalian PLC has four families based on sequence similarities: PLCβ (most significant), PLCY, PLCδ (prototype), and PLCE
  • PLCδ is considered as a prototype and the simplest PLC isoform
  • PLCδ is made up of a PH domain, EF-hand motif, C2 domain, and X-Y catalytic domains

PLC Family

  • PLCβ can be activated with Gq/11, Gi and Go
  • Gq/11 signaling is activated by binding to α-subunit (Gαq/11) (e.g., bradykinin receptor)
  • Gi and Go signaling is activated by binding to βγ-subunits (e.g., acetylcholine M2 receptor)
  • GPCRs coupled to Gαq triggers a cascade to PLC, IP3, and DAG, which results in PKC activation ultimately impacting smooth muscle contraction and vesicle release

PKC

  • PKC is cyclic nucleotide-independent protein kinase
  • First isolated from bovine cerebellum in the 1970s
  • PKC has 3 main subgroups: Conventional, Novel, Atypical
  • Conventional CPKC consists of 4 isoforms a, β1, β2, and γ
  • Novel nPKC contains 3 isoforms δ, ε, η and θ
  • Atypical aPKC includes 4 isoforms ι, ζ, μ, and λ

PKC Activation

  • PKC is activated by Diacylglycerol (DAG), Calcium (Ca2+), and phorbol esters (e.g., PMA)
  • Phosphorylation and autophosphorylation steps are part of the process of activation
  • PKC downregulation occurs through prolonged stimulation with phorbol esters leading to dephosphorylation and degradation of PKC; also with DAG kinases converting DAG to phosphatidic acid depleting DAG stores

Anchoring Proteins

  • Some PKCs cannot translocate to the plasma membrane by themselves, needing help of anchoring proteins
  • These proteins include PDZ-containing proteins or non-PDZ-containing proteins
  • These proteins help with the precise localization of PKCs

PKC Activation Sequence

  • PKC starts as an inactive precursor often associated to the cytoskeleton, readily accessible to ATP
  • Phosphorylation stages occur at Thr500, Thr641 and Ser660 leading to active C2 domain
  • Ca2+ binding helps PKC translocate to the membrane via phosphatidylserine (PS)
  • Other kinases participate in the release of pseudosubstrate domain for full activation

PKC Different States

  • Catylically incompetent
  • Catylically competent but inactive
  • Active

Latest Theory of PKC Activation

  • DAG and Ca2+ binding to PKC improves membrane association and increases its recognition by PDK1, enhancing subsequent activation of catalytic domain

Additional Information

  • PKC activation is modulated by anchoring proteins
  • Example of interaction with PDZ-based scaffold is the interaction of eye-PKC with InaD in the rhodopsin signaling pathway
  • Different receptor families are involved in PKC anchoring including RICK/AKAPS, STICKS and RACKS, and PICKS.
  • The final step leading to activation involves release of the pseudosubstrate domain from the N-terminal of PKC.

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BioC325 Lecture 5 2023 PDF

Description

Dive into the intricacies of effector systems and second messengers downstream of GPCRs in this quiz. Explore the PLC-(IP3 & DAG)-PKC pathway and its role in cellular signaling. Test your knowledge on phospholipase C families and their activation mechanisms.

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