Phospholipids and Phosphoinositide Signalling

Phospholipids and Phosphoinositide Signalling

Basic Biochemistry - Phospholipid Structure

Glycerol and Phosphoglycerides

Glycerol is a fundamental component of membrane lipids, particularly phospholipids (phosphoglycerides). These lipids are amphipathic, possessing both hydrophobic and hydrophilic regions.

• Glycerol Structure:

  • $CH_2OH$ at position 1

  • $CHOH$ at position 2

  • $CH_2OH$ at position 3

Fatty Acyl Chains

Fatty acyl chains contribute to the hydrophobic nature of phospholipids.

• Saturated Fatty Acids: Examples include $CH<em>3-CH</em>2-CH_2$ etc.

• Unsaturated Fatty Acids: Often found at the 2 position, examples include $CH<em>3-CH</em>2-CH=CH-CH_2$ etc.

Triacylglycerol (Triglyceride)

Triacylglycerol consists of a glycerol molecule esterified with three fatty acids. This is structurally different from phospholipids, which have a polar head group attached to the glycerol backbone.

• Structure:

  • $CH_2-O-C-R1$ at position 1

  • $CH-O-C-R2$ at position 2

  • $CH_2-O-C-R3$ at position 3

Phospholipid Structure

Phospholipids have a glycerol backbone, fatty acid chains, and a polar head group. The general structure includes:

• Glycerol backbone (hydrophilic)

• Two fatty acid chains

• A phosphate group linked to a polar head group

$O=C-O-1CH<em>2$ $O=C-O-2CH$ $H</em>2-3C-O-P-O-X$ where X is the polar head group

Polar Head Groups

Examples of polar head groups and their corresponding phospholipids:

• Choline: Forms phosphatidylcholine.

• Inositol: Forms phosphatidylinositol.

• Serine: Forms phosphatidylserine.

Phospholipid Classification and Composition

Classification

Phospholipids are classified based on their polar head group, leading to variations in membrane composition across different cell types.

Composition in Membranes

• Phosphatidylcholine: Approximately 50% of membrane lipids.

• Phosphatidylserine: Ranges from 2% to 10%.

• Phosphatidylethanolamine: Ranges from 15% to 35%.

• Phosphatidylinositol: Ranges from 5% to 10%.

Phosphatidylinositol (PI) and its Variants

Structure of Phosphatidylinositol

• Also known as PtdIns.

• Inositol ring structure with hydroxyl groups at various positions.

• Agranoff's 'turtle' representation highlights its orientation in the membrane, with the head group facing the cytosol.

Key Role in Signal Transduction

PI is a crucial phospholipid in cell signalling, existing in several 'flavours' due to phosphorylation at different positions on the inositol ring.

• Phosphatidylinositol (PI or PtdIns)

• Phosphatidylinositol 4-phosphate (PIP or PtdIns(4)P)

• Phosphatidylinositol (4,5)-bisphosphate (PIP2 or PtdIns(4,5)P2)

• Phosphatidylinositol (3,4,5)-trisphosphate (PIP3 or PtdIns(3,4,5)P3)

Other variations exist, but these are the primary ones involved in signalling or act as precursors.

Phosphatidylinositol 4,5-bisphosphate (PIP2)

• Structure includes phosphate groups at the 4 and 5 positions of the inositol ring.

• $1, 2, 3, 4, 5, 6$ positions on the inositol ring are key sites for phosphorylation.

Phospholipases

Action on Glycerophospholipids

Phospholipases are enzymes that act on glycerophospholipids, playing diverse roles in cell signalling.

Types of Phospholipases

• Phospholipase A1 (PLA1)

• Phospholipase A2 (PLA2)

• Phospholipase C (PLC)

• Phospholipase D (PLD)

These enzymes have various substrates, produce different signalling molecules, and result in diverse functional outcomes.

Phospholipase C (PLC)

Cleavage of PIP2

PLC cleaves PIP2 into two important second messengers:

• Inositol 1,4,5-trisphosphate (IP3 or Ins(1,4,5)P3)

• Diacylglycerol (DAG)

Receptor-Mediated Signalling

1. A stimulus activates a receptor (R).

2. The activated receptor stimulates PLC.

3. PLC hydrolyzes $PtdIns(4,5)P2$.

4. This hydrolysis generates the second messengers IP3 and DAG.

Isoforms of PLC

• Diversity: 6 families and 13 isoforms (with ~30 splice variants).

  • PLC-β (beta): 4 isoforms (~150kDa)

  • PLC-γ (gamma): 2 isoforms (~145kDa)

  • PLC-δ (delta): 3 isoforms (~85kDa)

  • PLC-ε (epsilon): 1 isoform (~230kDa)

  • PLC-ζ (zeta): 1 isoform (~70kDa)

  • PLC-η (eta): 2 isoforms (~120kDa)

• Rationale: This large number of isoforms allows variable but precise control over a multitude of functions.

PLC-β Isoforms

• Domains:

  • PH domain: Binds polyphosphoinositides (specifically $PtdIns(4,5)P2$), facilitating membrane localization.

  • EF-hand domains: Four tandem domains; function less clear but may bind $Ca^{2+}$.

  • X and Y domains: Catalytic domains with 60% homology between isoforms.

  • C2 domain: Binds $Ca^{2+}$.

  • CC (coiled-coil) domain: Protein-protein interaction domain.

  • PDZ domain: Protein-protein interaction domains.

• The core structure is common to all PLCs.

• PDZ domains mediate protein-protein interactions.

Tissue Distribution and Signalling

• Distribution:

  • β1 and β3: Fairly widespread.

  • β2: Immune/haematopoietic cells.

  • β4: Retina and certain neurons.

• Most commonly associated with signalling by GPCRs (G protein-coupled receptors).

• Multiple roles in $PtdIns(4,5)P2$ hydrolysis, producing DAG and $Ins(1,4,5)P3$.

Activation Mechanisms of PLC-β

• Activation:

  • $Gα<em>q$ interactions: Gβ2 > β3 >> β1 (not β4).

  • $Gβγ$ almost always from $Gα_{i/o}$ due to abundance.

• Other Regulatory Features:

  • PLC-β2 is stimulated by monomeric G-proteins Rac1, Rac2, and Cdc42 binding to the PH domain.

  • PLC-β1, -β2, and -β3 are inhibited by phosphorylation by PKA, PKC, PKG, and CamKII.

  • Protein-protein interactions influence subcellular localization and regulation via the PDZ domain.

Coincidence Detection in PLC-β3 Activation

• $Gα_q$ and $Gβγ$ act synergistically, providing variety in regulation.

• PLC-β isoforms act as GTPase activating proteins (GAPs) for $Gα<em>{q/11}$, increasing $Gα</em>q/11$ GAP activity by x1000 (compare RGS proteins).

Importance of PLC Activation

• Different PLC isoforms have varying activation mechanisms.

• They also exhibit different cell and subcellular distributions.

• This leads to signalling variety, specificity, and integration.

• Interactions with other signalling pathways are common.

• The production of $Ins(1,4,5)P_3$ and DAG leads to numerous signalling events with differences in space, time, and amplitude.

Central Role of $PtdIns(4,5)P_2$ in Cellular Signalling

Synthesis and Breakdown

• Synthesis:

  • PtdIns is converted to PtdIns(4)P by PtdIns 4-kinase.

  • PtdIns(4)P is converted to $PtdIns(4,5)P_2$ by PtdIns(4)P 5-kinase.

• Breakdown: PLC produces inositol trisphosphate and diacylglycerol (DAG).

Regulation and Downstream Effects

• $PtdIns(4,5)P_2$ regulates enzymes and structural proteins with C1 domains (e.g., PKC).

• DAG activates PKC.

• Phosphatidic acid is a major regulator.

• Increase in calcium concentration. stimulates multiple kinases, transcription, and RNA processing.

• Regulation of PKC, PLD, PLA2, actin polymerization/depolymerization, and some ion channels.

Phosphoinositide 3-Kinases (PI3Ks)

Function

• Act at the 3, 4, or 5 positions of phosphatidylinositol to generate multiple polyphosphoinositides.

• Phosphorylate the 3'-OH position of the inositol ring in phosphatidylinositol.

Multiple Isoforms

There are three main classes: I, II, and III.

• Class Ia (α, β, and δ) are the best understood.

• Activated by diverse cell surface receptors.

• $PtdIns(4,5)P<em>2$ (PIP2) is the preferred substrate in vivo, producing $PtdIns(3,4,5)P</em>3$ (PIP3).

Activation Mechanisms

PI3Ks, particularly those with intrinsic or associated tyrosine kinase activity but also including GPCRs (through Src transactivation of RTKs), are involved in phosphorylation.

Protein Domains and Activation of Class Ia PI3-Kinase

• Catalytic Subunit (p110): Contains HR1 (kinase core), HR2 (acts as scaffold), HR3 (C2-like, possibly plays role in membrane binding).

• Regulatory Subunit (p85): Contains SH2, SH2, and SH3 domains.

Activation Process

1. Recruitment to phosphorylated RTKs or adaptor proteins.

2. Alleviates inhibition of catalytic subunit.

3. Brings the catalytic domain to its preferred substrate, PIP2, allowing PIP3 generation.

Prototypical Activation via Growth Factor Receptors

1. Growth factor receptor activation causes autophosphorylation, producing docking sites for adaptor proteins and PI3-kinase.

2. p85 subunit binds to phosphorylated receptor.

3. Activated RAS interacts with the p110 subunit, recruiting PI3-kinase to the membrane.

4. PI3-kinase is activated, converting PIP2 to PIP3.

Downstream Signalling of PI3-Kinase Activation

• PIP3 acts as an anchor for a range of signalling proteins containing PH domains.

• Examples of PH domain-containing proteins include:

  • Rac and Rho GTPases (cytoskeletal changes and regulation of MAP kinases).

  • TEC tyrosine kinases (regulating gene expression).

  • ARF6 (endocytosis/trafficking).

  • PLCγ (phospholipase Cγ).

  • PKB (Akt) - a serine/threonine kinase; wide range of functions.

  • PDK1 - phosphoinositide-dependent kinase 1 (involved in activation of PKB).

• These proteins regulate cell growth, survival, and movement.

Activation of Protein Kinase B/Akt

• Akt1 is activated by PDK1 and mTORC1 (indirect activation).

• Phosphorylation at T308 (activation loop of catalytic/kinase domain) causes partial activation.

• Phosphorylation at S473 (regulatory domain) leads to full activation.

• Regulation of other kinases (e.g., PKCζ) occurs via PDK phosphorylation, which promotes autophosphorylation, generating a catalytically competent PKC that requires DAG/Ca2+ for full activation.

Function of PKB/Akt

• Generally associated with anti-apoptosis, growth, proliferation, and migration.

Termination of PI3-Kinase Signalling: Phosphatases

• SHIP activity removes binding sites for proteins with PIP3-selective PH domains but generates $PtdIns(3,4)P_2$ that PKB/Akt can bind to.

• PH domain of PKB/Akt binds $PtdIns(3,4)P<em>2$ and $PtdIns(3,4,5)P</em>3$ with equal affinity.

• PTEN removes both $PtdIns(3,4,5)P<em>3$ and $PtdIns(3,4)P</em>2$, inhibiting PKB/Akt.

Phosphoinositide 3-Kinase Signalling and Cancer

• Numerous oncogenes activate type IA PI3-kinase.

• Activating mutations of PI3-kinase (particularly in p110α catalytic subunit) are described in cancer.

• PTEN has tumour-suppressor properties through dephosphorylation of PIP3; mutations in PTEN are associated with cancers.

• Mutations in SHIP1 are recently associated with some leukaemias.

• Phospho-specific antibodies for PKB/Akt (activated) show increased activity in cancers including breast, colon, ovarian, pancreatic, and prostate cancers.

PI3-Kinase Inhibitors as Cancer Treatments

General Inhibitors

• Wortmannin:

  • From Penicillium wortmannin (1957).

  • Not specific; short half-life; causes liver dysfunction and hyperglycemia; not for clinical use; irreversible.

• LY294002:

  • Developed by Eli Lilly (1994).

  • Not specific; less potent than wortmannin; poor aqueous solubility; reversible.

Specific Inhibitors

• Many PI3K inhibitors have been developed for clinical use.

• Copanlisib: Pan-class I PI3K inhibitor (predom. α, δ); FDA approval 2017 (lymphoma).

• Alpelisib: Pan-class I PI3K inhibitor; FDA approval 2019 (advanced breast cancer).

• A number of others approved but subsequently withdrawn.

Selective Inhibitors

• Inhibitors of Akt (not yet approved).

• Inhibition of downstream signalling from PI3K activation may also be beneficial.

• Many inhibitors of Akt are available; some are currently in trials for cancer treatment:

  • GDC-0068 (Ipatasertib) - binds to the ATP binding site of Akt (breast cancer)

  • GSK2110183 (Afuresertib) - competitive inhibitor

  • Capivarsitib - competitive inhibitor

  • MK-2206 - allosteric inhibitor of Akt

• Isotype-Selective PI3K inhibitors:

  • Inhibitors targeting specific p110 catalytic subunits minimize side effects.

  • Idelalisib (trade name Zydelig) p110δ –selective (first in class) - important in anti-apoptotic signalling in some forms of leukaemia. Used with or after other drugs. FDA approved 2014 but subsequently withdrawn (toxicity).

Examination Questions

• Describe the structure, function, and regulation of phospholipase C-β isoforms.

• Describe the structure of phosphoinositide 3-kinase and its function in relation to cellular signalling.

• Discuss the regulation and roles of protein kinase B (Akt).