PI3K-MAPK-PLC I

• Definition and Activation of RTKs:

  • Receptor Tyrosine Kinases (RTKs) are a special group of proteins found in cell membranes that play a crucial role in communication between cells. They become activated when specific signaling molecules, known as ligands, bind to them. This binding is essential for starting many important cellular processes.

  • Examples of RTKs include the Insulin Receptor Kinase, which is involved in regulating glucose levels in the body, and the Epidermal Growth Factor Receptor (EGFR) kinase, which is important for cell growth and division.

  • Once a ligand binds to an RTK, it triggers a process called auto-phosphorylation, where the RTK adds phosphate groups to specific amino acids called tyrosines in its own structure. This modification is an important signal that prompts the next steps in cellular communication.

  • Docking Sites: The addition of phosphate groups creates special sites (called $phosphotyrosine$ sites) on the receptor where other proteins can attach. These docking points are essential for the next steps in the signaling pathway that controls cellular activities.

• Protein Interaction Domains:

  • The proteins that bind to these phosphotyrosine sites usually contain regions known as $SH2$ (src homology two) domains. These domains help the proteins recognize and bind to the phosphorylated parts of the RTK.

  • The recruitment of these proteins to the RTK is the first step toward launching various cellular signaling cascades, or sequences of biochemical events that lead to cellular responses. This means that when an RTK is activated, it can trigger multiple events in the cell, like growth, division, or survival.

The PI3 Kinase (Phosphatidylinositol 3-Kinase) Pathway

• Structure of PI3 Kinase:

  • The $PI3$ kinase ($PI3K$) is an important enzyme involved in cell signaling, and it consists of two main parts called subunits:

    • $p85$ Regulatory Subunit: This part acts like an adapter that helps the enzyme connect to the activated RTK. It has $SH2$ domains for binding to phosphotyrosine residues and also contains $pH$ domains that help with its function.

    • $p110$ Catalytic Subunit: This subunit is responsible for the actual phosphorylation process, meaning it adds phosphate groups to certain lipids, specifically to a lipid called Phosphatidylinositol $4,5$ bisphosphate (PIP2) at the $3'$ position of its inositol ring.

• Chemical Reaction and Lipids:

  • Substrate: The starting molecule for the reaction is Phosphatidylinositol $4,5$ bisphosphate (PIP2).

  • Product: The end product of this reaction is Phosphatidylinositol $3,4,5$ trisphosphate (PIP3).

  • Mechanism: The $PI3$ kinase modifies PIP2 by adding a phosphate group, transforming it into PIP3, which is crucial for further signaling within the cell.

• Downstream Recruitment via $PH$ Domains:

  • The creation of PIP3 leads to the formation of docking sites in the cell membrane for other proteins that have a special region called Pleckstrin Homology ($PH$) domains.

  • $AKT$ (also known as $PKB$): This is an important kinase (an enzyme that adds phosphate groups to proteins) with a $PH$ domain. When PIP3 levels rise, $AKT$ is directed to the plasma membrane, where it becomes further activated.

  • $PDK1$: Another kinase with a $PH$ domain that also gets recruited to the membrane when there are high levels of PIP3.

• Activation of $AKT$:

  • Usually, $PDK1$ and $AKT$ are found in the cytoplasm and don’t interact very often. However, when PIP3 increases, both proteins move to the plasma membrane.

  • Once at the membrane, $PDK1$ phosphorylates $AKT$ at a specific site, activating it so it can perform its functions.

  • $mTORC2$: A second kinase complex called $mTORC2$ also phosphorylates $AKT$ to fully activate it. This process is important for regulating many cellular functions, including metabolism and growth.

Biochemical Characteristics of Phospholipids and Kinase Specificity

• Phospholipid Structure:

  • Glycerol Backbone: Phospholipids, including those involved in the signaling pathway, have a structure consisting of a glycerol backbone made up of three carbon atoms. Two of these carbons are linked to fatty acids, while the third is connected to a phosphate group.

  • Polar Head Group: The phosphate group is also attached to another molecule, creating a polar head that is hydrophilic (water-attracting). In this pathway, the head group is linked to an Inositol ring.

  • Inositol Ring Structure: The Inositol ring is structured like a cyclohexane, where each carbon (numbered $1, 2, 3, 4, 5, 6$) has a hydroxyl group that can be modified by adding phosphate groups.

• Classification of $PI3$ Kinases:

  • Class I $PI3$ Kinase: These enzymes respond to signals from RTKs and typically add phosphates to lipids that already have phosphates at the $4$ and $5$ positions, such as converting PIP2 into PIP3. This action is important for activating pathways related to growth and survival.

  • Class III $PI3$ Kinase ($VPS34$): This type is involved in cellular processes such as autophagy, which is the process of degradation and recycling of cellular components. Class III $PI3$ kinases phosphorylate inositol lipids that do not have any added phosphates previously.

• Divergent Signaling of PIP2:

  • PIP2 serves as an important branching point for signaling:

    1. Phosphorylation: Driven by $PI3K$ to convert PIP2 to PIP3, leading to activation of $AKT$ which promotes cell survival and growth.

    2. Cleavage: By another enzyme called Phospholipase C ($PLC$), which cuts PIP2 to produce two different signaling molecules: Inositol trisphosphate (IP3) and Diacylglycerol ($DAG$).

       • IP3: This molecule stimulates the release of calcium ions from intracellular stores, which plays a critical role in many cellular functions.

       • $DAG$: This product activates Protein Kinase C ($PKC$), another important signaling protein that regulates various cellular activities.

Regulation and Oncogenic Alterations in PI3K Signaling

• The "Off Switch" - $p10$ Phosphatase:

  • The reaction catalyzed by $PI3K$ is dynamic, meaning it can be turned on and off. The process is reversible, and the enzyme $p10$ plays a crucial role in turning this pathway off.

  • $p10$: This phosphatase specifically removes the phosphate from the $3'$ position of PIP3, converting it back into PIP2 and stopping the signaling process.

• Cellular Function and Cancer:

  • The $PI3K/AKT$ signaling pathway is vital for promoting cell survival and proliferation, meaning it helps cells grow and divide. If this pathway is blocked, it can lead to cell death or stop the cells from dividing, which can have significant implications in various diseases, including cancer.

  • Cancerous Disruptions:

    • Loss of $p10$: If $p10$, the phosphatase that turns off the pathway, is lost, it can lead to an accumulation of PIP3, resulting in constant activation of oncogenic proteins like $AKT$. This uncontrolled signaling can contribute to the growth and spread of cancer cells.

    • $p110$ Mutations: Mutations in the catalytic subunit of the enzyme can cause it to be always active, meaning it does not require the RTK stimulation to function, which again can lead to uncontrolled cell growth and cancer.

• Specific Examples (Insulin and Leptin):

  • Insulin Signaling: When insulin binds to its receptor (the Insulin Receptor), it causes auto-phosphorylation of the receptor. This modification recruits a protein called $IRS$ (Insulin Receptor Substrate), which then recruits the $p85/p110$ complex to activate the signaling pathway. This process ultimately results in the movement of glucose transporters (like $GLUT4$) from inside the cell to the plasma membrane, allowing cells to take in glucose from the bloodstream.

  • Leptin Signaling: Although the leptin receptor is not a classical RTK, it gets phosphorylated on tyrosine residues and utilizes the same recruitment mechanism involving the $IRS/p85/p110$ complex to regulate glucose uptake and fat production.

The RAS-MAP Kinase Signaling Pathway

• Initiation:

  • Just like with RTKs, when a ligand binds to a receptor, it activates the receptor through auto-phosphorylation.

  • $GRAB2$: An important adapter protein that binds to the activated receptor using its $SH2$ domains, effectively "grabbing" it.

  • $SOS$ (Son of Sevenless): This protein binds to $GRAB2$ and acts as a Guanine Nucleotide Exchange Factor ($GEF$) for $RAS$, which is a critical signaling protein that operates downstream in this pathway.

• Activation of $RAS$:

  • In its inactive state, $RAS$ is bound to a molecule called $GDP$ (Guanosine diphosphate).

  • The binding of $GRAB2/SOS$ decreases $RAS$'s ability to hold onto $GDP$, allowing it to release $GDP$ and bind to $GTP$ instead (because there is always more $GTP$ present in the cell).

  • Once $RAS$ is bound to $GTP$, it changes shape and becomes activated, allowing it to bind to and activate other downstream signaling proteins.

Post-Translational Modification and Membrane Anchoring of RAS Proteins

• Structure and Isoforms:

  • There are three main types of $RAS$ proteins, known as isoforms: $KRAS$, $NRAS$, and $HRAS$.

  • $RAS$ proteins are not transmembrane proteins, meaning they don’t span the cell membrane, but they are attached to the membrane through a specific region at their end called the carboxy terminus ($C$-terminus).

  • The CaAX Box: This is a particular sequence found at the $C$-terminus where "C" refers to Cysteine, "A" stands for an aliphatic residue (an amino acid with a non-polar side chain), and "X" can be any residue. This motif is essential for membrane anchoring.

• Modification Steps:

  1. Farnesylation: An enzyme called farnesyltransferase attaches a farnesyl group (a hydrophobic anchor) to the cysteine, forming a stable carbon-sulfur bond.

  2. Proteolysis: A different enzyme removes the three terminal amino acids ($AAX$) from the sequence, helping to finalize the membrane attachment.

  3. Methylation: Another modification occurs where a methyltransferase adds a methyl group to the new end of the protein, neutralizing its charge and making it more hydrophobic (water-repelling), which helps it stay anchored in the membrane.

  4. Palmitoylation: Some $RAS$ isoforms may also undergo palmitoylation, where a 16-carbon fatty acid called palmitate is added to other cysteine residues. Unlike farnesylation, this modification can be reversed and allows flexibility in membrane association.

The RAS-GTPase Cycle and Kinase Cascades

• The Timer Function:

  • $RAS$ possesses a natural ability called intrinsic $GTPase$ activity, meaning it can gradually convert $GTP$ back into $GDP$ by hydrolysis over time.

  • $GAP$ (GTPase Activating Protein): This protein binds to the activated $RAS-GTP$ and significantly speeds up the conversion back to $GDP$, acting as a type of "off switch" to turn off the signaling.

• The Kinase Cascade (MAPK):

  • $RAF$ (MAP Kinase Kinase Kinase): Once $RAS$ is activated, it recruits $RAF$ to the membrane, where it gets activated.

  • $MEK$ (MAP Kinase Kinase): This protein is then phosphorylated and activated by $RAF$.

  • $ERK$ (MAP Kinase/Extracellular Regulated Kinase): Finally, $ERK$ is phosphorylated and activated by $MEK$, leading to various cellular responses such as growth and differentiation.

• Properties of $MEK$:

  • $MEK$ has a unique ability called dual-specificity kinase, which means it can phosphorylate both Serine/Threonine and Tyrosine residues on proteins, thus activating them.

  • To fully activate $ERK$, it must be phosphorylated on two specific residues (one Threonine and one Tyrosine) within a unique sequence motif known as the $TEY$ motif.

MAP Kinase Families and Functional Specialization

• Three Primary Modules in Mammals:

  1. $ERK1/2$ Pathway: This pathway is primarily activated by growth factors through RTKs and controls cell proliferation and division.

  2. Jun Kinase ($JNK$ or $JUNK$) Pathway: This pathway is activated by stress signals from the environment, like oxidative stress or inflammatory signals.

  3. $p38$ (or $HOG$ - High Osmolarity Glycerol) Pathway: Discovered in yeast and found in humans, this pathway responds to stress stimuli such as UV radiation and heat stress.

• Specificity and Regulatory Motifs:

  • Different types of MAPKs have unique regulatory motifs that can be identified by specific antibodies — useful for research and clinical diagnostics:

    • $ERK1/2$: Recognized by the $TEY$ motif in protein sequences.

    • $p38$: Recognized by the $TGY$ motif.

    • $JNK$: Recognized by the $TPY$ motif.

• Complex Signaling Integration:

  • $RAS$ has the ability to activate multiple signaling pathways at once, including the $RAF/MEK/ERK$ pathway, the $PI3K$ pathway, and phospholipase C (PLC) pathways simultaneously.

  • This complexity allows cells to respond to various growth factors distinctively, where different factors activate these pathways to various extents. For instance, insulin is a strong activator of the $PI3K$ pathway but only weakly activates the $RAS$ pathway, whereas ligands like $EGFR$ can strongly activate the $RAS$ pathway.

Questions & Discussion

• Dialogue/End of Session:

  • The speaker summarizes the key points of the $PI3$ kinase pathway, encouraging the audience to think a bout how this complex network functions together and highlighting the importance of understanding these processes in biological systems.

  • As the session wraps up, the lecturer suggests taking a break and later returning to conclude the discussion, inviting questions and clarifications to ensure understanding.