The PI3 – K/ Akt pathway

The PI3 – K/ Akt pathway

PI3K-Akt Pathway is an intracellular signal transduction pathway that promotes metabolism, proliferation, cell survival, growth and angiogenesis in response to extracellular signals. This is mediated through serine and/or threonine phosphorylation of a range of downstream substrates.

Signaling molecules for Akt pathway: The pathway can be activated by a range of signals, including hormones (Insulin), growth factors (VEGF, EGF, FGF Etc) and components of the extracellular matrix (ECM)

Receptor for Akt pathway: Receptor tyrosine kinase and GPCR

Key components of the pathway: Receptor tyrosine kinase (RTKs), phosphatidylinositol 3-kinase (PI3K), phosphatidylinositol-4, 5-bisphosphate (PIP2), phosphatidylinositol-3,4,5-bisphosphate (PIP3) and AKT/protein kinase B.

Upstream events of PI3K activation

1) When the receptor is RTK

Binding of an extracellular ligand to a receptor tyrosine kinase (RTK) in the plasma membrane causes receptor dimerization and cross-phosphorylation of tyrosine residues in the intracellular domains.

2) When the receptor is GPCR

Binding of ligand to the GPCR causes a conformational change in the GPCR, which allows it to act as a guanine nucleotide exchange factor (GEF). The GPCR can then activate an associated G protein by exchanging the GDP bound to the G protein for a GTP. Then G protein α subunit together with the bound GTP dissociate from the β and γ subunits.

 PI3K enzyme (Protein kinase B)

There are the 3 classes of PI3K enzyme. Among these Class I PI3Ks are heterodimeric receptor-regulated enzymes that preferentially phosphorylate PIP2.

 PI3K have 2 domains: Regulatory domain (p85) and catalytic domain (p110)

  • Their catalytic subunits interact with Ras .GTP via a Ras-binding domain (RBD) near their N-termini.
  • Their regulatory subunits are adaptor proteins that link the catalytic subunits to upstream signaling events and hence form two subclasses according to the type of upstream effectors with which they interact:

(a) The class IA PI3Ks is activated by RTKs via the mediation of the adaptor subunit p85, which contains SH2 and SH3 domains and may be phosphorylated on specific Tyr side chains.

(b) The class IB PI3K is activated by the Gβϒ dimers of heterotrimeric G proteins

PI3K have 2 major enzymatic activities:

  • Lipid kinase activities: ATP –dependent phosphorylations of various phosphoinositides
  • Ser/Thr protein kinase activity: Phosphorylation the OH group of serine or threonine

How PI3K are activated?

P13K activation can be accomplished by 4 different pathways

1) Receptor tyrosine kinase activated by growth factors

The regulatory subunit p85 binds to phosphorylated tyrosine residues on the activated receptor via its Src homology 2 (SH2) domains. Interaction with phosphorylated tyrosine residues alter the conformation of enzyme and allow the catalytic subunit to perform its function.

2) Adaptor molecules such as insulin receptor substrate (IRS) of insulin signaling pathway can also activate p110.

3) Ras-GTP can activates the p110 subunit of PI3K

4) PI3K can also be activated by G protein-coupled receptors (GPCR) via G-protein βγ dimers

Once PI3K gets activated, it migrates to the inner side of the membrane where their substrate is located.

Figure 1: PI3K activation by activated RTK, Ras.GTP and βϒ dimer of activated GPCR

What is the major function of activated P13K?

Activated PI3K phosphorylates lipids on the plasma membrane (PIP2), forming second messenger phosphatidylinositol 3, 4, 5 – trisphosphate (PIP3).

Phosphorylation site: 3’OH (hydroxyl) group of inositol ring in PIP2

These phosphorylated lipids are anchored to the plasma membrane where they function as second messengers by recruiting the proteins that bind them to the cytosolic surface of the plasma membrane. They can directly bind intracellular proteins containing a pleckstrin homology (PH).

The resulting colocalization of enzymes and substrates results in further signaling activity that controls such vital functions as cell survival, proliferation, cytoskeletal rearrangement, endocytosis, and vesicle trafficking.

(For example, PIP3 binds Akt and phosphoinositide-dependent kinase 1 (PDK1) so they accumulate in close proximity at the membrane).

Figure 2: Activity of PI 3-kinase, PI 3-kinase phosphorylates the 3 position of inositol, converting PIP2 to PIP3.

Pleckstrin homology domain (PH domain)

  • Pleckstrin homology domain (PH domain) is a protein domain of approximately 120 amino acids that occurs in a wide range of proteins involved in intracellular signaling or as constituents of the cytoskeleton.
  • PH domain can bind Phosphatidylinositol lipids within biological membranes (such as PIP3 and PIP2), the βγ-subunits of heterotrimeric G proteins, and protein kinase C.
  • Through these interactions, PH domains play a role in recruiting proteins to different membranes, thus targeting them to appropriate cellular compartments or enabling them to interact with other components of the signal transduction pathways.

Proteins with PH domain

  • Pleckstrin (the protein where this domain was first detected) is the major substrate of protein kinase C in platelets. Pleckstrin is one of the rare proteins to contain two PH domains
  • Ser/Thr protein kinases such as the Akt/Rac family, the beta-adrenergic receptor kinases, the mu isoform of PKC
  • Insulin Receptor Substrate 1 (IRS-1)
  • Cytoskeletal proteins such as dynamin
  • Mammalian phosphatidylinositol-specific phospholipase C (PI-PLC)

A key target of PIP3, which is critical for signaling cell proliferation and survival, is a protein serine/threonine kinase called Akt.

Akt is a proto oncogene product that is implicated in regulating multiple biological processes including gene expression, apoptosis, glucose uptake, and cellular proliferation, and hence phosphorylates many target proteins

Akt consists of an N-terminal PH domain that binds PIP3 and a C-terminal kinase domain for phosphorylation activity

Akt activation

  • Akt resides in the cytosol in an inactive conformation until the cell is stimulated.
  • Once the cell is stimulated, it translocates into the plasma membrane
  • Akt is recruited to the inner face of the plasma membrane by binding to PIP3 via its pleckstrin homology (PH) domain.

Interaction with PIP3 causes conformational changes in Akt. As a consequence phosphorylation sites on Akt (Thr308 in the kinase domain and Ser473 in the C-terminal domain) are exposed.

The full activation of Akt requires its phosphorylation at both its Ser 473 and Thr 308.

Phosphorylation of Thr308 (in the “activation loop”) by: PDK1 (phosphoinositide dependent kinase 1).

Phosphorylation of Ser473 (in the carboxy-terminal hydrophobic motif) by: mTOR –rictor complex

(The formation of PIP3 is essential for the colocalization both Akt and PDK1 with the plasma membrane. PDK1 also has PH domain for binding PIP3)

Figure 3: Activation of Akt by phosphorylation at ser473 (mTOR/rictor) and Thr308 (PDK1)

Downstream Effects

  • Once active, Akt translocates from the plasma membrane to the cytosol and nucleus, where many of its substrates reside.

Target protein includes: Proteins that are direct regulators of cell proliferation and survival, transcription factors, other protein kinases.

  • Akt regulates a wide range of proteins by phosphorylation.
  • Phosphorylation by Akt can be inhibitory (GSK, FOXO etc) or stimulatory (Mdm2, CREB etc), either suppressing or enhancing the activity of target proteins.
Figure 4: Examples of inhibitory and stimulatory phosphorylation by Akt

1) Cell survival and apoptosis

The Akt-PI3K pathway is essential for cell survival as activated Akt influences many factors involved in apoptosis, either by transcription regulation or direct phosphorylation.

In the nucleus, Akt inhibits transcription factors that promote the expression of cell death genes, and enhances transcription of anti-apoptotic genes

Example 1: Akt prevents tumor suppressor protein FOXO from inhibiting proliferation

  • The critical transcription factors targeted by Akt include members of the Fork head family transcription factors or FOXO family
  • FOXO proteins are directly phosphorylated by Akt. Phosphorylation of FOXO by Akt creates a binding site for cytosolic chaperone proteins (14-3-3 proteins) that sequester FOXO in an inactive form in the cytoplasm. Phosphorylated FOXO is a substrate for ubiquitin ligase. Eventually these FOXO proteins undergo degradation via the ubiquitin-proteasome pathway.
  • In the absence of growth factor signaling and Akt activity, FOXO is released from 14-3-3 chaperon protein and translocates to the nucleus, where they stimulate transcription of genes that inhibit cell proliferation or induce cell death.

(Eg: Proapoptotic genes, including the gene encoding the BH3-only protein, Bim)

Figure 5: Regulation of FOXO in the presence and absence of growth factors

Akt negatively regulates pro-apoptotic proteins by direct phosphorylation (Inhibition of apoptosis)

Example 1: Phosphorylation of Bad by Akt inhibits apoptosis and promotes cell survival.

  • Phosphorylation of BH3-only protein BAD (the Bcl-2 family member) by Akt on Ser136 maintain it in an inactive state and causes translocation from the mitochondrial membrane to the cytosol, where it is sequestered by 14-3-3 proteins.
  • In the absence of Akt signaling, activation of Bad (pro apoptotic protein) promotes apoptosis.
  • (Bad is similarly phosphorylated by protein kinases of other growth factor-induced signaling pathways, including the Ras/ Raf/MEK/ERK pathway, so it serves as a convergent regulator of growth factor signaling in mediating cell survival)

Example 2:  Akt phosphorylates Caspase-9 on Ser196, preventing a caspase cascade leading to cell death.

Example 3: Akt binds BAX and renders its ability to form holes in the mitochondrial membrane.

(In the absence of active Akt, these holes promote apoptosis via caspase cascade)

Akt also positively regulates (stimulatory phosphorylation) some transcription factors to allow expression of pro-survival genes.

(Akt could promote growth factor-mediated cell survival both directly and indirectly)

Example 1: Activation of NF -kB

  • In an inactivated state, NF-κB is located in the cytosol complexed with the inhibitory protein IκBα.
  • Akt can phosphorylate and activate the IκB kinase (IKK) which in turn, phosphorylates the IκBα protein. Phosphorylated IκBα dissociate from NF-κB and eventually degraded by the proteasome.
  • The activated NF-κB is then translocated into the nucleus where it binds to specific sequences of DNA called response elements (RE).
  • The DNA/NF-κB complex then recruits other proteins such as coactivators and RNA polymerase, which transcribe downstream DNA into mRNA.
  • NF-κB promotes expression of caspase inhibitors, c-Myb and Bcl-xL. Thus result in transcription of pro-survival genes.
  • In turn, mRNA is translated into protein, resulting in a change of cell function.

Example 2: Mdm2 phosphorylation

Akt phosphorylate Mdm2 (key regulator of DNA damage responses) at Ser166 and Ser186.

Phosphorylation of Mdm2 by Akt upregulates its ubiquitin-ligase activity, therefore indirectly suppressing p53-mediated apoptosis.

Example 3: Activation of CREB protein by Akt and promoting cell survival

Akt/PKB promotes phosphorylation of CREB (cAMP response element binding protein) at Ser133 and stimulates recruitment of CBP (CREB-binding protein) to the promoter of target genes which coactivates it, allowing it to switch certain genes on or off.

Figure 6: Downstream effects of Akt pathway preventing apoptosis and promoting cell survival

2) Cell cycle progression

Example 1:

  • Akt promotes G1-S phase cell cycle progression by phosphorylating and inactivating glycogen synthase kinase 3 (GSK-3) at Ser9.
  • This prevents the phosphorylation and degradation of cyclin D1. Therefore, Akt promotes G1 phase progression in a positive feedback loop.

(GSK-3/3 phosphorylates metabolic enzymes like glycogen synthase, transcription factors like p53, and the translation initiation factor eIF-2B).

Example 2:

Akt both indirectly and directly regulates cyclin-dependent kinase (CDK) inhibitors p21 and p27 allowing cell cycle progression.

Figure 7

3) Cell migration:

Akt phosphorylates many proteins involved in polymerization and stabilization of the actin cytoskeleton.

4) Metabolism

Akt2 is required for the insulin-induced translocation of glucose transporter 4 (GLUT4) to the plasma membrane.

Glycogen synthase kinase 3 (GSK-3) could be inhibited upon phosphorylation by Akt, which results in increase of glycogen synthesis by glycogen synthase.

5) Activation of protein synthesis

mTOR Pathway

The mTOR pathway is a central regulator of cell growth that couples the control of protein synthesis to the availability of growth factors, nutrients, and energy

Cellular energy level is low – AMPK active – It activate TSC1/2 complex – this complex will inhibit Rheb – . mTOR/raptor kinase complex not active Translation inhibition

Growth factor stimulation – activation of Akt –inhibition of TC1/2 complex – Rheb protein become active –activate mTOR/raptor complexTranslation stimulation

Link: http://easylifescienceworld.com/mtor-pathway/

Figure 8: Downstream effectors of Akt signaling pathway

Regulation of PI3K-AKT signaling

The PI3K-Akt pathway has many downstream effects and must be carefully regulated.

Negative regulation of PI3K-AKT pathway can be achieved at to target:

1) The PIP3 level

Phosphatase and tensin homolog (PTEN) is a main down regulation protein which can convert PIP3 into PIP2.

2) The inactivation of AKT protein

  • Achieved by dephosphorylation of Akt.

Protein phosphatase 2A (PP2A): dephosphorylates Akt at Thr308

Phosphatase PHLPP: Dephosphorylates Akt at Ser473

Addition to this regulation by protein, the pathway itself also has feedback mechanisms:

Positive feedback:

Transcription factor NF-κB (activated by Akt) regulates peroxisome proliferator – activated receptor delta (PPARβ/δ) agonists and tumor necrosis factor α (TNFα), which in turn repress PTEN expression as a positive feedback

Negative feedback:

Negative feedback loop is functioned by mTORC1 and S6K1 activation. S6K1 is able to phosphorylate IRS-1 at multiple serine residues, preventing binding to RTKs, resulting the suppression of PI3K activation

Figure 9: The positive and negative feedback mechanism of PI3K-AKT pathway.

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