TheAkt signaling pathway orPI3K-Akt signaling pathway is asignal transduction pathway that promotes survival and growth in response to extracellular signals. Keyproteins involved are PI3K (phosphatidylinositol 3-kinase) and Akt (protein kinase B).
Initial stimulation by one of the growth factors causes activation of a cell surface receptor andphosphorylation of PI3K. Activated PI3K then phosphorylates lipids on the plasma membrane, formingsecond messengerphosphatidylinositol (3,4,5)-trisphosphate (PIP3). Akt, aserine/threonine kinase, is recruited to the membrane by interaction with these phosphoinositide docking sites, so that it can be fully activated.[1]Activated Akt mediates downstream responses, including cell survival, growth,proliferation,cell migration andangiogenesis, by phosphorylating a range of intracellular proteins. The pathway is present in all cells of highereukaryotes and is highly conserved.[2]
The pathway is highly regulated by multiple mechanisms, often involving cross-talk with other signaling pathways. Problems with PI3K-Akt pathway regulation can lead to an increase in signaling activity. This has been linked to a range of diseases such ascancer andtype 2 diabetes. A major antagonist of PI3K activity isPTEN (phosphatase and tensin homolog), a tumour suppressor which is often mutated or lost in cancer cells. Akt phosphorylates as many as 100 different substrates, leading to a wide range of effects on cells.[3]
There are multiple types ofphosphoinositide 3-kinase but only class I are responsible for lipid phosphorylation in response to growth stimuli. Class 1 PI3Ks are heterodimers composed of a regulatorysubunit p85 and a catalytic subunit p110, named by their molecular weights.[4]
The pathway can be activated by a range of signals, includinghormones,growth factors and components of theextracellular matrix (ECM).[5] It is stimulated by binding of an extracellular ligand to areceptor tyrosine kinase (RTK) in the plasma membrane, causing receptor dimerization and cross-phosphorylation of tyrosine residues in the intracellular domains. The regulatory subunit p85 binds to phosphorylated tyrosine residues on the activated receptor via itsSrc homology 2 (SH2) domain. It then recruits the catalytic subunit p110 to form the fully active PI3K enzyme. Alternatively, adaptor moleculeGrb2 binds to phospho-YXN motifs of the RTK and recruits p85 viaGrb2-associated binding (GAB) scaffold protein.[6]
The p110 subunit can also be recruited independently of p85. For example, Grb2 can also bind the Ras-GEF Sos1, leading to activation ofRas. Ras-GTP then activates the p110 subunit of PI3K. Other adaptor molecules such asinsulin receptor substrate (IRS) can also activate p110.[7]
PI3K can also be activated byG protein-coupled receptors (GPCR), via G-protein βγ dimers or Ras which bind PI3K directly. In addition, the Gα subunit activates Src-dependentintegrin signaling which can activate PI3K.[8]
Activated PI3K catalyses the addition of phosphate groups to the 3'-OH position the inositol ring ofphosphoinositides (PtdIns), producing three lipid products, PI(3)P, PI(3,4)P2 and PI(3,4,5)P3:
Phosphatidylinositol (PI) →PI 3-phosphate, (PI(4)P) →PI 3,4-bisphosphate, (PI(4,5)P2) →PI 3,4,5-triphosphate[9]
These phosphorylated lipids are anchored to the plasma membrane, where they can directly bind intracellular proteins containing apleckstrin homology (PH) orFYVE domain. For example, the triphosphate form (PI(3,4,5)P3) binds Akt andphosphoinositide-dependent kinase 1 (PDK1) so they accumulate in close proximity at the membrane.[1][10]
Akt resides in the cytosol in an inactive conformation, until the cell is stimulated and it translocates to the plasma membrane. The Akt PH domain has a high affinity for second messenger PI(3,4,5)P3, binding to it preferentially over other phosphoinositides.[11] Thus PI3K activity is essential for translocation of Akt to the membrane. Interaction withPI(3,4,5)P3 causesconformational changes and exposure of phosphorylation sites Thr308 in the kinase domain and Ser473 in the C-terminal domain. Akt is partially activated by phosphorylation of T308 by PDK1. Full activation requires phosphorylation of S473, which can be catalysed by multiple proteins, including phosphoinositide-dependent kinase 2 (PDK2),integrin-linked kinase (ILK),[1]mechanistic target of rapamycin complex complex 2 (mTORC2) andDNA-dependent protein kinase (DNA-PK).[12][7][13] The regulation of Ser473 phosphorylation is not fully understood but may also be influenced by autophosphorylation after Thr308 phosphorylation. After stimulation, the levels of PIP3 decrease and Akt activity is attenuated by dephosphorylation by serine/threoninephosphatases.[5]
Although PI3K is the major mode of Akt activation, other tyrosine or serine/threonine kinases have been shown to activate Akt directly, in response to growth factors, inflammation or DNA damage. These can function even when PI3K activity is inhibited.[14]Other studies have shown Akt can be activated in response toheat shock[15] or increases in cellularCa2+ concentration, via Ca2+/Calmodulin-dependent protein kinase kinase (CAMKK).[13][16]
| Activating Kinase | Akt Phosphorylation Site | Details |
|---|---|---|
| Activated CDC42 kinase 1 (Ack1) | Tyr176 | Akt binds preferentially tophosphatidic acid (PA) instead of PIP3 allowing translocation to the plasma membrane.[17] |
| Src | Tyr315, Tyr326 | Requires interaction of the SrcSH3 domain and proline-rich region at the C-terminal of Akt.[18] |
| Protein tyrosine kinase 6 (PTK6) | Tyr215, Tyr315 and Tyr326 | Activates Akt in response toepidermal growth factor (EGF)[19] |
| TANK-binding kinase 1 (TBK1) | Thr195, Ser378 and Ser473 | In response toToll-like receptor activation in macrophages.[20] |
| DNA-dependent protein kinase (DNA-PK) | Ser473 | Activated by double-strand DNA breaks formed by ionizing radiation.[21] |
The PI3K-Akt pathway has many downstream effects and must be carefully regulated. One of the ways the pathway is negatively regulated is by reducing PIP3 levels.Phosphatase and tensin homolog (PTEN) antagonises PI3K by converting PI(3,4,5)P3 into PI(4,5)P2. Loss of PTEN function leads to over-activation of Akt and is common in cancer cells (PTEN is atumour suppressor).SH2-containing Inositol Phosphatase (SHIP) also dephosphorylates PI(3,4,5)P3, at the 5' position of the inositol ring.[22] The PI3K-Akt pathway regulates PTEN levels by affecting its transcription and activity. Transcription factorNF-κB, activated by Akt, regulatesperoxisome proliferator-activated receptor delta (PPARβ/δ) agonists andtumour necrosis factor α (TNFα), which in turn repress PTEN expression.[3] NEDD4-1, an E3 ligase that recognises PTEN for degradation is up-regulated by the PI3K pathway. Therefore, when Akt is activated, PTEN is further repressed in apositive feedback loop.[23]
The pathway is also controlled byprotein phosphatase 2A (PP2A), which dephosphorylates Akt at Thr308 and phosphatasePHLPP dephosphorylates Akt at Ser473.[3] Another protein important in Akt attenuation is Carboxy Terminal Modulator Protein (CTMP). CTMP binds to the regulatory domain of Akt, blocking its phosphorylation and activation.[1]
When the pathway is activated byinsulin,insulin receptor substrate 1 (IRS-1) transcription is down-regulated, in anegative feedback loop via mTORC1 and S6K1 activation. S6K1 is also able to phosphorylate IRS-1 at multiple serine residues, preventing binding to RTKs.[24] Another negative feedback control mechanism regulating the pathway involvesFoxO transcription factors. Activated Akt causes FoxO degradation, so it can no longer inhibit PP2A, thus leading to a decrease in Akt phosphorylation.[3]
Once active, Akt translocates from the plasma membrane to thecytosol andnucleus, where many of its substrates reside.[13] Akt regulates a wide range of proteins by phosphorylation. Akt target substrates contain a minimumconsensus sequence R-X-R-X-X-[Ser/Thr]-Hyd, where Hyd is a hydrophobicamino acid, although other factors such as sub-cellular localisation and 3-dimensional structure are important.[5] Phosphorylation by Akt can be inhibitory or stimulatory, either suppressing or enhancing the activity of target proteins.
The Akt-PI3K pathway is essential for cell survival as activated Akt influences many factors involved inapoptosis, either bytranscription regulation or direct phosphorylation.[5] In the nucleus, Akt inhibits transcription factors that promote the expression of cell death genes, and enhances transcription of anti-apoptotic genes. A well studied example is theForkhead family transcription factors (FoxO/FH), of whichFKHR/FoxO1,FKHRL1/FoxO3 andAFX/FoxO4 are directly phosphorylated by Akt.[13][25]This phosphorylation induces export to the cytosol where they are sequestered by14-3-3 proteins and eventually undergo degradation via theubiquitin-proteasome pathway.[2][26]
Akt also positively regulates some transcription factors to allow expression of pro-survival genes. Akt can phosphorylate and activate theIκB kinase IKKα, causing degradation ofIκB and nuclear translocation ofNF-κB where it promotes expression of caspase inhibitors,c-Myb andBcl-xL.[2][13] Also promoting cell survival,cAMP response element binding protein (CREB) is phosphorylated by Akt at Ser133, stimulating recruitment ofCREB-binding protein (CBP) to the promoter of target genes, such asBcl-2.[27] Akt has also been shown to phosphorylatemurine double minute 2 (Mdm2), a key regulator of DNA damage responses, at Ser166 and Ser186. Phosphorylation of Mdm2 by Akt upregulates its ubiquitin-ligase activity, therefore indirectly suppressingp53-mediated apoptosis.[25] Another target of Akt is theYes-associated protein (YAP), phosphorylated at Ser127 leading to 14-3-3 binding and cytosolic localisation. Therefore, it cannot co-activatep73-mediated apoptosis in response to DNA damage.[28]
Akt negatively regulates pro-apoptotic proteins by direct phosphorylation. For example, phosphorylation ofBAD, the Bcl-2 family member, on Ser136 causes translocation from the mitochondrial membrane to the cytosol, where it is sequestered by14-3-3 proteins.[27] Akt phosphorylatesCaspase-9 on Ser196, preventing acaspase cascade leading to cell death.[2][13] Akt also phosphorylatesMAP kinase kinase kinases (MAPKKK) upstream of the stress-activated protein kinase (SAPK) pathway. Phosphorylation ofapoptosis signal-regulating kinase 1 (ASK1) on Ser83 andmixed lineage kinase 3 (MLK3) on Ser674 inhibits their activity and prevents MAP kinase induced apoptosis.[25]
Akt regulatesTFEB, a master controller of lysosomal biogenesis,[29] by direct phosphorylation ofTFEB at serine 467.[30] Phosphorylated TFEB is excluded from the nucleus and less active.[30] Pharmacological inhibition of Akt promotes nuclear translocation ofTFEB, lysosomal biogenesis and autophagy.[30]
Akt promotes G1-S phasecell cycle progression by phosphorylating and inactivatingglycogen synthase kinase 3 (GSK-3) at Ser9. This prevents the phosphorylation and degradation ofcyclin D1.[31]Therefore, Akt promotes G1 phase progression in a positive feedback loop. Akt promotes cyclin D1 translation via indirect activation ofmTOR. mTOR increases translation of cyclin D1 by activating ribosomal proteinS6K, and inhibitingeukaryotic translation initiation factor 4E-binding protein (4E-BP), thus increasingeIF4e activity.[5][32]
Akt both indirectly and directly regulatescyclin-dependent kinase (CDK) inhibitorsp21Cip1 andp27Kip1, allowing cell cycle progression. Akt phosphorylates p27Kip1 at Thr157, preventing its nuclear import.[33] In addition, Akt phosphorylates Thr145 and Ser146 of p21Cip1, preventingPCNA binding and decreasing stability.[34] Akt phosphorylation ofFoxo transcription factors also affects the cell cycle, as inhibitory phosphorylation ofFoxO4 (also named AFX) prevents p27 gene expression.[35]
Akt phosphorylates many proteins involved in polymerisation and stabilisation of theactincytoskeleton. In normal cells, this can either increase the stability of cytoskeleton components or promotemigration via remodelling. Examples are listed below:
Akt promotes cell migration by interacting with other cytoskeleton components. The type IIIintermediate filamentVimentin is phosphorylated by Akt1 at Ser39, preventing its degradation. In normal cells, this maintains tissue stability.S-phase kinase-associated protein 2 (Skp2) - Ser72 phosphorylation enhancesE3 ligase activity and cytosolic localisation, promoting cell motility. Akt phosphorylatesGSK3 beta, indirectly activatingmicrotubule binding proteinadenomatous polyposis coli (APC).Endothelial nitric oxide synthase (eNOS) is phosphorylated at Ser1177, leading to NO synthesis and endothelial cell migration.[43] In addition, the pro-migratoryGTPase-activating proteinRhoGAP22 is phosphorylated at Ser16.[36]
Under oxidative stress, miR-126 promotes Akt/PKB signaling pathway activation. This increases the biological function of cells under oxidative stress. This is important inendothelial progenitor cell transplantation to treatacute myocardial infarction (AMI) and may serve as a new therapeutic approach to treat AMI.[44]
Aberrant activation of Akt, either via PI3K or independently of PI3K, is often associated with malignancy.[14] Studies have identifiedgene amplification of the Akt isoforms in many types of cancer, includingglioblastoma,ovarian,pancreatic andbreast cancers. Akt is also up-regulated in terms of mRNA production in breast andprostate cancer. Functional inactivation of PTEN, the major PI3K antagonist, can occur in cancer cells bypoint mutation, gene deletion orepigenetic mechanisms.[1] Mutation in the pathway can also affect receptor tyrosine kinases, growth factors, Ras and the PI3K p110 subunit, leading to abnormal signaling activity. Therefore, many of the proteins in the pathway are targets for cancer therapeutics.[45] In addition to its effects on cell survival and cell cycle progression, the PI3K-Akt pathway promotes othercharacteristics of cancer cells. Hyperactivity of the pathway promotes theepithelial-mesenchymal transition (EMT) andmetastasis due to its effects on cell migration.[36]
Angiogenesis, the formation of new blood vessels, is often critical for tumour cells to survive and grow in nutrient-depleted conditions. Akt is activated downstream ofvascular endothelial growth factor (VEGF) inendothelial cells in the lining of blood vessels, promoting survival and growth. Akt also contributes to angiogenesis by activatingendothelial nitric oxide synthase (eNOS), which increases production ofnitric oxide (NO). This stimulates vasodilation and vascular remodelling.[2]Signaling through the PI3K-Akt pathway increases translation ofhypoxia-inducible factor α (HIF1α and HIF2α)transcription factors via mTOR.[46] HIF promotes gene expression of VEGF andglycolytic enzymes, allowing metabolism in oxygen-depleted environments.[47]
In cancer cells, an increase in Akt signaling correlates with an increase in glucose metabolism, compared to normal cells. Cancer cells favourglycolysis for energy production over mitochondrialoxidative phosphorylation, even when oxygen supply is not limited. This is known as theWarburg effect, or aerobic glycolysis. Akt affects glucose metabolism by increasing translocation of glucose transportersGLUT1 andGLUT4 to the plasma membrane, increasinghexokinase expression and phosphorylatingGSK3 which stimulatesglycogen synthesis.[5] It also activates glycolysis enzymes indirectly, via HIF transcription factors and phosphorylation ofphosphofructokinase-2 (PFK2) which activatesphosphofructokinase-1 (PFK1).[48]