AKT/PKB Interactive Pathway and Related Products
- Interactive Pathway
- Akt Wall Poster
- Technical Overview
- Akt Assay Kits
- Akt Antibodies & Blocking Peptides
- Akt Inhibitors
- Akt Substrates/Enzymes
- Related Resources
| Related Literature | ||
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Akt (protein kinase B), a serine/threonine kinase, has emerged as a critical enzyme in signal transduction pathways involved in cell proliferation, apoptosis, angiogenesis, and diabetes. In mammals three isoforms of Akt (α, β, γ or Akt 1, 2, 3) are reported that exhibit a high degree of homology, but differ slightly in the localization of their regulatory phosphorylation sites. Aktα is the predominant isoform in most tissues, whereas the highest expression of Aktβ is observed in the insulin-responsive tissues, and Aktγ is abundant in brain tissue. Each Akt isoform is composed of three functionally distinct regions: an N-terminal pleckstrin homology (PH) domain that provides a lipid-binding module to direct Akt to PIP2 and PIP3, a central catalytic domain, and a C-terminal hydrophobic motif.
Akt is constitutively phosphorylated at Ser124, in the region between the PH and catalytic domains, and on Thr450, in the C-terminal region (in Aktα, the most widely studied isoform) in unstimulated cells. Activation of Akt involves growth factor binding to a receptor tyrosine kinase and activation of PI 3-K, which phosphorylates membrane bound PIP2 to generate PIP3. The binding of PIP3 to the PH domain anchors Akt to the plasma membrane and allows its phosphorylation and activation by PDK1. Akt is fully activated following its phosphorylation at two regulatory residues, a threonine residue on the kinase domain and a serine residue on the hydrophobic motif, which are structurally and functionally conserved within the AGC kinase family. Phosphorylation at Thr308 and Ser473 is required for the activation of Aktα, while phosphorylation at Thr309 and Ser474 activates Aktβ. Phosphorylation at Thr305 activates Aktγ. Phosphorylation of a threonine residue on the kinase domain, catalyzed by PDK1, is essential for Akt activation. It causes a charge-induced conformational change, allowing substrate binding and increased rate of catalysis. Akt activity is augmented about 10-fold by phosphorylation at the serine residue by PDK2. DNA-PK and PKCβII are reported to phosphorylate the serine residue on the regulatory subunit. Without threonine phosphorylation, the hydrophobic motif of Akt is more susceptible to the action of phosphatases; however, the dually phosphorylated and fully active enzyme is stable, allowing its localization to the nucleus and other sites. The activity of Akt is negatively regulated by PTEN and SHIP.
The principal role of Akt is to facilitate growth factor-mediated cell survival and to block apoptotic cell death. This is achieved by phosphorylating and deactivating pro-apoptotic factors such as Bad, caspase-9, and Forkhead transcription factors (AFX, Daf-16, FKHR). The phosphorylation of Bad at Ser136 promotes its association with 14-3-3 proteins in the cytosol, which prevents Bad from localizing at the mitochondria to induce apoptosis. Akt is also known to promote cell survival by inactivating caspase-9 through phosphorylating it at Ser196. Likewise, activated Akt phosphorylates Forkhead family members, resulting in their sequestration in the cytoplasm. In the absence of survival factors and Akt activity, Forkhead family members translocate to the nucleus, where they initiate a program of gene expression (e.g., FasL) that promotes cell death. Akt is also reported to phosphorylate IKKα at Thr23 and activate it. The activated IKKα, in turn, phosphorylates IkB, targeting it for ubiquitination and proteasomal degradation. This leads to the activation and nuclear translocation of NF-kB, and transcription of NF-kB-dependent pro-survival genes, including Bcl-xL and caspase inhibitors. Akt also phosphorylates and inactivates GSK-3, allowing the activation of glycogen synthase to proceed. An important point to note is that phosphorylation of cyclin D by GSK-3 targets it for proteolysis; hence the inactivation of GSK-3 may promote the up-regulation of cyclin D and enhance cell cycling. Recently it has been shown that when Chk1, a DNA damage effector kinase, is phosphorylated by Akt at Ser280 it can no longer be phosphorylated by ATM/ATR at Ser345 to undergo activation. This may be of therapeutic significance as Chk1 inhibition is shown to enhance sensitization of tumors to chemotherapeutic agents. Akt also phosphorylates Cdc25B on Ser353, resulting in its cytoplasmic accumulation. Cdc25B undergoes activation during S-phase and plays a role in activating the mitotic kinase Cdk1/cyclinB in the cytoplasm. In relocating Cdc25B to the cytoplasm, Akt regulates its function and participates in controlling the entry of cells into mitosis.
A number of oncogenes and tumor suppressor genes that function upstream of Akt influence cancer progression by regulating Akt. Aktα is expressed to various degrees in breast cancer cell lines and is important in estrogen-stimulated growth. Treatment of multiple myeloma cell lines with the Akt inhibitor, 1L-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Cat. No. 124005), results in reduced survival of both drug resistant and drug sensitive cells. Akt plays a critical role in tumorigenesis, becoming activated when tumor suppressors such as p27 and PTEN lose their functions. Phosphorylation of p27 at Thr157 by Akt impairsits nuclear import. Cytoplasmic mislocalization of p27 has been strongly linked to loss of differentiation and poor outcome in breast cancer. Akt is also reported to physically associate with endogenous p21, a cell cycle inhibitor, and phosphorylate it at Thr145, causing its localization to the cytoplasm and subsequent degradation.
Akt and p53 play opposing roles in signaling pathways that determine cell survival and the interaction between these two molecules is becoming an important area of study. Under conditions where the apoptotic effect of p53 is dominant, destruction of Akt plays a role in accelerating the apoptotic process. In apoptosis-prone cells, p53-dependent signaling enables downregulation of Akt, which predisposes cells to rapid apoptosis in response to stress signals. Under certain circumstances Akt activation may overcome the death promoting effects of p53 and may rescue cells from apoptosis. It has been reported that Akt can phosphorylate Mdm2 on Ser166 and Ser188 and promote its translocation to the nucleus where it destabilizes p53 and enhances its degradation via the proteasomal pathway.
| Product | Format | Form | Sensitivity | Assay Range | Assay Time | Sample Type | Cat. No. |
|---|---|---|---|---|---|---|---|
| Akt Activity Immunoassay Kit | Immunoblot | 40 Tests | 4 h | Cell lysates, tissue extracts | 124007 | ||
| K-LISA™ Akt Activity Kit | 96-well plate | 96 Tests | 0.05 mUnits | 6-100 mU/well | 3 h | Cell lysates, tissue extracts, purified enzyme | CBA019 |
| PhosphoDetect™ Akt (pSer473) ELISA Kit | 96-well plate | 96 Tests | ≤0.8 units/ml | 1.6-100 units/ml | 4 h | Cell lysates | CBA005 |
| PhosphoDetect™ Akt (pThr308) ELISA Kit | 96-well plate | 96 Tests | ≤0.8 units/ml | 1.6-100 units/ml | 4 h | Cell lysates | CBA004 |
Antibodies & Blocking Peptides
| Product | Application | Species Reactivity | Cat. No. |
|---|---|---|---|
| Anti-Akt1 (88-100) Rabbit pAb | ELISA (see comments) Immunoblotting (1 µg/ml) Immunoprecipitation (see comments) |
<i>Xenopus</i>, human, mouse, rat | 530311 |
| Anti-Akt1 (Ab-1) (135-145) Rabbit pAb | Immunocytochemistry (1:5000) Immunoblotting (not recommended) Paraffin Sections (not recommended) |
human, mouse | PC510 |
| Anti-Akt2 Rabbit pAb | ELISA (1:10,000-1:100,000) Immunoblotting (1:1000-1:5000, chemiluminescence) |
human, mouse, rat | 124002 |
| Anti-Akt, PH Domain Mouse mAb (SKB1) | Flow Cytometry (see comments) Immunoblotting (0.5-2 µg/ml, see application references) Immunoprecipitation (4 µg/0.5 mg total protein; see comments and see application references) |
human, mouse, rat | ST1088 |
| PhosphoDetect™ Anti-Akt1 (pSer473) Mouse mAb (11E6) | ELISA (phosphopeptide; see comments) Immunoblotting (1 µg/ml colorimetric or 0.5 µg/ml chemiluminescence) |
human, mouse | 124003 |
| PhosphoDetect™ Anti-Akt1 (pThr308) Rabbit pAb | Immunoblotting (1:1000) | human, mouse | 124001 |
