The serine–threonine kinase Akt (also known as protein kinase B (PKB) plays a critical role in a broadly influential signaling network. Akt activation serves as a master switch for cellular signaling pathways by generating a multitude of intracellular responses through downstream targets and interacting partners. Akt has been implicated in diseases ranging from cancer and diabetes to neurodegeneration. Akt is one of the most actively studied kinases in both basic research and drug development. Thermo Scientific™ has a wide range of products to help with Akt research. 


Key Akt/PKB Pathway Targets

Akt/PKB is expressed as three isoforms [1]

AKT1/PKBα AKT2/ PKBβ AKT3/PKBγ

Common targets in the Akt/PKB Pathway:

4E-BP1 GβL PDK1
AR GSK-3β PI3K
ASK-1 IκB PIP2
BAD IKK PIP3
Β-CTN MDM2 PP2A
CASP9 mTOR PRAS40
CCND1 NFAT PTEN
eIF2B NFκB Raptor
eIF4A p110 Rheb
eIF4B p21 Rictor
eIF4E p27 RTK
FKBP p53 SK6
FoxO1 p85 TSC1
GATA4 PDCD4 TSC2

An amino terminal pleckstrin homology (PH) domain, a central serine–threonine catalytic domain, and a small carboxy terminal regulatory domain characterize all the three isoforms. The PH domain binds to phosphatidylinositol-3,4-bisphosphate (PIP2) and phosphatidylinositol-3,4,5-trisphosphate (PIP3), products of PI3K. This binding causes Akt to locate to the plasma membrane, where it becomes phosphorylated by phosphoinositide-dependent kinase 1 (PDK1) on Thr308 in the activation loop of the catalytic domain. This phosphorylation leads to activation. Full activation requires phosphorylation at a second site (Ser473).

The mTOR–rictor complex (mTORC2) is the primary kinase for the second phosphorylation event, although other kinases like Ilk (integrin linked kinase) [2, 3], PDK1 [4, 5], DNA-dependent protein kinase (DNA-PK) [6, 7] and ATM (ataxia telangiectasia mutated) have also been identified [8].

Abnormal activation or blockage of the Akt/PKB pathway is implicated in tumor cell survival and proliferation. Thus, the Akt pathway is an attractive target for anticancer drug discovery [9]. Key targets for tumor suppression include PH domain Leucine-rich repeat Protein Phosphatase (PHLPP), phosphatase and tensin homolog (PTEN) and tuberous sclerosis complex (TSC) [10]. These targets provide suppression at multiple points throughout the pathway. PTEN dephosphorylates PIP3 and stops activation of Akt. PHLPP1/PHLPP2 negatively regulates PI3K and, in turn, regulates Akt [10]. TSC1 and TSC2 are activated by Akt and then hinder mTOR downstream [11].

Data

Thermo Scientific™ offers antibodies, ELISAs, Luminex® multiplex assays and growth factors for key targets in the Akt signaling pathway. 

Featured below is western blot, ELISA, and Luminex® data using Thermo Scientific™ products.

Western blot analysis of AKT [pS473]

 

Western blot analysis of AKT [pS473] was performed by loading 20 µg of NIH/3T3 (lane1), NIH/3T3 treated for 10 minutes with 25 ng/ml of PDGF (lane2) and U-87 MG (lane3) cell lysates. Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. AKT [pS473] was detected at ~55 kDa using ABfinity™ AKT [pS473] Recombinant Rabbit Monoclonal Antibody (Cat. No. 700256) at 0.5 µg-1 µg/ml in 2.5 % skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with phosphopeptide (10 µg/mL) as shown in the corresponding blot on right. Goat Anti-Rabbit IgG-HRP Secondary Antibody (Cat. No. G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Cat. No. WP20005).


Immunofluorescent analysis of AKT (pT308)

Immunofluorescent analysis of AKT (pT308) was performed on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0. 25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ABfinity™ AKT (pT308) recombinant rabbit monoclonal antibody (Cat. No. 701052) at a dilution of 1:500 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (Cat. No. A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Cat. No. S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 phalloidin (Cat. No. A12381). Panel d is a merged image showing cytoplasmic and perinuclear localization of phosphorylated AKT (pT308). Panel e shows competition with the phospho-AKT (pT308) peptide. 

References

  1. Vivanco, I., et al. (2002) The phosphatidylinosital 3-kinase AKT pathway in human cancer. Nat Rev Cancer 7: 489-501.
  2. Persad, S., et al. (2000) Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells. Proc Natl Acad Sci USA 97: 3207-3212.
  3. Lee, S.L., et al. (2013) Functional role of mTORC2 versus integrin-linked kinase in mediating Ser743-Akt phosphorylation in PTEN-negative prostate and breast cancer cells lines. PLoS ONE 8: e67149.
  4. Balendram, A., et al. (1999) PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr Biol 9: 393-404.
  5. Rong, J., Simons, M. (2012) Syndecan 4 regulation of PDK1-dependent Akt activation. Cellular Signaling 25: 101-105.
  6. Feng, et al. (2004) Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem 279: 41189-41196.
  7. Yikun, L., et al. (2013) Protein phosphatase 2A and DNA-dependent protein kinase are involved in mediating rapamycin-induced Akt phosphorylation. J Biol Chem 288: 13215-13224.
  8. Dong, L.Q., Liu, F. (2005) PDK2: the mission piece in the receptor tyrosine kinase signaling pathway puzzle. Am J Physiol Endocrinol Metab 289: E187-E196.
  9. Cheng, J.Q., et al. (2005) The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 24: 7482-7492.
  10. Newton, A. & Trotman, L. (2014) Turning off AKT: PHLPP as a drug target. Annu Rev Pharmacol Toxicol 54: 537-558.
  11. Hay, N. (2005) The Akt-mTOR tango and its relevance to cancer. Cancer Cell 8: 179-183.

Featured Products

Product Name Catalog Number
MDM2 Antibody 337100
GSK3 alpha + beta Antibody 44610
p53 Antibody MA512557
Phospho-PRAS40 pThr246 Antibody 441100G
PI3 Kinase p110-alpha Antibody MA514870
FoxO1 Antibody MA514846
p21 / Cip1 Antibody 337000
Tuberin / TSC2 Antibody AHO1422
Raptor Antibody 424000
PTEN Antibody 325800
AKT1 + AKT2 + AKT3 Antibody MA514999
PDK1 Antibody MA515797
RICTOR Antibody MA515681
PP2A C Subunit Antibody PA517510
Bad Antibody A16707
P27 Kip1 Antibody PA527188
IKK alpha Antibody MA516157
PDCD4 Antibody PA528150
TSC1 Antibody PA520131
Phospho-EIF4EBP1pThr36 Antibody PA512847

* This promotion is open to customers in the US and Canada. Discount will apply to orders received by Life Technologies no later than July 31, 2013, or until promotional supplies are depleted, whichever comes first. Customer can use the discount only once. Discount only applies to a maximum of 1 kit per customer. Discount applies to list price in effect at the time order is received by Life Technologies. Cannot be combined with other discounts or promotions. Offer void where prohibited, licensed, or restricted by federal, state, provincial, or local laws or regulation or agency/institutional policy. Other restrictions may apply.