AKT3 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

AKT (also known as protein kinase B (PKB) and RAC (related to A and C kinases)) is a critical intracellular serine/threonine kinase that translates signals from extracellular stimuli including growth factors, cytokines and neurotransmitters (1). AKT signaling plays critical roles in cell growth, proliferation, survival and differentiation (1). It is also involved in organogenesis, angiogenesis and metabolism. Three mammalian AKT isoforms have been identified. The AKT pathway can be activated by any of the three members who share a high level of protein homology but are independently encoded by AKT1 (PKB alpha; 14q32.32), AKT2 (PKB beta; 19q13.2), or AKT3 (PKB gamma; 1q44) (1, 2). Each AKT family member contains an N-terminal pleckstrin homology (PH) domain, a central kinase domain, and a C-terminal regulatory domain. AKT mediates many of the downstream events of phosphatidylinositol 3-kinase (PI3-K), a lipid kinase activated by growth factors, cytokines and insulin. PI3-K recruits AKT to the membrane, where it is activated by PDK1 phosphorylation. AKT has two main phosphorylation sites (Ser473 and Thr308, predicted molecular weight 56 kDa) (3, 4). Once phosphorylated, AKT dissociates from the membrane and phosphorylates targets in the cytoplasm and the cell nucleus including mammalian target of rapamycin (mTOR).

The main function of AKT is to control inhibition of apoptosis and promote cell proliferation. Survival factors can activate AKT Ser473 and Thr308 phosphorylation sites in a transcription-independent manner, resulting in the inactivation of apoptotic signaling transduction through the tumor suppressor PTEN, an antagonist to PI3-K (5). PTEN exerts enzymatic activity as a phosphatidylinositol-3,4,5-trisphosphate (PIP3) phosphatase, opposing PI3K activity by decreasing availability of PIP3 to proliferating cells, leading to overexpression and inappropriate activation of AKT noted in many types of cancer.

AKT1 function has been linked to overall physiological growth and function (2). AKT1 has been correlated with proteus syndrome, a rare disorder characterized by overgrowth of various tissues caused by a mosaic variant in the AKT1 gene in humans.

AKT2 is strongly correlated with Type II diabetes, including phenotypes of insulin resistance, hyperglycemia and atherosclerosis (2, 6).

The function of AKT3 is specifically associated to brain development, where disruptions to AKT3 are correlated with microcephaly, hemimegalencephaly, megalencephaly and intellectual disabilities (2).

References

1. Ersahin, T., Tuncbag, N., & Cetin-Atalay, R. (2015). The PI3K/AKT/mTOR interactive pathway. Mol Biosyst, 11(7), 1946-1954. doi:10.1039/c5mb00101c

2. Cohen, M. M., Jr. (2013). The AKT genes and their roles in various disorders. Am J Med Genet A, 161a(12), 2931-2937. doi:10.1002/ajmg.a.36101

3. Georgescu, M. M. (2010). PTEN Tumor Suppressor Network in PI3K-Akt Pathway Control. Genes Cancer, 1(12), 1170-1177. doi:10.1177/1947601911407325

4. Mishra, P., Paital, B., Jena, S., Swain, S. S., Kumar, S., Yadav, M. K., . . . Samanta, L. (2019). Possible activation of NRF2 by Vitamin E/Curcumin against altered thyroid hormone induced oxidative stress via NFkB/AKT/mTOR/KEAP1 signalling in rat heart. Sci Rep, 9(1), 7408. doi:10.1038/s41598-019-43320-5

5. Wedel, S., Hudak, L., Seibel, J. M., Juengel, E., Oppermann, E., Haferkamp, A., & Blaheta, R. A. (2011). Critical analysis of simultaneous blockage of histone deacetylase and multiple receptor tyrosine kinase in the treatment of prostate cancer. Prostate, 71(7), 722-735. doi:10.1002/pros.21288

6. Rotllan, N., Chamorro-Jorganes, A., Araldi, E., Wanschel, A. C., Aryal, B., Aranda, J. F., . . . Fernandez-Hernando, C. (2015). Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis. Faseb j, 29(2), 597-610. doi:10.1096/fj.14-262097

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-07444

• AKT3 Antibodies
• AKT3 ELISA Kits
• AKT3 Lysates
• AKT3 Proteins