The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway controls apoptosis, cell proliferation, and the cell cycle (Rodon et al. 2013). The serine/threonine kinase AKT is a downstream effector of phosphatidylinositol 3-OH kinase (PI3K) (Burgering and Coffer 1995). Extracellular signals activate PI3K to generate phosphatidylinositol 3′ phosphate (PIP3). The tumor suppressor PTEN is a major negative regulator of the pathway by dephoshorylating the 3’phosphate of PIP3 (Sulis and Parsons 2003). AKT has a PH domain at the N-terminus which is required for the binding of PIP3 (Bhaskar and Hay 2007) (Figure 1). In mammals, there are three AKT isoforms (AKT1, AKT2, and AKT3). Phosphorylation of Thr308 and Ser473 are required for full activation of AKT1. Thr308 phosphorylation is mediated by phosphoinositide-dependent kinase-1 (PDK1) (Vanhaesebroeck and Alessi 2000), whereas Ser473 is phosphorylated by the mammalian target of rapamycin complex 2 (mTORC2) (Sarbassov et al. 2005, Laplante and Sabatini 2012) (Figure 1). AKT activity is inhibited by dephosphorylation by phosphatases including protein phosphatase type 2A (PP2A) (Millward et al. 1999) and pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) (Brognard et al. 2007).
Fig. 1. Structure of AKT1. Position of pThr308 and pSer473 are indicated (Manning and Toker 2017, Kitagishi et al. 2012).
When activated, AKT impacts many cellular processes. It activates the mammalian target of rapamycin complex 1 (mTORC1), which results in increased protein synthesis and cell survival (Aoki et al. 2001). AKT phosphorylates and inhibits Forkhead box O (FOXO) transcription factors, resulting in their degradation in the cytoplasm (Zhang et al. 2011). Phosphorylation of the BCL2 Antagonist of Cell Death (BAD) suppresses apoptosis and promotes cell survival (Datta et al. 1997). Moreover, AKT activates inhibitor of nuclear factor (NF) kappa-B kinase (IKK), activating the NF-kB pathway, and resulting in transcription of antiapoptotic genes (Pommier et al. 2004). AKT also phosphorylates MDM2, allowing its translocation to the nucleus where it targets p53 for degradation (Zhou and Hung 2002). AKT also potently induces Ser-133 phosphorylation of cyclic AMP-response element binding protein (CREB). This phosphorylation promotes the recruitment of co-activator protein CREB binding antibody (CBP) (Du and Montminy 1998) and transcription of genes containing CREs (cAMP-responsive elements) in the promoter.
The PI3K/AKT pathway is also important for the regulation of cell cycle progression. AKT inactivates glycogen synthase kinase 3β (GSK3β), leading to increased cyclin D1 (Liang and Slingerland 2003). It also phosphorylates cell cycle inhibitors p21Waf1/Cip1 and p27Kip1 in order to induce their cytoplasmic retention (Testa and Bellacosa 2001).
Dysfunction of the pathway has been implicated in various pathological conditions including cancer, cardiovascular disease, type 2 diabetes, and autoimmune and neurological disorders (Manning and Toker 2017). Dissecting the complexity of PI3K/AKT signaling offers valuable insights into the development of novel therapeutics for treatment of various disorders.
We have compiled key antibodies involved in the PI3K/AKT signaling pathway into one handy poster you can download for your reference.
Click the pathway components within the images below to discover the key antibodies that Bio-Rad provides.
The Rabbit Anti-AKT E17K Mutant Antibody (MCA6297, Figure 2) detects AKT1 kinase mutated at E17K, common in tumorigenesis, where glutamate is substituted for lysine at the 17th amino acid position, in its pleckstrin homology (PH) domain (Carpten et al. 2007). The E17K mutation activates AKT1 by means of pathological localization to the plasma membrane, and stimulates downstream signaling, leading to cellular transformation and tumor formation (Carpten et al. 2007, Brugg et al. 2007). AKT1 E17K mutant triggers phosphorylation and cytoplasmic delocalization of the cyclin-dependent kinase inhibitor p27, and promotes cell proliferation (De Marco et al. 2015). AKT E17K mutant was also found to display higher levels of ubiquitination than AKT wild type. Interestingly, blocking this ubiquitination results in reduction in AKT membrane recruitment and phosphorylation (Yang et al. 2009, Restuccia and Hemmings 2009).
Fig. 2. Western blot analysis of a cell lysate mixture of untransfected 293T and 293T transfected with a DNA construct encoding the AKT E17K mutant and probed with Rabbit Anti-AKT (E17K Mutant) (MCA6297) or Rabbit Anti-AKT (MCA6307).
The mutation is present in multiple cancers, including human breast, ovarian, colorectal (Carpten et al. 2007), lung (Malanga et al. 2008), and endometrial cancer (Cohen et al. 2010). AKT1 E17K may predict clinical response to capivasertib (AZD5363), a drug targeting the AKT signaling pathway (Tamura et al. 2016).
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