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Overview and Functions of Akt Protein

Akt (also known as PKB or Rac) plays a critical role in regulating cell survival and apoptosis[1]. This protein kinase can be activated by insulin, various growth factors, and survival factors, functioning through a wortmannin-dependent pathway involving PI3 kinase[2].

Activation of Akt requires phospholipid binding and phosphorylation at two sites: phosphorylation at the catalytic domain Thr308 mediated by PDK1[3], and phosphorylation at the carboxyl terminal Ser473. The previously unknown PDK2 responsible for phosphorylating Akt at Ser473 has been identified as mTOR (mammalian target of rapamycin), which exists in a rapamycin-insensitive complex containing rictor and Sin1[4].

Akt inhibits apoptosis and promotes cell survival by phosphorylating and inactivating multiple target proteins, including Bad[5], forkhead transcription factors, c-Raf, and caspase-9. PTEN phosphatase is a major negative regulator of the PI3K/Akt signaling pathway[6-9]; LY294002 is a specific PI3 kinase inhibitor[10].

Another important function of Akt is regulating glycogen synthesis by phosphorylating and inactivating GSK-3α and GSK-3β[10], and it may also be involved in insulin-stimulated glucose transport[11]. In addition to regulating survival and glycogen synthesis, Akt participates in cell cycle regulation through: 1) preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1[12], and 2) negatively regulating cyclin-dependent kinase inhibitors p27 Kip1[14] and p21 Waf1/Cip1[15].

Akt also plays a key role in cell growth: it can directly phosphorylate mTOR in the rapamycin-sensitive complex containing raptor[16]; more importantly, Akt can phosphorylate and inactivate tuberous sclerosis protein 2 (TSC2/tuberin) — a negative regulator of mTOR in the mTOR-raptor complex[17,18].

Mechanisms of Akt Signaling Regulation

A. Growth factor-mediated Akt activation: In response to various growth factors and cytokines, Akt is activated downstream of PI3 kinase through a multi-step mechanism. Activated PI3 kinase converts PIP2 to PIP3, providing recruitment sites on the cell membrane for PH domain-containing proteins (including Akt and PDK1 kinases). Following translocation to the cell membrane, Akt's catalytic domain is phosphorylated by PDK1 and its hydrophobic motif by mTORC2, ultimately conferring catalytic activity. Activated Akt regulates multiple cellular functions by phosphorylating various downstream target proteins within the cell.

B. Overlapping and specific functions of Akt family members: Phenotypic analysis of single/double Akt isoform knockout mice has elucidated shared and unique functions of Akt isoforms, including:    
   - Akt1: Skin, bone, and muscle development, postnatal survival, cell growth, angiogenesis    
   - Akt2: Glucose homeostasis, adipogenesis    
   - Akt3: Neuronal development, overall body weight and size, embryonic development and survival

C. Potential mechanisms determining Akt isoform functional specificity: These involve tissue distribution, regulatory partners, stimulating signals, subcellular localization, specific regulation, and substrate specificity.

Specific Signaling of Akt2 in Insulin-Mediated Glucose Uptake

Following insulin binding to the insulin receptor (IR) on adipocyte surfaces, the receptor is activated and undergoes autophosphorylation, subsequently recruiting and phosphorylating insulin receptor substrate proteins (IRS). Phosphorylated IRS provides docking sites for PI3 kinase, which converts PIP2 to PIP3, thereby creating sites for Akt kinase recruitment to the cell membrane.

In response to PI3 kinase activation, both Akt1 and Akt2 translocate to the cell membrane and are activated through phosphorylation by PDK1 and mTORC2. Using Akt1/Akt2 reporter genes and total internal reflection fluorescence (TIRF) microscopy, it was observed that Akt2 accumulates to a greater extent than Akt1 in the membrane region (TIRF zone) following insulin stimulation. This unique subcellular distribution facilitates Akt2-mediated phosphorylation and inactivation of the Rab GAP protein AS160, which is likely located on GLUT4-containing vesicles; upon AS160 inactivation, GLUT4 vesicles can dock and fuse with the cell membrane, ultimately allowing glucose entry into the cell.

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