Akt-GSK3β Pro-survival Signaling Pathway in Cerebral Ischemic Injury

Introduction

The balance between apoptotic signals and survival signals determines the fate of neurons after cerebral ischemia. Unobstructed activation of caspases in pathological conditions will lead to cell death; however, activation of phosphoinositide 3-kinase (PI3K)/Akt signaling pathway may counteract the apoptotic process and support neuron survival and recovery from ischemic insults. Akt, also called protein kinase B (PKB), is a 57kD serine/threonine protein kinase that is composed of an N-terminal pleckstrin homology (PH) domain, a central catalytic domain, and a C-terminal regulatory domain.

Akt Activation and Regulation

Full activation of Akt occurs by successive phosphorylation of Akt by serine/threonine kinases at Thr-308 within the catalytic domain and at Ser-473 within the C-terminus. This requires the upstream spatial coordination of kinases with Akt, and is largely mediated by the availability of the membrane phospholipid phosphoinositol-(3,4,5)-phosphate (PIP ₃ ). By binding Akt via its PH domain, PIP ₃ recruits both Akt and upstream kinases, including the serine/threonine kinase 3-phosphoinositide-dependent kinase-1 (PDK1), to plasma membrane. Once in close proximity to Akt, PDK1 can directly phosphorylate Akt on Thr308. In addition to the dominant role of PDK1, Akt phosphorylation on Thr308 may also be mediated by unidentified non-PDK1 kinases or autophosphorylation . Phosphorylation of Akt at Ser-473 is largely dependent on PI3K family activity, and may serve as a regulatory step to Thr308 phosphorylation. Importantly, and as mentioned later, phosphorylation of Akt at Ser-473 does not always correlate with kinase activity.

Phosphatidylserine (PS) is a membrane phospholipid that regulates the Akt phosphorylation process by interacting with both Akt and PDK1 at the cytoplasmic membrane. PS, the major acidic phospholipid class in the cerebral cortex, is exclusively localized in the inner leaflet of membrane where proteins—including Akt—dock for activation. Akt contains the basic residues R15 and K20 that bind to PS in anionic domains of the cytoplasmic leaflet, and appear to be required for Akt membrane binding and conformational changes that promote activation. PS is also specifically required for the association of PDK1 with the plasma membrane and its subsequent activation of Akt. Disruption of the PS-PDK1 interaction blocks membrane localization of PDK1 and leads to diminished phosphorylation of Akt at Thr308. In addition to PDK1 phosphorylation of Akt at Thr308, the binding between the basic residues in the regulatory domain of Akt and PS facilitates phosphorylation of Ser473 by mTOR. Although phosphorylation of only Ser473 is not sufficient to stimulate its cell survival activity, full Akt activity is classically regulated by both phosphorylation sites (Thr308 and Ser473). Externalization of PS induced by cell death stimuli, including cerebral ischemia, impedes the activation of Akt and stimulates microglia phagocytosis of apoptotic cells.

Regulation of Akt activation is also highly controlled by the bioavailability of PIP ₃ . PIP ₃ is generated by phosphorylation of phosphatidylinositol phosphate 2 (PIP ₂ ) via PI3K. PI3K is normally present in cytosol and can be activated directly by recruitment to an activated Trk receptor. Active PI3K then phosphorylates phosphatidylinositol phosphate 2 (PIP ₂ ) to PIP ₃ , which in turn can bind to Akt. Counter to the activity of PI3K, phosphatase and tensin homolog (PTEN) dephosphorylates PIP ₃ back to PIP ₂ , leading to the subsequent inactivation of Akt. Phosphorylation of PTEN blocks its ability to interact with PIP, and thus is permissive to Akt activation. In addition to the regulation of PTEN by phosphorylation, S-nitrosylation of PTEN at Cys83 inhibits the phosphatase activity of PTEN, thereby preventing dephosphorylation of PIP ₃ and allowing the recruitment and activation of Akt .