Molecular, cellular and genetic analysis of protein kinase B (PKB/Akt) mediated intracellular signal transduction.

Description

The control of complex cellular events requires acute orchestration of many signaling processes. There are at least 518 human protein kinases and a few hundred phosphatases that simultaneously target a huge variety of highly interconnected signaling molecules, and regulate their activation state to determine the dynamics and responsiveness of cell behaviors.In an effort to identify novel targets of this protein kinase, genetic mapping for mutations within the Drosophila Dakt1 locus was carried out. A null allele was identified and subsequently exploited in an enhancer screen of a P-element library. Among several genes that genetically interacted with PKB was trachealess (trh), which encodes a bHLH-PAS domain transcription factor required for development of the trachea and other tubular organs. Using a combination of biochemical and transgenic approaches, we demonstrated that direct Trachealess phosphorylation by PKB at serine 665 is essential for nuclear localization and functional activation of this master regulator of tubular morphogenesis.Aberrant PKB activity has been implicated in tumorigenesis. To assess cellular responses to chronic PKB signaling, we sought to develop an experimental system where PKB activity could be arbitrarilly induced. To this end, a conditionally activated allele of PKBbeta (PKB-ER) was stably introduced into tissue culture cells. Chronic activation of PKB lead to extensive multinucleation and rampant cell fusion events; both phenotypes associated with tumorigenesis that possibly contribute to genomic instability and invasion.Protein kinase B (PKB/Akt) is a protein-serine/threonine kinase that translocates to the plasma membrane via its binding to phosphoinositides generated by growth factor stimulation of phosphoinositide 3' kinase (PI3'K). PKB activation requires phosphorylation of two conserved residues. We examined the properties of a series of point mutations within the ATP binding cleft and the regulatory phosphorylation sites. While several independent mutations generated inactive protein kinases, co-expression of two distinct active mutants resulted in reconstitution of activity. These results suggest a model whereby a PKB multimer is normally held in an inactive state through intermolecular inhibition.

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