Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses.

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects.

CodY-mediated regulation of the Staphylococcus aureus Agr system integrates nutritional and population density signals.

The Staphylococcus aureus Agr system regulates virulence gene expression by responding to cell population density (quorum sensing). When an extracellular peptide signal (AIP-III in strain UAMS-1, used for these experiments) reaches a concentration threshold, the AgrC-AgrA two-component regulatory system is activated through a cascade of phosphorylation events, leading to induction of the divergently transcribed agrBDCA operon and the RNAIII gene. RNAIII is a posttranscriptional regulator of numerous metabolic and pathogenesis genes. CodY, a global regulatory protein, is known to repress agrBDCA and RNAIII transcription during exponential growth in rich medium, but the mechanism of this regulation has remained elusive. Here we report that phosphorylation of AgrA by the AgrC protein kinase is required for the overexpression of the agrBDCA operon and the RNAIII gene in a codY mutant during the exponential-growth phase, suggesting that the quorum-sensing system, which normally controls AgrC activation, is active even in exponential-phase cells in the absence of CodY. In part, such premature expression of RNAIII was attributable to higher-than-normal accumulation of AIP-III in a codY mutant strain, as determined using ultrahigh-performance liquid chromatography coupled to mass spectrometry. Although CodY is a strong repressor of the agr locus, CodY bound only weakly to the agrBDCA-RNAIII promoter region, suggesting that direct regulation by CodY is unlikely to be the principal mechanism by which CodY regulates agr and RNAIII expression. Taken together, these results strongly suggest that cell population density signals inducing virulence gene expression can be overridden by nutrient availability, a condition monitored by CodY.