Akt is a protein serine/threonine kinase that plays an important role in the mitogenic responses of cells to variable stimuli. Akt contains a pleckstrin homology (PH) domain and is activated by phosphorylation at threonine 308 and serine 473. Binding of 3′-OH phosphorylated phosphoinositides to the PH domain results in the translocation of Akt to the plasma membrane where it is activated by upstream kinases such as (phosphoinositide-dependent kinase-1 (PDK1). Over-expression of constitutively active forms of Akt promotes cell proliferation and survival, and also stimulates p70 S6 kinase (p70S6K). In many cells, an increase in levels of intracellular cyclic AMP (cAMP) diminishes cell growth and promotes differentiation, and in certain conditions cAMP is even antagonistic to the effect of growth factors. Here, we show that cAMP has inhibitory effects on the phosphatidylinositol 3-kinase/PDK/Akt signaling pathway. cAMP potently inhibits phosphorylation at threonine 308 and serine 473 of Akt, which is required for the protein kinase activities of Akt. cAMP also negatively regulates PDK1 by inhibiting its translocation to the plasma membrane, despite not affecting its protein kinase activities. Furthermore, when we co-expressed myristoylated Akt and PDK1 mutants which constitutively co-localize in the plasma membrane, Akt activity was no longer sensitive to raised intracellular cAMP concentrations. Finally, cAMP was also found to inhibit the lipid kinase activity of PI3K and to decrease the levels of phosphatidylinositol 3,4,5-triphosphate in vivo, which are required for the membrane localization of PDK1. Collectively, these data strongly support the theory that the cAMP-dependent signaling pathway inhibits Akt activity by blocking the coupling between Akt and its upstream regulators, PDK, in the plasma membrane.

We have studied a possible role of extracellular zinc ion in the activation of p70S6k, which plays an important role in the progression of cells from the G(1) to S phase of the cell cycle. Treatment of Swiss 3T3 cells with zinc sulfate led to the activation and phosphorylation of p70S6k in a dose-dependent manner. The activation of p70S6k by zinc treatment was biphasic, the early phase being at 30 min followed by the late phase at 120 min. The zinc-induced activation of p70S6k was partially inhibited by down-regulation of phorbol 12-myristate 13-acetate-responsive protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate, but this was not significant. Moreover, Go6976, a specific calcium-dependent PKC inhibitor, did not significantly inhibit the activation of p70S6k by zinc. These results demonstrate that the zinc-induced activation of p70S6k is not related to PKC. Also, extracellular calcium was not involved in the activation of p70S6k by zinc. Further characterization of the zinc-induced activation of p70S6k using specific inhibitors of the p70S6k signaling pathway, namely rapamycin, wortmannin, and LY294002, showed that zinc acted upstream of mTOR/FRAP/RAFT and phosphatidylinositol 3-kinase (PI3K), because these inhibitors caused the inhibition of zinc-induced p70S6k activity. In addition, Akt, the upstream component of p70S6k, was activated by zinc in a biphasic manner, as was p70S6k. Moreover, dominant interfering alleles of Akt and PDK1 blocked the zinc-induced activation of p70S6k, whereas the lipid kinase activity of PI3K was potently activated by zinc. Taken together, our data suggest that zinc activates p70S6k through the PI3K signaling pathway.

Phosphoinositide-dependent kinase-1 (PDK-1) is a central mediator of the cell signaling between phosphoinositide 3-kinase (PI3K) and various intracellular serine/threonine kinases including Akt/protein kinase B (PKB), p70 S6 kinases, and protein kinase C. Recent studies with cell transfection experiments have implied that PDK-1 may be involved in various cell functions including cell growth and apoptosis. However, despite its pivotal role in cellular signalings, the in vivo functions of PDK-1 in a multicellular system have rarely been investigated. Here, we have isolated Drosophila PDK-1 ( dPDK-1 ) mutants and characterized the in vivo roles of the kinase. Drosophila deficient in the dPDK-1 gene exhibited lethality and an apoptotic phenotype in the embryonic stage. Conversely, overexpression of dPDK-1 increased cell and organ size in a Drosophila PI3K-dependent manner. dPDK-1 not only could activate Drosophila Akt/PKB ( Dakt1 ), but also substitute the in vivo functions of its mammalian ortholog to activate Akt/PKB. This functional interaction between dPDK-1 and Dakt1 was further confirmed through genetic analyses in Drosophila . On the other hand, cAMP-dependent protein kinase, which has been proposed as a possible target of dPDK-1 , did not interact with dPDK-1 . In conclusion, our findings provide direct evidence that dPDK-1 regulates cell growth and apoptosis during Drosophila development via the PI3K-dependent signaling pathway and demonstrate our Drosophila system to be a powerful tool for elucidating the in vivo functions and targets of PDK-1.

Mitophagy has been implicated in mitochondrial quality control and in various human diseases. However, the study of in vivo mitophagy remains limited. We previously explored in vivo mitophagy using a transgenic mouse expressing the mitochondria‐targeted fluorescent protein Keima (mt‐Keima). Here, we generated mt‐Keima Drosophila to extend our efforts to study mitophagy in vivo . A series of experiments confirmed that mitophagy can be faithfully and quantitatively measured in mt‐Keima Drosophila . We also showed that alterations in mitophagy upon environmental and genetic perturbation can be measured in mt‐Keima Drosophila . Analysis of different tissues revealed a variation in basal mitophagy levels in Drosophila tissues. In addition, we found a significant increase in mitophagy levels during Drosophila embryogenesis. Importantly, loss‐of‐function genetic analysis demonstrated that the phosphatase and tensin homolog‐induced putative kinase 1 (PINK1)‐Parkin pathway is essential for the induction of mitophagy in vivo in response to hypoxic exposure and rotenone treatment. These studies showed that the mt‐Keima Drosophila system is a useful tool for understanding the role and molecular mechanism of mitophagy in vivo . In addition, we demonstrated the essential role of the PINK1‐Parkin pathway in mitophagy induction in response to mitochondrial dysfunction.—Kim, Y. Y., Um, J.‐H., Yoon, J.‐H., Kim, H., Lee, D.‐Y., Lee, Y. J., Jee, H. J., Kim, Y. M., Jang, J. S., Jang, Y.‐G., Chung, J., Park, H. T., Finkel, T., Koh, H., Yun, J. Assessment of mitophagy in mt‐Keima Drosophila revealed an essential role of the PINK1‐Parkin pathway in mitophagy induction in vivo . FASEB J. 33, 9742–9751 (2019). www.fasebj.org