PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival.

Protein kinase B (PKB/Akt) is a well-established regulator of several essential cellular processes. Here, we report a route by which activated PKB promotes survival in response to DNA insults in vivo. PKB activation following DNA damage requires 3-phosphoinositide-dependent kinase 1 (PDK1) and DNA-dependent protein kinase (DNA-PK). Active PKB localizes in the nucleus of gamma-irradiated cells adjacent to DNA double-strand breaks, where it colocalizes and interacts with DNA-PK. Levels of active PKB inversely correlate with DNA damage-induced apoptosis. A significant portion of p53- and DNA damage-regulated genes are misregulated in cells lacking PKBalpha. PKBalpha knockout mice show impaired DNA damage-dependent induction of p21 and increased tissue apoptosis after single-dose whole-body irradiation. Our findings place PKB downstream of DNA-PK in the DNA damage response signaling cascade, where it provides a prosurvival signal, in particular by affecting transcriptional p21 regulation. Furthermore, this function is apparently restricted to the PKBalpha isoform.

Protein kinase B (PKB/Akt), a key mediator of the PI3K signaling pathway.

Protein kinase B (PKB/Akt) is a serine/threonine protein kinase that created serious interest when it was revealed as a mediator of the PI3K pathway. It comprises three isoforms that play both unique and redundant roles. Upon binding to phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) generated by PI3K, PKB is phosphorylated by PDK1 at T308. To achieve full kinase activity, PKB needs to be phosphorylated at a second key residue, S473, by members of the PI3K-related kinase family mTORC2 or DNA-PK, depending on the stimulus and the context. Besides, a number of phosphatases and interacting partners have been shown to further modulate its subcellular localization, phosphorylation, and kinase activity. This review aims at illustrating the remarkable complexity in the regulation of PKB signaling downstream of PI3K. Such regulation could be attributed to the specific roles of the PKB isoforms, their expression pattern, subcellular localization, targets, phosphorylation by upstream kinases in a stimulus- and context-dependent manner and by phosphatases, and interaction with binding partners. This allows this key kinase to fulfill physiological functions in numerous processes, including embryonic development, thymocyte development, adipocyte differentiation, glucose homeostasis, and to avoid pathological loss of control such as tumor formation.

Meeting report: targeting the kinome--20 years of tyrosine kinase inhibitor research in Basel.

With basic and pharmaceutical researchers, chemists, pharmacologists, and clinicians, the Targeting the Kinome meeting brought together a tremendous group of scientists to discuss the past, present, and future of protein kinase research, with a special emphasis on cancer therapy development. The topics ranged from the kinases themselves as drug targets through the interactions that the actions of these enzymes promote, to the physiological and pathological processes they regulate. Attendees gained insight into drug development, as well as increased understanding of how normal cellular processes are subverted during oncogenesis, which may lead to new targets for intervention.

PIKKing on PKB: regulation of PKB activity by phosphorylation.

Ser/Thr protein kinase PKB/Akt is a key regulator of a wide range of cellular processes including growth, proliferation and survival. PKB is clearly a crucial signaling molecule and extensive research efforts aim to understand its regulation and action. Recent studies of the regulation of PKB activity by hydrophobic motif phosphorylation have yielded several exciting findings about members of the PI3-kinase-like family of kinases (PIKKs) acting as PKB regulators. Mammalian target of rapamycin complex 2 (mTORC2) and DNA-dependent protein kinase (DNA-PK) can both phosphorylate Ser473 and activate PKB. This present review concerns PKB regulation by mTORC2 and DNA-PK in a stimulus-dependent and context-dependent manner and the possible implications of this for PKB activity, substrate specificity and therapeutic intervention.

Carboxy-Terminal Modulator Protein (CTMP) is a mitochondrial protein that sensitizes cells to apoptosis.

The Carboxy-Terminal Modulator Protein (CTMP) protein was identified as a PKB inhibitor that binds to its hydrophobic motif. Here, we report mitochondrial localization of endogenous and exogenous CTMP. CTMP exhibits a dual sub-mitochondrial localization as a membrane-bound pool and a free pool of mature CTMP in the inter-membrane space. CTMP is released from the mitochondria into the cytosol early upon apoptosis. CTMP overexpression is associated with an increase in mitochondrial membrane depolarization and caspase-3 and polyADP-ribose polymerase (PARP) cleavage. In contrast, CTMP knock-down results in a marked reduction in the loss of mitochondrial membrane potential as well as a decrease in caspase-3 and PARP activation. Mutant CTMP retained in the mitochondria loses its capacity to sensitize cells to apoptosis. Thus, proper maturation of CTMP is essential for its pro-apoptotic function. Finally, we demonstrate that CTMP delays PKB phosphorylation following cell death induction, suggesting that CTMP regulates apoptosis via inhibition of PKB.

In vivo analysis of protein kinase B (PKB)/Akt regulation in DNA-PKcs-null mice reveals a role for PKB/Akt in DNA damage response and tumorigenesis.

Full activation of protein kinase B (PKB/Akt) requires phosphorylation on Thr-308 and Ser-473. It is well established that Thr-308 is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1). Ser-473 phosphorylation is mediated by both mammalian target of rapamycin-rictor complex (mTORC2) and DNA-dependent protein kinase (DNA-PK) depending on type of stimulus. However, the physiological role of DNA-PK in the regulation of PKB phosphorylation remains to be established. To address this, we analyzed basal, insulin-induced, and DNA damage-induced PKB Ser-473 phosphorylation in DNA-PK catalytic subunit-null DNA-PKcs(-/-) mice. Our results revealed that DNA-PK is required for DNA damage-induced phosphorylation but dispensable for insulin- and growth factor-induced PKB Ser-473 phosphorylation. Moreover, DNA-PKcs(-/-) mice showed a tissue-specific increase in basal PKB phosphorylation. In particular, persistent PKB hyperactivity in the thymus apparently contributed to spontaneous lymphomagenesis in DNA-PKcs(-/-) mice. Significantly, these tumors could be prevented by deletion of PKBalpha. These findings reveal stimulus-specific regulation of PKB activation by specific upstream kinases and provide genetic evidence of PKB deregulation in DNA-PKcs(-/-) mice.