Thomas F. Franke

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

IGF-I promotes Schwann cell motility and survival via activation of Akt.

We previously reported insulin-like growth factor-I (IGF-I) promotes Schwann cell (SC) motility and rescues SC from apoptosis induced by serum deprivation. This effect is mediated by phosphatidylinositol-3 (PI-3) kinase. In the current study, we examined the role of Akt, a downstream kinase of PI-3K, in SC motility and IGF-I mediated protection from apoptosis. IGF-I induces Akt phosphorylation at Ser473, an event which may be blocked by pretreatment with a PI-3K inhibitor, LY294002. In dominant negative K179M Akt (K179M) transfected SC, however, Akt is not activated in response to IGF-I. In addition, IGF-I is unable to promote SC motility and survival in K179M SC. These results suggest a critical role for Akt in IGF-I mediated motility and survival in SC.

Erratum: Regulation of endothelium-derived nitric oxide production by the protein kinase Akt (Nature (1999) 399 (597-601))

This corrects the article DOI: 21218

Characterization of a novel isoform of caspase-9 that inhibits apoptosis.

We have identified a novel isoform of rat caspase-9 in which the C terminus of full-length caspase-9 is replaced with an alternative peptide sequence. Casp-9-CTD (where CTD is carboxyl-terminal divergent) is expressed in multiple tissues, with the relative highest expression observed in ovary and heart. Casp-9-CTD was found primarily in the cytoplasm and was not detected in the nucleus. Structural predictions suggest that in contrast to full-length caspase-9, casp-9-CTD will not be processed. Our model is supported by reduced protease activity of casp-9-CTD preparations in vitro and by the lack of detectable processing of casp-9-CTD proenzyme or the induction of cell death following transfection into cells. Both neuronal and non-neuronal cell types transfected with casp-9-CTD were resistant to death evoked by trophic factor deprivation or DNA damage. In addition, cytosolic lysates prepared from cells permanently expressing exogenous casp-9-CTD were resistant to caspase induction by cytochrome c in reconstitution assays. Taken together, our observations indicate that casp-9-CTD acts as a dominant-negative variant. Its expression in various tissues indicates a physiological role in regulating cell death.

Roles of extracellular signal‐regulated kinase and Akt signaling in coordinating nuclear transcription factor‐κB‐dependent cell survival after serotonin 1A receptor activation

To investigate the functional consequences of cross‐talk between multiple effectors of serotonin (5‐HT) 1A receptor, we employed transfected Chinese hamster ovary cells. Activation of 5‐HT1A receptor stimulated extracellular signal‐regulated kinase (ERK)1/2, Akt and nuclear transcription factor‐κB (NF‐κB). Stimulation of cells with 5‐HT1A receptor agonist induced a rapid but transient ERK1/2 phosphorylation followed by increased phosphorylation of Akt. Elevated Akt activity in turn suppressed Raf activity and induced a decline in ERK activation. The activation of ERK and Akt downstream of 5‐HT1A receptor was sensitive to inhibitors of Ras, Raf and phosphatidylinositol 3‐kinase (PI3K). Stimulation of 5‐HT1A receptor also resulted in activation of NF‐κB through a decrease in inhibitor of nuclear transcription factor‐κB. In support of the importance of 5‐HT1A receptor signaling for cell survival, inhibition of NF‐κB facilitated caspase 3 activation and cleavage of poly (ADP‐ribose) polymerase, while treatment of cells with agonist inhibited caspase 3, DNA fragmentation and cell death. Both agonist‐dependent NF‐κB activation and cell survival were decreased by Akt Inhibitor II or by overexpression of dominant‐negative Akt. These findings suggest a role for 5‐HT1A receptor signaling in the Ras/Raf‐dependent regulation of multiple intracellular signaling pathways that include ERK and PI3K/Akt. Of these, only PI3K/Akt and NF‐κB activation were required for 5‐HT1A receptor‐dependent cell survival, implying that the relative distribution of signals between competing transduction pathways determines the functional outcome of 5‐HT1A receptor activation.

Context-dependent expression of a conditionally-inducible form of active Akt

Akt kinases are key signaling components in proliferation-competent and post-mitotic cells. Here, we sought to create a conditionally-inducible form of active Akt for both in vitro and in vivo applications. We fused a ligand-responsive Destabilizing Domain (DD) derived from E. coli dihydrofolate reductase to a constitutively active mutant form of Akt1, Akt(E40K). Prior work indicated that such fusion proteins may be stabilized and induced by a ligand, the antibiotic Trimethoprim (TMP). We observed dose-dependent, reversible induction of both total and phosphorylated/active DD-Akt(E40K) by TMP across several cellular backgrounds in culture, including neurons. Phosphorylation of FoxO4, an Akt substrate, was significantly elevated after DD-Akt(E40K) induction, indicating the induced protein was functionally active. The induced Akt(E40K) protected cells from apoptosis evoked by serum deprivation and was neuroprotective in two cellular models of Parkinsons disease (6-OHDA and MPP+ exposure). There was no significant protection without induction. We also evaluated Akt(E40K) induction by TMP in mouse substantia nigra and striatum after neuronal delivery via an AAV1 adeno-associated viral vector. While there was significant induction in striatum, there was no apparent induction in substantia nigra. To explore the possible basis for this difference, we examined DD-Akt(E40K) induction in cultured ventral midbrain neurons. Both dopaminergic and non-dopaminergic neurons in the cultures showed DD-Akt(E40K) induction after TMP treatment. However, basal DD-Akt(E40K) expression was 3-fold higher for dopaminergic neurons, resulting in a significantly lower induction by TMP in this population. Such findings suggest that dopaminergic neurons may be relatively inefficient in protein degradation, a property that could relate to their lack of apparent DD-Akt(E40K) induction in vivo and to their selective vulnerability in Parkinsons disease. In summary, we generated an inducible, biologically active form of Akt. The degree of inducibility appears to reflect cellular context that will inform the most appropriate applications for this and related reagents.