Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.     

An acetylation site in the middle domain of Hsp90 regulates chaperone function

Heat-shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.     

Function of Akt/PKB signaling to cell motility,invasion and the tumor stroma in cancer

The serine/threonine protein kinase Akt is a major signal transducer of the phosphoinositide 3-kinase (PI 3-K) pathway in all cells and tissues and plays a pivotal role in the maintenance of cellular processes including cell growth, proliferation, survival and metabolism. The frequent aberrant activation of the PI 3-K/Akt pathway in human cancer has made it an attractive therapeutic target. Numerous studies have provided a comprehensive understanding of the specific functions of Akt signaling in cancer cells as well as the surrounding tumor microenvironment and this has informed and enabled the development of therapeutic drugs to target both PI 3-K and Akt. However, recent studies have provided evidence for distinct functions of the three mammalian Akt isoforms, particularly with respect to the regulation of cell motility and metastasis of breast cancer. Here we discuss the mechanisms by which Akt signaling contributes to invasive migration and tumor metastasis, and highlight recent advances in our understanding of the contribution of the Akt pathway in the tumor-associated stroma.     

Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function

Hsp90 is a chaperone required for the conformational maturation of certain signaling proteins including Raf, cdk4, and steroid receptors. Natural products and synthetic small molecules that bind to the ATP-binding pocket in the amino-terminal domain of Hsp90 inhibit its function and cause the degradation of these client proteins. Inhibition of Hsp90 function in cells causes down-regulation of an Akt kinase-dependent pathway required for D-cyclin expression and retinoblastoma protein-dependent G(1) arrest. Intracellular Akt is associated with Hsp90 and Cdc37 in a complex in which Akt kinase is active and regulated by phosphatidylinositol 3-kinase. Functional Hsp90 is required for the stability of Akt in the complex. Occupancy of the ATP-binding pocket by inhibitors is associated with the ubiquitination of Akt and its targeting to the proteasome, where it is degraded. This results in a shortening of the half-life of Akt from 36 to 12 h and an 80% reduction in its expression. Akt and its activating kinase, PDK1, are the only members of the protein kinase A/protein kinase B/protein kinase C-like kinase family that are affected by Hsp90 inhibitors. Thus, transduction of growth factor signaling via the Akt and Raf pathways requires functional Hsp90 and can be coordinately blocked by its inhibition.     

Hsp90 regulates the activity of wild type p53 under physiological and elevated temperatures

The activity and structural integrity of the tumor suppressor protein p53 is of crucial importance for the prevention of cancer. p53 is a conformational flexible and labile protein, in which structured and unstructured regions function in a synergistic manner. The molecular chaperone Hsp90 is known to bind to mutant and wild type p53 in vivo. Using highly purified proteins we analyzed the interaction and the binding sites between both proteins in detail. Our results demonstrate that Hsp90 binds to a folded, native-like conformation of p53 in vitro with micromolar affinity. Specifically, the DNA-binding domain of p53 and the middle and carboxy-terminal domains of Hsp90 are responsible for this interaction, which is essential to stabilize p53 at physiological temperatures and to prevent it from irreversible thermal inactivation. Our results are in agreement with a model in which Hsp90 is required to maintain the folded, active state of p53 by a reversible interaction, thus introducing an additional level of regulation.     

Methylation of p53 by Set7/9 mediates p53 acetylation and activity in vivo

The protein methyltransferase Set7/9 was recently shown to regulate p53 activity in cancer cells. However, the impact of Set7/9 on p53 function in vivo is unclear. To explore these issues, we created a null allele of Set7/9 in mice. Cells from Set7/9 mutant mice fail to methylate p53 K369, are unable to induce p53 downstream targets upon DNA damage, and are predisposed to oncogenic transformation. Importantly, we find that methylation of p53 by Set7/9 is required for the binding of the acetyltransferase Tip60 to p53 and for the subsequent acetylation of p53. We provide the first genetic evidence demonstrating that lysine methylation of p53 by Set7/9 is important for p53 activation in vivo and suggest a mechanistic link between methylation and acetylation of p53 through Tip60.     

Gibberellic acid stabilises microtubules in maize suspension cells to cold and stimulates acetylation of alpha-tubulin

Gibberellic acid is known to stabilise microtubules in plant organs against depolymerisation. We have now devised a simplified cell system for studying this. Pretreatment of a maize cell suspension with gibberellic acid for just 3 h stabilised protoplast microtubules against depolymerisation on ice. In other eukaryotes, acetylation of alpha-tubulin is known to correlate with microtubule stabilisation but this is not established in plants. By isolating the polymeric tubulin fraction from maize cytoskeletons and immunoblotting with the antibody 6-11B-1, we have demonstrated that gibberellic acid stimulates the acetylation of alpha-tubulin. This is the first demonstrated link between microtubule stabilisation and tubulin acetylation in higher plants.     

p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB

Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the alpha6beta4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:20733-20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of alpha6beta4 in carcinoma cells that express wild-type p53. In contrast, alpha6beta4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the alpha6beta4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of alpha6beta4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.     

Phosphorylated Hsp27 activates ATM-dependent p53 signaling and mediates the resistance of MCF-7 cells to doxorubicin-induced apoptosis

DNA damage activates p53 and its downstream target genes, which further leads to apoptosis or survival either by the cell cycle arrest or by DNA repair. In many tumors, the heat shock protein 27 (Hsp27) is expressed at high levels to provide protection against anticancer drugs. However, the roles of Hsp27 in p53-mediated cellular responses to DNA damage are controversial. Here, we investigated the interplay between the phosphorylation status of Hsp27 and p53 in kidney 293A (HEK293A) cells and found that over-expressing phosphorylated Hsp27 mimics (Hsp27-3D) activated p53/p21 in an ATM-dependent manner. In addition, incubation with doxorubicin (Dox), an anticancer drug, induced Hsp27 phosphorylation in human adenocarcinoma cells (MCF-7). In contrast, inhibition of Hsp27 phosphorylation retarded both p53 induction and p21 accumulation, and led to cell apoptosis. Furthermore, phosphorylated Hsp27 increased p53 nuclear importing and its downstream target gene expression such as p21 and MDM2, while de-phosphorylated Hsp27 impeded this procession. Taken together, our data suggest that Hsp27, in its phosphorylated or de-phosphorylated status, plays different roles in regulating p53 pathway and cell survival.     

Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis.

The phosphoinositide 3-OH kinase (PI3K)-PKB/Akt signaling pathway has been shown to mediate both Ras- and cytokine-induced protection from apoptosis. In addition, apoptosis induced by the p53 tumor suppressor protein can be inhibited by Ras- and cytokine-mediated signaling pathways. It was therefore of interest to determine if the PI3K-PKB/Akt signaling pathway was capable of conferring protection from apoptosis induced by p53. We demonstrate in this report that constitutively active PI3K and PKB/Akt are capable of significantly delaying the onset of p53-mediated apoptosis. This was manifested as a delay in the kinetics of DNA degradation and cell death as well as a profound attenuation in the accumulation of cells with a sub-G(1) DNA content. Moreover, we found that this effect is mediated in the absence of changes in expression of Bcl-2, Bcl-Xl, and the pro-apoptotic protein Bax. Our results provide the first direct and unambiguous link between p53-mediated apoptosis and the PI3K-PKB/Akt signaling pathway.     

Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling

PI3k-Akt and p53 pathways are known to play anti- and pro-apoptotic roles in cell death, respectively. Whether these pathways are recruited in influenza virus infection in highly productive monkey (CV-1) and canine (MDCK) kidney cells was studied here. Phosphorylation of Akt (Akt-pho) was found to occur only early after infection (5–9 h.p.i). Nuclear accumulation and phosphorylation of p53 (p53-pho), and expression of its natural target p21/waf showed low constitutive levels at this period, whereas all three parameters were markedly elevated at the late apoptotic stage (17–20 h.p.i.). Up-regulation of Akt-pho and p53-pho was not induced by UV-inactivated virus suggesting that it required virus replication. Also, mRNAs of p53 and its natural antagonist mdm2 were not increased throughout infection indicating that p53-pho was up-regulated by posttranslational mechanisms. However, p53 activation did not seem to play a leading role in influenza-induced cell death: (i) infection of CV1 and MDCK cells with recombinant NS1-deficient virus provoked accelerated apoptotic death characterized by the lack of p53 activation; (ii) mixed apoptosis-necrosis death developed in influenza-infected human bronchial H1299 cells carrying a tetracycline-regulated p53 gene did not depend on p53 gene activation by tetracycline. Virus-induced apoptosis and signaling of Akt and p53 developed in IFN-deficient VERO cells with similar kinetics as in IFN-competent CV1-infected cells indicating that these processes were endocrine IFN-independent. Apoptosis in influenza-infected CV-1 and MDCK cells was Akt-dependent and was accelerated by Ly294002, a specific inhibitor of PI3k-Akt signaling, and down-regulated by the viral protein NS1, an inducer of host Akt. The obtained data suggest that influenza virus (i) initiates anti-apoptotic PI3k-Akt signaling at early and middle phases of infection to protect cells from fast apoptotic death and (ii) provokes both p53-dependent and alternative p53-independent apoptotic and/or necrotic (in some host systems) cell death at the late stage of infection. These data have been partially presented at The 3rd Orthomyxovirus Research Conference (sponsored by ESWI and NIH). Abstr. p. 23 entitled: “Influenza virus-specific up-regulation of Akt and Mdm2 in infected cells” by Zhirnov O.P., and Klenk H.D., July 28–21, 2005. Queen’s College, Cambridge, United Kingdom; and at The Annual Meeting of Virology in Munich, March 15–18 (2006)—“Influenza virus-specific up-regulation of Akt, Mdm2, and p53 in infected cells” by O. P. Zhirnov and H. D. Klenk; Book of abstracts, p. 339     

Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery

Hsp90 is a molecular chaperone essential for the activation and assembly of many key eukaryotic signalling and regulatory proteins. Hsp90 is assisted and regulated by co-chaperones that participate in an ordered series of dynamic multiprotein complexes, linked to Hsp90 conformationally coupled ATPase cycle. The co-chaperones Aha1 and Hch1 bind to Hsp90 and stimulate its ATPase activity. Biochemical analysis shows that this activity is dependent on the N-terminal domain of Aha1, which interacts with the central segment of Hsp90. The structural basis for this interaction is revealed by the crystal structure of the N-terminal domain (1-153) of Aha1 (equivalent to the whole of Hch1) in complex with the middle segment of Hsp90 (273-530). Structural analysis and mutagenesis show that binding of N-Aha1 promotes a conformational switch in the middle-segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N-terminal nucleotide-binding domain of the chaperone.