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Results for “PI3K/AKT1 signalling”

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PubMed2002

Wortmannin-sensitive pathway is required for insulin-stimulated phosphorylation of inhibitor kappaBalpha.

The aim of this study was to examine the signaling pathways by which insulin promotes activation of nuclear factor kappaB (NFkappaB) through the regulation of inhibitor kappaBalpha (IkappaBalpha). We show here that although insulin increased kappaB-dependent reporter gene expression and augmented nuclear translocation of the p65/RelA subunit of NFkappaB and its DNA binding, it was able to induce a time-dependent accumulation of phosphorylated and ubiquitinated IkappaBalpha without its proteolytic degradation. In contrast, cell stimulation with the cytokine TNFalpha allowed activation of NFkappaB through phosphorylation, ubiquitination, and subsequent degradation of IkappaBalpha. Immunofluorescence studies revealed the presence of a large pool of phosphorylated IkappaBalpha in the nucleus of unstimulated and insulin-treated cells. IkappaB kinase alpha and beta, central players in the phosphorylation of IkappaBalpha, were rapidly induced following exposure to TNFalpha but not insulin. Furthermore, insulin-stimulated IkappaBalpha phosphorylation did not depend on activation of the Ras/ERK cascade. Expression of a dominant-negative mutant of Akt1 or class I PI3K inhibited the insulin stimulation of PI3K/Akt1 signaling without affecting phosphorylation of IkappaBalpha. Interestingly, the PI3K inhibitors wortmannin and LY294002 blocked insulin-stimulated class I PI3K-dependent events at much lower doses than that required to inhibit phosphorylation of IkappaBalpha. These data demonstrate that insulin regulates IkappaBalpha function through a distinct low-affinity wortmannin-sensitive pathway.

Androstadienes↗

PubMed2006

Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region.

BACKGROUND: p27kip1 (p27) is a multifunctional protein implicated in regulation of cell cycling, signal transduction, and adhesion. Its activity is controlled in part by Phosphatylinositol-3-Kinase (PI3K)/Akt1 signaling, and disruption of this regulatory connection has been identified in human breast cancers. The serine/threonine protein kinase Akt1 directly phosphorylates p27, so identifying the modified residue(s) is essential for understanding how it regulates p27 function. Various amino acids have been suggested as potential targets, but recent attention has focused on threonine 157 (T157) because it is located in a putative Akt1 consensus site. However, T157 is not evolutionarily conserved between mouse and human. We therefore re-evaluated Akt1 phosphorylation of p27 using purified proteins and in cells. RESULTS: Here we show purified Akt1 phosphorylates human and mouse p27 equally well. Phospho-peptide mapping indicates Akt1 targets multiple sites conserved in both species, while phospho-amino acid analysis identifies the targeted residues as serine rather than threonine. P27 deletion mutants localized these sites to the N-terminus, which contains the major p27 phosphorylation site in cells (serine 10). P27 phosphorylated by Akt1 was detected by a phospho-S10 specific antibody, confirming this serine was targeted. Akt1 failed to phosphorylate p27S10A despite evidence of a second site from mapping experiments. This surprising result suggested S10 phosphorylation might be required for targeting the second site. We tested this idea by replacing S10 with threonine, which as expected led to the appearance of phospho-threonine. Phospho-serine was still present, however, confirming Akt1 sequentially targets multiple serines in this region. We took two approaches in an attempt to explain why different residues were previously implicated. A kinetic analysis revealed a putative Akt1 binding site in the C-terminus, which may explain why mutations in this region affect p27 phosphorylation. Furthermore, commercially available recombinant Akt1 preparations exhibit striking differences in substrate specificity and site selectivity. To confirm S10 is a relevant site, we first showed that full-length wild type Akt1 purified from mammalian cells phosphorylates both human and mouse p27 on S10. Finally, we found that in cultured cells under physiologically relevant conditions such as oxidative stress or growth factor deprivation, endogenous Akt1 causes p27 accumulation by phosphorylating S10. CONCLUSION: Identifying where Akt1 phosphorylates p27 is essential for understanding its functional implications. We found that full-length wild type Akt1--whether purified, transiently overexpressed in cells, or activated in response to cellular stress--phosphorylates p27 at S10, a noncanonical but evolutionarily conserved site known to regulate p27 activity and stability. Using recombinant Akt1 recapitulating this specificity, we showed modification of p27S10 also leads to phosphorylation of an adjacent serine. These results integrate PI3K/Akt1 signaling in response to stress with p27 regulation through its major phosphorylation site in cells, and thus identify new avenues for understanding p27 deregulation in human cancers.

Journal Article↗

PubMed2026

Proteomic regulation of anti-proliferative and anti-migratory activity by potent phytochemicals from Pistacia integerrima J.L. Steward Ex Brandis via PI3K, AKT1, and KRAS for Lung Cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of mortality worldwide and remains a major therapeutic challenge due to high metastasis, drug resistance and limited treatments. Pistacia integerrima J.L. Steward Ex Brandis (PI) consists of flavonoids, steroids, terpenoids and phenolic compounds reported for pharmacological activities. The efficacy of potent bioactives from P. integerrima may be ascertained employing cytotoxic, antiproliferative, anti-migratory, and anti-metastatic evaluations in A549 NSCLC cells with proteomic profiling, molecular docking, and dynamics simulation study. METHODS AND RESULTS: PI EtAc produced significant dose-dependent cytotoxicity in A549 cells (100 µg/mL, p < 0.0001 in the MTT assay. There was a pronounced decrease in colony formation after treatment with EtAc, with 18.41% (p < 0.002), and markedly. Furthermore. PI EtAC markedly inhibited cell migration emphasized by wound healing and Transwell migration (p < 0.01) assays, indicating reduced metastatic migratory potential. Proteomic analysis demonstrated significant downregulation of Endoglin (CD105), KLK5 and MMP-2, indicating suppression of angiogenic and metastatic signalling pathways in the Human XL Oncology protein array. The interaction of major PI phytochemicals with key NSCLC-associated targets was recorded in Molecular docking, revealing favourable binding affinities of kaempferol, β- sitosterol, luteolin, and quercetin towards several oncogenic targets, including AKT1(- 7.6 kcal/mol), PI3K(- 9.4 kcal/mol), KRAS (- 8.5 kcal/mol) and MMP9 (- 8.1 kcal/mol). Molecular dynamics simulation confirmed the structural stability of the kaempferol -AKT1 complex throughout the 100 ns simulation. CONCLUSION: Pistacia integerrima bioactives exhibited significant anti-proliferative, anti-migratory, and anti-metastatic activities in vitro, which may provide scientific rationale identifying newer promising candidates for NSCLC.

Humans↗

PubMed2004

Chemokine receptor CCR7 induces intracellular signaling that inhibits apoptosis of mature dendritic cells.

Acquisition of CCR7 expression is an important phenotype change during dendritic cell (DC) maturation that endows these cells with the capability to migrate to lymph nodes. We have analyzed the possible role of CCR7 on the regulation of the survival of DCs. Stimulation with CCR7 ligands CCL19 and CCL21 inhibits apoptotic hallmarks of serum-deprived DCs, including membrane phosphatidylserine exposure, loss of mitochondria membrane potential, increased membrane blebs, and nuclear changes. Both chemokines induced a rapid activation of phosphatidylinositol 3'-kinase/Akt1 (PI3K/Akt1), with a prolonged and persistent activation of Akt1. Interference with PI3K, Gi, or G protein betagamma subunits abrogated the effects of the chemokines on Akt1 activation and on survival. In contrast, inhibition of extracellular signal-related kinase 1/2 (Erk1/2), p38, or c-Jun N-terminal kinase (JNK) was ineffective. Nuclear factor-kappaB (NFkappaB) was involved in the antiapoptotic effects of chemokines because inhibition of NFkappaB blunted the effects of CCL19 and CCL21 on survival. Furthermore, chemokines induced down-regulation of the NFkappaB inhibitor IkappaB, an increase of NFkappaB DNA-binding capability, and translocation of the NFkappaB subunit p65 to the nucleus. In summary, in addition to its well-established role in chemotaxis, we show that CCR7 also induces antiapoptotic signaling in mature DCs.

Apoptosis↗

PubMed2004

Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism.

In response to growth factors, mTOR (mammalian target of rapamycin) has been identified as a central component of the signalling pathways that control the translational machinery and cell growth. Signalling through mTOR has also been shown to be necessary for the mechanical load-induced growth of cardiac and skeletal muscles. Although the mechanisms involved for mechanically induced activation of mTOR are not known, it has been suggested that activation of PI3K (phosphoinositide 3-kinase) and protein kinase B (Akt), via the release of locally acting growth factors, underlies this process. In the present study, we show that mechanically stimulating (passive stretch) the skeletal muscle ex vivo results in the activation of mTOR-dependent signalling events. The activation of mTOR-dependent signalling events was necessary for an increase in translational efficiency, demonstrating the physiological significance of this pathway. Using pharmacological inhibitors, we show that activation of mTOR-dependent signalling occurs through a PI3K-independent pathway. Consistent with these results, mechanically induced signalling through mTOR was not disrupted in muscles from Akt1-/- mice. In addition, ex vivo co-incubation experiments, along with in vitro conditioned-media experiments, demonstrate that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for the activation of the mTOR pathway. Taken together, our results demonstrate that mechanical stimuli can activate the mTOR pathway independent of PI3K/Akt1 and locally acting growth factors. Thus mechanical stimuli and growth factors provide distinct inputs through which mTOR co-ordinates an increase in the translational efficiency.

Animals↗