In Vivo Regulation of Extracellular Signal-Regulated Protein Kinase (ERK) and Protein Kinase B (Akt) Phosphorylation by Acute and Chronic Morphine

Abstract Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis, stimulating growth, preventing apoptosis, and promoting differentiation of red blood cell progenitors. The critical importance of EPO, EPO receptor (EPO-R) and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops in EPO, EPO-R or JAK2 knockout mice. Intracellular signal transduction pathways regulating growth, differentiation and cell survival downstream of the EPO-R and JAK2 are well documented. However, activation of the EPO-R is transient and down regulated by several negative regulators including tyrosine phosphatases, inositol phosphatases and ubiquitin ligases. The negative regulator, Cbl, has been implicated as a tumour suppressor in murine sarcoma, B cell leukemia, and erythroleukemia. More recently, Cbl was found in a de novo form of acute myeloid leukemia and has been implicated in the formation of gastric tumours. Cbl is known to bind, ubiquitinate, and downregulate signaling from numerous activated hematopoietic and non-hematopoietic receptors. The discovery that Cbl is a target of EPO-dependent tyrosine phosphorylation, together with the finding that the EPO-R is ubiquitinated in vivo, led us to hypothesize that Cbl deficiency leads to altered murine erythropoiesis. Resting C57Bl/6 Cbl-/- mice display normal hematologic parameters with the exception of an increased platelet count. However, Cbl deficient mice respond to phenylhydrazine-mediated anemia with increased reticulocyte production and hematocrit recovery. The hypersensitivity of Cbl deficient mice to anemia may be explained by a three-fold enhancement of splenic colony forming unit-erythroid (CFU-E) and an overall increase in burst forming unit-erythroid (BFU-E) and CFU-E. Furthermore, the elevated sensitivity of Cbl deficient mice to anemia is echoed by increased EPO-R and protein kinase B (PKB)/Akt phosphorylation in splenic erythroblasts at high levels of EPO stimulation. Erythrocyte differentiation was examined by monitoring the expression of the erythroid markers CD71 (Transferrin Receptor) and Ter119. Cbl deficient mice do not have significantly more proerythroblasts than wild-type mice. Interestingly, Cbl deficient mice show impaired erythroid maturation with a 1.7 fold decrease in orthochromatophilic erythroblast levels, elevated erythroid apoptosis in the bone marrow, and no compensation by splenic erythroblast production. These data (as well as earlier studies from our laboratory with STAT1-/- and SHIP-1-/- mice) illustrate the remarkable ability of the spleen to compensate for alterations in bone marrow erythropoiesis. It also suggests that Cbl regulates pathway(s) associated with regulation of erythroid cell survival through regulation of the PI 3 kinase, PKB/Akt signaling cascade.

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Protein Kinase B and Extracellular Signal-Regulated Kinase Inactivation is Associated with Regorafenib-Induced Inhibition of Osteosarcoma Progression In Vitro and In Vivo

Journal of Clinical Medicine ◽

10.3390/jcm8060900 ◽

2019 ◽

Vol 8 (6) ◽

pp. 900 ◽

Cited By ~ 11

Author(s):

Po-Jung Pan ◽

Yu-Chang Liu ◽

Fei-Ting Hsu

Keyword(s):

Protein Kinase ◽

Kinase Inhibitor ◽

Protein Kinase B ◽

Akt Inhibitor ◽

Inhibitory Protein ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Apoptosis Protein

Osteosarcoma is the most common type of bone cancer. Multimodality treatment involving chemotherapy, radiotherapy and surgery is not effective enough to control osteosarcoma. Regorafenib, the oral multi-kinase inhibitor, has been shown to have positive efficacy on disease progression delay in chemotherapy resistant osteosarcoma patients. However anti-cancer effect and mechanism of regorafenib in osteosarcoma is ambiguous. Thus, the aim of this study is to investigate the efficacy and molecular mechanism of regorafenib on osteosarcoma in vitro and in vivo. Human osteosarcomas U-2 OS or MG-63 were treated with regorafenib, miltefosine (protein kinase B (AKT) inhibitor), or PD98059 (mitogen-activated protein/extracellular signal-regulated kinase (MEK) pathway inhibitor) for 24 or 48 h. Cell viability, apoptotic signaling transduction, tumor invasion, expression of tumor progression-associated proteins and tumor growth after regorafenib treatment were assayed by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, transwell assay, Western blotting assay and in vivo animal experiment, respectively. In these studies, we also indicated that regorafenib suppressed cell growth by prompting apoptosis of osteosarcoma cells, which is mediated through inactivation of ERK and AKT signaling pathways. After regorafenib treatment, downregulation of related genes in invasion (vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9)), proliferation (CyclinD1) and anti-apoptosis (X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia-1 (MCL-1), and cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (C-FLIP)) were found. Moreover, upregulation of caspase-3 and caspase-8 cleavage were also observed. In sum, we suggest that regorafenib has potential to suppress osteosarcoma progression via inactivation of AKT and ERK mediated signaling pathway.

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The Phosphoinositol-3-Kinase–Protein Kinase B/Akt Pathway Is Critical forPseudomonas aeruginosaStrain PAK Internalization

Molecular Biology of the Cell ◽

10.1091/mbc.e04-08-0717 ◽

2005 ◽

Vol 16 (5) ◽

pp. 2577-2585 ◽

Cited By ~ 91

Author(s):

A. Kierbel ◽

A. Gassama-Diagne ◽

K. Mostov ◽

J. N. Engel

Keyword(s):

Protein Kinase ◽

Epithelial Cells ◽

Mdck Cells ◽

Protein Kinase B ◽

Pi3k Signaling ◽

Dependent Manner ◽

Akt Phosphorylation ◽

Pi3k Signaling Pathway

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.

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Fucoidans from Cucumaria frondosa ameliorates renal interstitial fibrosis via inhibition of PI3K/Akt/NF-κB signaling pathway

Food & Function ◽

10.1039/d1fo03067a ◽

2022 ◽

Author(s):

Zhuo-yue Song ◽

Mengru Zhu ◽

Jun Wu ◽

Tian Yu ◽

Yao Chen ◽

...

Keyword(s):

Protein Kinase ◽

Signaling Pathway ◽

Interstitial Fibrosis ◽

Protein Kinase B ◽

Nuclear Factor Κb ◽

Nuclear Factor ◽

Renal Interstitial Fibrosis ◽

Cucumaria Frondosa

The effects of Cucumaria frondosa polysaccharides (CFP) on renal interstitial fibrosis via regulating phosphatidylinositol-3-hydroxykinase/protein kinase-B/Nuclear factor-κB (PI3K/AKT/NF-κB) signaling pathway were investigated in vivo and in vitro in this research. A...

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Inhibition of growth-factor-induced phosphorylation and activation of protein kinase B/Akt by atypical protein kinase C in breast cancer cells

Biochemical Journal ◽

10.1042/bj3520475 ◽

2000 ◽

Vol 352 (2) ◽

pp. 475-482 ◽

Cited By ~ 34

Author(s):

Muling MAO ◽

Xianjun FANG ◽

Yiling LU ◽

Ruth LAPUSHIN ◽

Robert C. BAST ◽

...

Keyword(s):

Breast Cancer ◽

Protein Kinase C ◽

Growth Factor ◽

Protein Kinase ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Protein Kinase B ◽

Akt Phosphorylation ◽

Kinase C ◽

Pkc Inhibitors

The protein kinase B/Akt serine/threonine kinase, located downstream of phosphoinositide 3-kinase (PI-3K), is a major regulator of cellular survival and proliferation. Atypical protein kinase C (aPKC) family members are activated by PI-3K and also contribute to cell proliferation, suggesting that Akt and aPKC might interact to activate signalling through the PI-3K cascade. Here we demonstrate that blocking PKC activity in MDA-MB-468 breast cancer cells increased the phosphorylation and activity of Akt. Functional PI-3K was required for the PKC inhibitors to increase Akt phosphorylation and activation, potentially owing to the activation of specific PKC isoforms by PI-3K. The concentration dependence of the action of the PKC inhibitors implicates aPKC in the inhibition of Akt phosphorylation and activity. In support of a role for aPKC in the regulation of Akt, Akt and PKCζ or PKCλ/ℓ were readily co-precipitated from the BT-549 breast cancer cell line. Furthermore, the overexpression of PKCζ inhibited growth-factor-induced increases in Akt phosphorylation and activity. Thus PKCζ associates physically with Akt and decreases Akt phosphorylation and enzyme activity. The effects of PKC on Akt were transmitted through the PI-3K cascade as indicated by changes in p70 s6 kinase (p70s6k) phosphorylation. Thus PKCζ, and potentially other PKC isoenzymes, regulate growth-factor-mediated Akt phosphorylation and activation, which is consistent with a generalized role for PKCζ in limiting growth factor signalling through the PI-3K/Akt pathway.

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Muscle Adaptation to Short-Term Fasting in Healthy Lean Humans

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2008-0250 ◽

2008 ◽

Vol 93 (7) ◽

pp. 2900-2903 ◽

Cited By ~ 24

Author(s):

Maarten R. Soeters ◽

Hans P. Sauerwein ◽

Peter F. Dubbelhuis ◽

Johanna E. Groener ◽

Mariëtte T. Ackermans ◽

...

Keyword(s):

Insulin Resistance ◽

Protein Kinase ◽

Glucose Uptake ◽

Protein Kinase B ◽

Muscle Adaptation ◽

Short Term ◽

Akt Phosphorylation ◽

Induce Insulin Resistance ◽

Before And After ◽

Muscle Biopsies

Abstract Context: It has been demonstrated repeatedly that short-term fasting induces insulin resistance, although the exact mechanism in humans is unknown to date. Intramyocellular sphingolipids (i.e. ceramide) have been suggested to induce insulin resistance by interfering with the insulin signaling cascade in obesity. Objective: Our objective was to study peripheral insulin sensitivity together with muscle ceramide concentrations and protein kinase B/AKT phosphorylation after short-term fasting. Main Outcome Measures and Design: After 14- and 62-h fasting, glucose fluxes were measured before and after a hyperinsulinemic euglycemic clamp. Muscle biopsies were performed in the basal state and during the clamp to assess muscle ceramide and protein kinase B/AKT. Results: Insulin-mediated peripheral glucose uptake was significantly lower after 62-h fasting compared with 14-h fasting. Intramuscular ceramide concentrations tended to increase during fasting. During the clamp the phosphorylation of protein kinase B/AKT at serine473 in proportion to the total amount of protein kinase B/AKT was significantly lower. Muscle ceramide did not correlate with plasma free fatty acids. Conclusions: Fasting for 62 h decreases insulin-mediated peripheral glucose uptake with lower phosphorylation of AKT at serine473. AKT may play a regulatory role in fasting-induced insulin resistance. Whether the decrease in AKT can be attributed to the trend to higher muscle ceramide remains unanswered.

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Domain Swapping Used To Investigate the Mechanism of Protein Kinase B Regulation by 3-Phosphoinositide-Dependent Protein Kinase 1 and Ser473 Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.5061 ◽

1999 ◽

Vol 19 (7) ◽

pp. 5061-5072 ◽

Cited By ~ 75

Author(s):

Mirjana Andjelković ◽

Sauveur-Michel Maira ◽

Peter Cron ◽

Peter J. Parker ◽

Brian A. Hemmings

Keyword(s):

Protein Kinase ◽

Kinase Inhibitors ◽

Protein Kinase B ◽

Second Messengers ◽

Activation Mechanism ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Phosphoinositide 3 Kinase ◽

Regulatory Sites

ABSTRACT Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.

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