Статті в журналах: "AKT PHOSPHORYLATION"

1

Ono, Hiraku, Hideyuki Sakoda, Midori Fujishiro, Motonobu Anai, Akifumi Kushiyama, Yasushi Fukushima, Hideki Katagiri, et al. "Carboxy-terminal modulator protein induces Akt phosphorylation and activation, thereby enhancing antiapoptotic, glycogen synthetic, and glucose uptake pathways." American Journal of Physiology-Cell Physiology 293, no. 5 (November 2007): C1576—C1585. http://dx.doi.org/10.1152/ajpcell.00570.2006.

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Carboxy-terminal modulator protein (CTMP) was identified as binding to the carboxy terminus of Akt and inhibiting the phosphorylation and activation of Akt. In contrast to a previous study, we found CTMP overexpression to significantly enhance Akt phosphorylation at both Thr308and Ser473as well as the kinase activity of Akt, while phosphatidylinositol 3-kinase (PI3-kinase) activity was unaffected. Translocation of Akt to the membrane fraction was also markedly increased in response to overexpression of CTMP, with no change in the whole cellular content of Akt. Furthermore, the phosphorylations of GSK-3β and Foxo1, well-known substrates of Akt, were increased by CTMP overexpression. On the other hand, suppression of CTMP with small interfering RNA partially but significantly attenuated this Akt phosphorylation. The cellular activities reportedly mediated by Akt activation were also enhanced by CTMP overexpression. UV-B-induced apoptosis of HeLa cells was significantly reversed not only by overexpression of the active mutant of Akt (myr-Akt) but also by that of CTMP. Increases in glucose transport activity and glycogen synthesis were also induced by overexpression of either myr-Akt or CTMP in 3T3-L1 adipocytes. Taking these results into consideration, it can be concluded that CTMP induces translocation of Akt to the membrane and thereby increases the level of Akt phosphorylation. As a result, CTMP enhances various cellular activities that are principally mediated by the PI3-kinase/Akt pathway.

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2

Hausenloy, Derek J., A. Tsang, Mihaela M. Mocanu, and Derek M. Yellon. "Ischemic preconditioning protects by activating prosurvival kinases at reperfusion." American Journal of Physiology-Heart and Circulatory Physiology 288, no. 2 (February 2005): H971—H976. http://dx.doi.org/10.1152/ajpheart.00374.2004.

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Pharmacological activation of the prosurvival kinases Akt and ERK-1/2 at reperfusion, after a period of lethal ischemia, protects the heart against ischemia-reperfusion injury. We hypothesized that ischemic preconditioning (IPC) protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion. In isolated perfused Sprague-Dawley rat hearts subjected to 35 min of lethal ischemia, the phosphorylation states of Akt, ERK-1/2, and p70 S6 kinase (p70S6K) were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a biphasic response in Akt and ERK-1/2 phosphorylation during the preconditioning and reperfusion phases after the period of lethal ischemia. IPC induced a fourfold increase in Akt, ERK-1/2, and p70S6K phosphorylation at reperfusion and reduced the infarct risk-to-volume ratio (56.9 ± 5.7 and 20.9 ± 3.6% for control and IPC, respectively, P < 0.01). Inhibiting the IPC-induced phosphorylation of Akt, ERK-1/2, and p70S6K at reperfusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 or the MEK-1/2 inhibitor PD-98059 abrogated IPC-induced protection (46.3 ± 5.8, 49.2 ± 4.0, and 20.9 ± 3.6% for IPC + LY-294002, IPC + PD-98059, and IPC, respectively, P < 0.01), demonstrating that the phosphorylation of these kinases at reperfusion is required for IPC-induced protection. In conclusion, we demonstrate that the reperfusion phase following sustained ischemia plays an essential role in mediating IPC-induced protection. Specifically, we demonstrate that IPC protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion.

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3

Ashcroft, Margaret, Robert L. Ludwig, Douglas B. Woods, Terry D. Copeland, H. Oliver Weber, Elizabeth J. MacRae, and Karen H. Vousden. "Phosphorylation of HDM2 by Akt." Oncogene 21, no. 13 (March 2002): 1955–62. http://dx.doi.org/10.1038/sj.onc.1205276.

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4

Kim, Albert H., Gus Khursigara, Xuan Sun, Thomas F. Franke, and Moses V. Chao. "Akt Phosphorylates and Negatively Regulates Apoptosis Signal-Regulating Kinase 1." Molecular and Cellular Biology 21, no. 3 (February 1, 2001): 893–901. http://dx.doi.org/10.1128/mcb.21.3.893-901.2001.

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ABSTRACT The Akt family of serine/threonine-directed kinases promotes cellular survival in part by phosphorylating and inhibiting death-inducing proteins. Here we describe a novel functional interaction between Akt and apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase. Akt decreased ASK1 kinase activity stimulated by both oxidative stress and overexpression in 293 cells by phosphorylating a consensus Akt site at serine 83 of ASK1. Activation of the phosphoinositide 3-kinase (PI3-K)/Akt pathway also inhibited the serum deprivation-induced activity of endogenous ASK1 in L929 cells. An association between Akt and ASK1 was detected in cells by coimmunoprecipitation. Phosphorylation by Akt inhibited ASK1-mediated c-Jun N-terminal kinase and activating transcription factor 2 activities in intact cells. Finally, activation of the PI3-K/Akt pathway reduced apoptosis induced by ASK1 in a manner dependent on phosphorylation of serine 83 of ASK1. These results provide the first direct link between Akt and the family of stress-activated kinases.

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5

Romic, Snjezana, Snezana Tepavcevic, Zorica Zakula, Tijana Milosavljevic, Mojca Stojiljkovic, Maja Zivkovic, Milan Popovic, Aleksandra Stankovic, and Goran Koricanac. "Does oestradiol attenuate the damaging effects of a fructose-rich diet on cardiac Akt/endothelial nitric oxide synthase signalling?" British Journal of Nutrition 109, no. 11 (October 16, 2012): 1940–48. http://dx.doi.org/10.1017/s0007114512004114.

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Fructose-rich diets (FRD) cause cardiac insulin resistance manifested by impairment of Akt/endothelial NO synthase (eNOS) signalling. In contrast, oestradiol (E2) activates this signalling pathway in the heart. To study the ability of E2 to revert the detrimental effect of fructose on cardiac Akt/eNOS, female rats were subjected to a FRD and ovariectomy followed with or without E2 replacement. We also analysed the effects of the FRD and E2 on cardiac extracellular signal-regulated kinase (Erk 1/2) signalling related to their role in cardiac hypertrophy development. Expression of Akt, eNOS and Erk 1/2, as well as regulatory phosphorylations of these molecules were determined. The protein expression of cardiac Akt and eNOS was not affected by the diet or E2 treatment. However, the FRD was accompanied by a decrease in Akt phosphorylation at Ser473 and Thr308, and eNOS at Ser1177, while the phosphorylation of eNOS at Thr495 was increased. E2 replacement in ovariectomised fructose-fed rats caused a reversion of the diet effect on Akt and eNOS serine phosphorylation, but mostly had no effect on threonine phosphorylation of the molecules. The FRD and E2 treatment did not influence Erk 1/2 expression and phosphorylation and heart mass as well. The data show that E2 selectively suppress the negative effects of a FRD on Akt/eNOS signalling and probably point to the different effects of E2 on kinase/phosphatase pathways responsible for phosphorylation/dephosphorylation of Akt and eNOS. Furthermore, the results suggest that the heart of females in the reproductive period is partially protected against the damaging effects of increased fructose intake.

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6

Dunn, Ewan F., Rachel Fearns, and John H. Connor. "Akt Inhibitor Akt-IV Blocks Virus Replication through an Akt-Independent Mechanism." Journal of Virology 83, no. 22 (September 9, 2009): 11665–72. http://dx.doi.org/10.1128/jvi.01092-09.

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ABSTRACT Many viruses activate the phosphatidylinositol 3′-kinase (PI3k)/Akt intracellular signaling pathway to promote viral replication. We have analyzed whether a rapidly replicating rhabdovirus, vesicular stomatitis virus (VSV), requires the PI3k/Akt signaling pathway for its replication. Through the use of chemical inhibitors of PI3k and Akt, we show that VSV replication and cytopathic effects do not require activation of these kinases. Inhibitors that block the activating phosphorylations of Akt at threonine 308 (Thr308) and serine 473 (Ser473) did not inhibit VSV protein expression or the induction of the cytopathic effects of VSV. One compound, Akt inhibitor Akt-IV, inhibited the replication of VSV, respiratory syncytial virus, and vaccinia virus but increased the phosphorylation of Akt at positions Thr308 and Ser473 and did not inhibit Akt kinase activity in vitro. Together, our data suggest that the PI3k/Akt pathway is of limited relevance to the replication of VSV but that Akt inhibitor Akt-IV is a novel broad-spectrum antiviral compound with a mechanism differing from that of its previously reported effect on the PI3k/Akt pathway. Identification of other targets for this compound may define a new approach for blocking virus replication.

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7

Kim, Ji-Eun, Duk-Shin Lee, Tae-Hyun Kim, Hana Park та Tae-Cheon Kang. "Distinct Roles of CK2- and AKT-Mediated NF-κB Phosphorylations in Clasmatodendrosis (Autophagic Astroglial Death) within the Hippocampus of Chronic Epilepsy Rats". Antioxidants 12, № 5 (28 квітня 2023): 1020. http://dx.doi.org/10.3390/antiox12051020.

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The downregulation of glutathione peroxidase-1 (GPx1) plays a role in clasmatodendrosis (an autophagic astroglial death) in the hippocampus of chronic epilepsy rats. Furthermore, N-acetylcysteine (NAC, a GSH precursor) restores GPx1 expression in clasmatodendritic astrocytes and alleviates this autophagic astroglial death, independent of nuclear factor erythroid-2-related factor 2 (Nrf2) activity. However, the regulatory signal pathways of these phenomena have not been fully explored. In the present study, NAC attenuated clasmatodendrosis by alleviating GPx1 downregulation, casein kinase 2 (CK2)-mediated nuclear factor-κB (NF-κB) serine (S) 529 and AKT-mediated NF-κB S536 phosphorylations. 2-[4,5,6,7-Tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazole-1-yl]acetic acid (TMCB; a selective CK2 inhibitor) relieved clasmatodendritic degeneration and GPx1 downregulation concomitant with the decreased NF-κB S529 and AKT S473 phosphorylations. In contrast, AKT inhibition by 3-chloroacetyl-indole (3CAI) ameliorated clasmatodendrosis and NF-κB S536 phosphorylation, while it did not affect GPx1 downregulation and CK2 tyrosine (Y) 255 and NF-κB S529 phosphorylations. Therefore, these findings suggest that seizure-induced oxidative stress may diminish GPx1 expression by increasing CK2-mediated NF-κB S529 phosphorylation, which would subsequently enhance AKT-mediated NF-κB S536 phosphorylation leading to autophagic astroglial degeneration.

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8

Xiang, Binggang, Guoying Zhang, Junling Liu, Andrew J. Morris, Susan S. Smyth, T. Kent Gartner, and Zhenyu Li. "A novel P2Y12-Independent Signaling Pathway Mediating Akt Phosphorylation In Response to Thrombin Receptors." Blood 116, no. 21 (November 19, 2010): 3191. http://dx.doi.org/10.1182/blood.v116.21.3191.3191.

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Abstract Abstract 3191 The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. It has been previously shown that Akt phosphorylation in response to thrombin receptors is dependent on Gi signaling activated primarily by secreted ADP through its receptor P2Y12. By using P2Y12 knockout mice combined with recombinant CHO models, we demonstrate a Gi-independent pathway for Akt phosphorylation in response to thrombin. Although Akt phosphorylation in response to low dose thrombin or the PAR4 thrombin receptor peptide AYPGKF was abolished in P2Y12 deficient platelets, at high concentrations, they stimulated substantial phosphorylation of both Akt residues Thr308 and Ser473 in P2Y12 deficient platelets, demonstrating that there are P2Y12-dependent and -independent pathways contributing to Akt phosphorylation in response to thrombin receptors. Thrombin or AYPGKF repressed forskolin-induced cAMP production in the wild type mouse platelets but not in the P2Y12 deficient platelets, suggesting that Gi activation by thrombin or AYPGKF is dependent on ADP receptor P2Y12. Therefore, thrombin- or AYPGKF-induced Akt phosphorylation in P2Y12 deficient platelets is Gi independent. AYPGKF-induced Akt phosphorylation was enhanced by expression of recombinant PAR4 cDNA in CHO cells, demonstrating that stimulation of thrombin receptor PAR4 is able to elicit Akt phosphorylation in the absence of platelet secretion. It is unlikely that PAR4-induced Akt phosphorylation in CHO cells involves the P2Y12 pathway, because CHO cells apparently do not express functional P2Y12. This conclusion is supported by the observation that ADP failed to stimulate Akt phosphorylation and inhibit forskolin-induced cAMP production in CHO cells. Unlike thrombin, U46619-induced Akt phosphorylation was dramatically decreased by P2Y12 deficiency, suggesting that TXA2-induced Akt phosphorylation is largely P2Y12 dependent. In addition, P2Y12-independent Akt phosphorylation was not inhibited by the integrin inhibitor peptide RGDS or integrin β3 deficiency, demonstrating that integrin αIIbβ3 outside-in signaling is not required for thrombin-induced, P2Y12-independent, Akt phosphorylation. Furthermore, Akt phosphorylation in response to thrombin or AYPGKF in P2Y12 deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, or PI3K inhibitors, but was not affected by PKC inhibitors. Thus, our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors. Disclosures: No relevant conflicts of interest to declare.

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9

Kim, Soochong, Jianguo Jin, and Satya P. Kunapuli. "Relative contribution of G-protein-coupled pathways to protease-activated receptor-mediated Akt phosphorylation in platelets." Blood 107, no. 3 (February 1, 2006): 947–54. http://dx.doi.org/10.1182/blood-2005-07-3040.

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AbstractProtease-activated receptors (PARs) activate Gq and G12/13 pathways, as well as Akt (protein kinase B [PKB/Akt]) in platelets. However, the relative contribution of different G-protein pathways to Akt phosphorylation has not been elucidated. We investigated the contribution of Gq and G12/13 to Gi/Gz-mediated Akt phosphorylation downstream of PAR activation. Selective G12/13 activation failed to cause Akt phosphorylation in human and Gαq-deficient mouse platelets. However, supplementing Gi/Gz signaling to G12/13 caused significant increase in Akt phosphorylation, confirming that G12/13 potentiates Akt phosphorylation. Inhibition of PAR-mediated Akt phosphorylation in the presence of the Gq-selective inhibitor YM-254890 was restored to the normal extent achieved by PAR agonists if supplemented with Gi signaling, indicating that Gq does not have any direct effect on Akt phosphorylation. Selective G12/13 activation resulted in Src kinase activation, and Akt phosphorylation induced by costimulation of G12/13 and Gi/Gz was inhibited by a Src kinase inhibitor but not by a Rho kinase inhibitor. These data demonstrate that G12/13, but not Gq, is essential for thrombin-induced Akt phosphorylation in platelets, whereas Gq indirectly contributes to Akt phosphorylation through Gi stimulation by secreted ADP. G12/13 activation might mediate its potentiating effect through Src activation, and Src kinases play an important role in thrombin-mediated Akt phosphorylation.

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10

Gayer, Christopher P., Lakshmi S. Chaturvedi, Shouye Wang, Brittany Alston, Thomas L. Flanigan, and Marc D. Basson. "Delineating the signals by which repetitive deformation stimulates intestinal epithelial migration across fibronectin." American Journal of Physiology-Gastrointestinal and Liver Physiology 296, no. 4 (April 2009): G876—G885. http://dx.doi.org/10.1152/ajpgi.90648.2008.

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Repetitive strain stimulates intestinal epithelial migration across fibronectin via focal adhesion kinase (FAK), Src, and extracellular signal-related kinase (ERK) although how these signals act and interact remains unclear. We hypothesized that PI3K is central to this pathway. We subjected Caco-2 and intestinal epithelial cell-6 cells to 10 cycles/min deformation on flexible fibronectin-coated membranes, assayed migration by wound closure, and signaling by immunoblots. Strain stimulated PI3K, AKT, glycogen synthase kinase (GSK), and p38 phosphorylation. Blocking each kinase prevented strain stimulation of migration. Blocking PI3K prevented strain-stimulated ERK and p38 phosphorylation. Blocking AKT did not. Downstream, blocking PI3K, AKT, or ERK inhibited strain-induced GSK-Ser9 phosphorylation. Upstream of AKT, reducing FAK or Rac1 by siRNA blocked strain-stimulated AKT phosphorylation, but inhibiting Src by PP2 or siRNA did not. Transfection with FAK point mutants at Tyr397, Tyr576/577, or Tyr925 demonstrated that only FAK925 phosphorylation is required for strain-stimulated AKT phosphorylation. Myosin light chain activation by strain required FAK, Rac1, PI3K, AKT, GSK, and ERK but not Src or p38. Finally, blebbistatin, a nonmuscle myosin II inhibitor, blocked the motogenic effect of strain downstream of myosin light chain. Thus strain stimulates intestinal epithelial migration across fibronectin by a complex pathway including Src, FAK, Rac1, PI3K, AKT, GSK, ERK, p38, myosin light chain, and myosin II.

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11

Wei, Yingze, Jianyun Zhou, Haiyan Yu, and Xiaoxia Jin. "AKT phosphorylation sites of Ser473 and Thr308 regulate AKT degradation." Bioscience, Biotechnology, and Biochemistry 83, no. 3 (November 29, 2018): 429–35. http://dx.doi.org/10.1080/09168451.2018.1549974.

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12

Kim, Soochong, Jianguo Jin, and Satya P. Kunapuli. "G12/13 Pathways Potentiate Akt Phosphorylation in Platelets Mediated by Gi/Gz Pathways in a Src Kinase-Dependent Manner." Blood 104, no. 11 (November 16, 2004): 1570. http://dx.doi.org/10.1182/blood.v104.11.1570.1570.

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Akt is a serine/threonine kinase that is activated by various agonists including thrombin and ADP in platelets, and activation of Akt in platelets is known to require Gi signaling pathways. Even though thrombin-induced Akt phosphorylation depends on secretion/Gi pathways, thrombin caused much stronger Akt phosphorylation than ADP and epinephrine. In this study, we investigated the contribution of G12/13 pathways to Akt phosphorylation mediated by Gi or Gz pathways. We used selective agonists to activate different G protein pathways. PAR4-activating peptide (AYPGKF) and thrombin failed to induce Akt phosphorylation in Gαq-deficient platelets, but Akt phosphorylation was restored to the levels achieved by AYPGKF and thrombin in wild-type platelets by selective supplement of either Gi or Gz signaling with 2-MeSADP and epinephrine, respectively. This phosphorylation of Akt was dramatically inhibited in the presence of PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src family tyrosine kinase, but not by PP3, an inactive structural analog. Importantly, AYPGKF and thrombin induced the activation of Src kinase in Gαq-deficient platelets, suggesting the involvement of Src-dependent pathways in the G12/13 signaling in potentiation of Akt phosphorylation. When human platelets were stimulated with low concentrations of YFLLRNP, a PAR1-specific partial agonist, to selectively activate the G12/13 signaling cascade, Akt phosphorylation did not occur. However, combined stimulation of YFLLRNP-mediated G12/13 signaling and selective activation of Gi pathways caused the Akt phosphorylation. This Akt phosphorylation was blocked by the P2Y12 receptor antagonist AR-C69931MX, or the PI 3-kinase inhibitor LY294002. Platelet aggregation induced by co-activation of both G12/13 and Gi signaling was dramatically inhibited by ML-9 (1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride), an Akt selective inhibitor, suggesting an important role of Akt in platelet aggregation stimulated by combined G12/13 and Gi pathways. These data demonstrate that G12/13 signaling alone does not cause Akt phosphorylation in platelets, but G12/13 signaling has a significant role in potentiating Akt phosphorylation mediated by selective Gi or Gz signaling in human as well as mouse platelets. Finally, we conclude that Src family kinases play an important role in this Akt phosphorylation in platelets.

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13

Hart, Jonathan R., and Peter K. Vogt. "Phosphorylation of AKT: a Mutational Analysis." Oncotarget 2, no. 6 (June 10, 2011): 467–76. http://dx.doi.org/10.18632/oncotarget.293.

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14

BASINI, G., S. E. SANTINI, S. BUSSOLATI, and F. GRASSELLI. "Sanguinarine Inhibits VEGF-Induced Akt Phosphorylation." Annals of the New York Academy of Sciences 1095, no. 1 (January 1, 2007): 371–76. http://dx.doi.org/10.1196/annals.1397.040.

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15

Burchfield, James G., Alecia J. Lennard, Sakura Narasimhan, William E. Hughes, Valerie C. Wasinger, Garry L. Corthals, Tomohiko Okuda, Hisato Kondoh, Trevor J. Biden, and Carsten Schmitz-Peiffer. "Akt Mediates Insulin-stimulated Phosphorylation ofNdrg2." Journal of Biological Chemistry 279, no. 18 (February 24, 2004): 18623–32. http://dx.doi.org/10.1074/jbc.m401504200.

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16

Bayascas, Jose R., and Dario R. Alessi. "Regulation of Akt/PKB Ser473 Phosphorylation." Molecular Cell 18, no. 2 (April 2005): 143–45. http://dx.doi.org/10.1016/j.molcel.2005.03.020.

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17

Su, Chih-Hao, Keng-Hsin Lan, Chung-Pin Li, Yee Chao, Han-Chieh Lin, Shou-Dong Lee, and Wei-Ping Lee. "Phosphorylation accelerates geldanamycin-induced Akt degradation." Archives of Biochemistry and Biophysics 536, no. 1 (August 2013): 6–11. http://dx.doi.org/10.1016/j.abb.2013.04.015.

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18

Gosmanov, Aidar R., Guillermo E. Umpierrez, Ana H. Karabell, Ruben Cuervo, and Donald B. Thomason. "Impaired expression and insulin-stimulated phosphorylation of Akt-2 in muscle of obese patients with atypical diabetes." American Journal of Physiology-Endocrinology and Metabolism 287, no. 1 (July 2004): E8—E15. http://dx.doi.org/10.1152/ajpendo.00485.2003.

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Although a pharmacological dose of insulin produces a dramatic increase in phosphorylation and activity of Akt isoforms 1 and 2 in mammalian skeletal muscle, few studies have examined the effect of physiological concentrations of insulin on the phosphorylation of Akt-1 and -2 in normal and diabetic tissue. This study examined the patterns of insulin-stimulated Akt isoform phosphorylation and protein expression in muscle biopsies obtained from obese patients with atypical diabetes immediately after a hyperglycemic crisis and again after near-normoglycemic remission. In obese patients with new-onset diabetes mellitus presenting with hyperglycemic crisis (plasma glucose 30.5 ± 4.8 mM), in vitro stimulation of vastus lateralis muscle biopsies with 100 μU/ml (0.6 nM) insulin increased insulin receptor phosphorylation threefold and Akt-1 phosphorylation on Ser473 twofold, whereas Akt-2 phosphorylation was not stimulated. After 10-wk intensive insulin therapy that led to near-normoglycemic remission and discontinuation of insulin therapy, both Akt-2 expression and insulin-stimulated Akt-2 Ser474 phosphorylation doubled. Hyperglycemic crisis did not affect insulin-stimulated threonine phosphorylation of either Akt-1 or Akt-2. The decreased Akt-2 expression at presentation was accompanied by reduced GLUT4 protein expression and increased expression of enzymes counterregulatory to insulin action. Thus a physiological concentration of insulin stimulated Akt-1 and Akt-2 phosphorylation in human skeletal muscle in the absence of hyperglycemia, but Akt-2 expression and stimulation appeared to be impaired in muscle of obese patients with atypical diabetes presenting with severe hyperglycemia.

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19

Sharma, Naveen, Edward B. Arias, Abhijit D. Bhat, Donel A. Sequea, Steve Ho, Kelsey K. Croff, Mini P. Sajan, Robert V. Farese, and Gregory D. Cartee. "Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats." American Journal of Physiology-Endocrinology and Metabolism 300, no. 6 (June 2011): E966—E978. http://dx.doi.org/10.1152/ajpendo.00659.2010.

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Calorie restriction [CR; ∼65% of ad libitum (AL) intake] improves insulin-stimulated glucose uptake (GU) and Akt phosphorylation in skeletal muscle. We aimed to elucidate the effects of CR on 1) processes that regulate Akt phosphorylation [insulin receptor (IR) tyrosine phosphorylation, IR substrate 1-phosphatidylinositol 3-kinase (IRS-PI3K) activity, and Akt binding to regulatory proteins (heat shock protein 90, Appl1, protein phosphatase 2A)]; 2) Akt substrate of 160-kDa (AS160) phosphorylation on key phosphorylation sites; and 3) atypical PKC (aPKC) activity. Isolated epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles from AL or CR (6 mo duration) 9-mo-old male F344BN rats were incubated with 0, 1.2, or 30 nM insulin and 2-deoxy-[3H]glucose. Some CR effects were independent of insulin dose or muscle type: CR caused activation of Akt (Thr308and Ser473) and GU in both muscles at both insulin doses without CR effects on IRS1-PI3K, Akt-PP2A, or Akt-Appl1. Several muscle- and insulin dose-specific CR effects were revealed. Akt-HSP90 binding was increased in the epitrochlearis; AS160 phosphorylation (Ser588and Thr642) was greater for CR epitrochlearis at 1.2 nM insulin; and IR phosphorylation and aPKC activity were greater for CR in both muscles with 30 nM insulin. On the basis of these data, our working hypothesis for improved insulin-stimulated GU with CR is as follows: 1) elevated Akt phosphorylation is fundamental, regardless of muscle or insulin dose; 2) altered Akt binding to regulatory proteins (HSP90 and unidentified Akt partners) is involved in the effects of CR on Akt phosphorylation; 3) Akt effects on GU depend on muscle- and insulin dose-specific elevation in phosphorylation of Akt substrates, including, but not limited to, AS160; and 4) greater IR phosphorylation and aPKC activity may contribute at higher insulin doses.

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20

Hiraoka, D., E. Okumura, and T. Kishimoto. "Turn motif phosphorylation negatively regulates activation loop phosphorylation in Akt." Oncogene 30, no. 44 (May 16, 2011): 4487–97. http://dx.doi.org/10.1038/onc.2011.155.

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21

Ding, Jixin, and Keyong Du. "ClipR-59 Interacts with Akt and Regulates Akt Cellular Compartmentalization." Molecular and Cellular Biology 29, no. 6 (January 12, 2009): 1459–71. http://dx.doi.org/10.1128/mcb.00754-08.

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ABSTRACT Akt is activated on the plasma membrane and its substrates are distributed throughout various cellular compartments. To phosphorylate its substrates, Akt needs to be recruited to specific intracellular compartments. Thus, regulation of Akt cellular compartmentalization constitutes an important mechanism to specify Akt signaling. Here, we report the identification of ClipR-59 as an Akt interaction protein. We show that the interaction of ClipR-59 with Akt is mediated by the CAP-Gly domain of ClipR-59 and kinase domain of Akt and is regulated by Akt phosphorylation. We demonstrate that ClipR-59 regulates the Akt membrane association through its interaction with Akt and membrane localization and, by modulating Akt cellular compartmentalization, differentially modulates phosphorylation of Akt substrates in adipocytes. Finally, we provide evidence that one of the Akt substrates whose phosphorylation is upregulated by ClipR-59 is AS160, a negative regulator of adipocyte glucose transport. Accordingly, ectopic expression of ClipR-59 enhances, whereas knockdown of ClipR-59 suppresses, adipocyte glucose transport. We suggest that ClipR-59 functions as a scaffold protein that interacts with phospho-Akt and recruits active Akt on the membrane and may play an important role in adipocyte glucose transport.

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22

Lee, Jin Hee, and Louis Ragolia. "AKT phosphorylation is essential for insulin-induced relaxation of rat vascular smooth muscle cells." American Journal of Physiology-Cell Physiology 291, no. 6 (December 2006): C1355—C1365. http://dx.doi.org/10.1152/ajpcell.00125.2006.

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Insulin resistance, a major factor in the development of type 2 diabetes, is known to be associated with defects in blood vessel relaxation. The role of Akt on insulin-induced relaxation of vascular smooth muscle cell (VSMC) was investigated using siRNA targeting Akt (siAKTc) and adenovirus constructing myristilated Akt to either suppress endogenous Akt or overexpress constitutively active Akt, respectively. siAKTc decreased both basal and insulin-induced phosphorylations of Akt and glycogen synthase kinase 3β, abolishing insulin-induced nitric oxide synthase (iNOS) expression. cGMP-dependent kinase 1α (cGK1α) and myosin-bound phosphatase (MBP) activities, both downstream of iNOS, were also decreased. siAKTc treatment resulted in increased insulin and ANG II-stimulated phosphorylation of contractile apparatus, such as MBP substrate (MYPT1) and myosin light chain (MLC20), accompanied by increased Rho-associated kinase α (ROKα) activity, demonstrating the requirement of Akt for insulin-induced vasorelaxation. Corroborating these results, constitutively active Akt upregulated the signaling molecules involved in insulin-induced relaxation such as iNOS, cGK1α, and MBP activity, even in the absence of insulin stimulation. On the contrary, the contractile response involving the phosphorylation of MYPT1 and MLC20, and increased ROKα activity stimulated by ANG II were all abolished by overexpressing active Akt. In conclusion, we demonstrated here that insulin-induced VSMC relaxation is dependent on Akt activation via iNOS, cGK1α, and MBP activation, as well as the decreased phosphorylations of MYPT1 and MLC20 and decreased ROKα activity.

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Lawlor, Margaret A., and Dario R. Alessi. "PKB/Akt." Journal of Cell Science 114, no. 16 (August 15, 2001): 2903–10. http://dx.doi.org/10.1242/jcs.114.16.2903.

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The serine/threonine protein kinase PKB (also known as Akt) is thought to be a key mediator of signal transduction processes. The identification of PKB substrates and the role PKB phosphorylation plays in regulating these molecules have been a major focus of research in recent years. A recently developed motif-profile scoring algorithm that can be used to scan the genome for potential PKB substrates is therefore a useful tool, although additional considerations, such as the evolutionary conservation of the phosphorylation site, must also be taken into account. Recent evidence indicates that PKB plays a key role in cancer progression by stimulating cell proliferation and inhibiting apoptosis and is also probably a key mediator of insulin signalling. These findings indicate that PKB is likely to be a hot drug target for the treatment of cancer, diabetes and stroke. There are, however, a number of pitfalls of methodologies currently employed to study PKB function, and therefore caution should be used in interpretation of such experiments.

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24

Song, Ping, Yong Wu, Jian Xu, Zhonglin Xie, Yunzhou Dong, Miao Zhang, and Ming-Hui Zou. "Reactive Nitrogen Species Induced by Hyperglycemia Suppresses Akt Signaling and Triggers Apoptosis by Upregulating Phosphatase PTEN (Phosphatase and Tensin Homologue Deleted on Chromosome 10) in an LKB1-Dependent Manner." Circulation 116, no. 14 (October 2, 2007): 1585–95. http://dx.doi.org/10.1161/circulationaha.107.716498.

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Background— Oxidative stress plays a causal role in vascular injury in diabetes mellitus, but the mechanisms and targets remain poorly understood. Methods and Results— Exposure of cultured human umbilical vein endothelial cells to either peroxynitrite (ONOO − ) or high glucose significantly inhibited both basal and insulin-stimulated Akt phosphorylation at Ser473 and Akt activity in parallel with increased apoptosis, phosphorylation, and activity of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Furthermore, protein kinase B/Akt inhibition induced by ONOO − or high glucose and apoptosis triggered by high glucose could be abolished by transfection of PTEN-specific small interfering RNA, suggesting that PTEN mediated the Akt inhibition by ONOO − . In addition, exposure of human umbilical vein endothelial cells to ONOO − or high glucose remarkably increased Ser428 phosphorylation of LKB1, a tumor suppressor. Interestingly, the ONOO − -enhanced PTEN phosphorylation and Akt inhibition can be blocked by LKB1-specific small interfering RNA. Consistently, LKB1 phosphorylated PTEN at Ser380/Thr382/383 in vitro, suggesting that LKB1 might act as an upstream kinase for PTEN. Compared with nondiabetic mice, the levels of PTEN, LKB1-Ser428 phosphorylation, and 3-nitrotyrosine (a biomarker of ONOO − ) were significantly increased in the aortas of streptozotocin-induced diabetic mice, which was in parallel with a reduction in Akt-Ser473 phosphorylation and an increase in apoptosis. Furthermore, administration of PTEN-specific small interfering RNA suppressed diabetes-enhanced apoptosis and Akt inhibition. Finally, treatment with Tempol, a superoxide dismutase mimetic, and insulin, both of which reduced the ONOO − formation, markedly reduced diabetes-enhanced LKB1-Ser428 phosphorylation, PTEN, and apoptosis in the endothelium of mouse aortas. Conclusion— We conclude that hyperglycemia triggers apoptosis by inhibiting Akt signaling via ONOO − -mediated LKB1-dependent PTEN activation.

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25

MAO, Muling, Xianjun FANG, Yiling LU, Ruth LAPUSHIN, Robert C. BAST, and Gordon B. MILLS. "Inhibition of growth-factor-induced phosphorylation and activation of protein kinase B/Akt by atypical protein kinase C in breast cancer cells." Biochemical Journal 352, no. 2 (November 24, 2000): 475–82. http://dx.doi.org/10.1042/bj3520475.

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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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26

Imai, Norikazu, Masato Shikami, Hiroshi Miwa, Akiko Hattori, Akihito Hiramatsu, Masaya Watarai, Atsushi Satoh, et al. "Serum Dependency of t(8;21) AML Cell Line Is Associated with VEGF/VEGFR Pathway and Early Phosphorylation of Akt." Blood 106, no. 11 (November 16, 2005): 4571. http://dx.doi.org/10.1182/blood.v106.11.4571.4571.

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Abstract Most human leukemia cell lines are dependent on serum supplementation (usually fetal calf serum (FCS)), although the extent of serum dependency differs among each cell line. Kasumi-1, a t(8;21) AML cell line is one of the most serum-dependent cell lines. Since growth and survival of many leukemia cell lines are associated with phosphorylation of Akt, we examined the Akt phosphorylation by FCS treatment. In Kasumi-1, Akt was phosphorylated by culture with FCS in a dose-dependent manner, although no such Akt phosphorylation was observed in NB-4, a t(15;17) cell line. By FCS stimulation, Akt (Thr308, Ser473) was phosphorylated from 0.5 hr and the phosphorylation sustained until 48 hours in Kasumi-1. Then, we tested the effect of VEGF/VEGFR signaling in phosphorylation of Akt by FCS. The addition of VEGFR1/Fc and VEGFR2/Fc (which bind external VEGF and abrogate its function) inhibited the Akt phosphorylation from 2 hours until 10 hours, although the growth of Kasumi-1 was not inhibited. The addition of VEGFR2 kinase inhibitor (which inhibits internal VEGF signal) inhibited the Akt phosphorylation from 0.5 hr until 2 hours, and the growth of Kasumi-1 was greatly inhibited. Taken together, it is suggested that serum dependency of Kasumi-1 is at least in part attributed to VEGF/VEGFR pathway. Then, both external and internal VEGF/VEGFR pathways work in Kasumi-1, which in turn phosphorylate Akt. However, blockade of only internal VEGF signal (by VEGFR2 kinase inhibitor) inhibit the early Akt phosphorylation (0.5 hr), which resulted in growth inhibition, indicating the importance of early Akt phosphorylation.

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Katayama, Kazuhiro, Naoya Fujita, and Takashi Tsuruo. "Akt/Protein Kinase B-Dependent Phosphorylation and Inactivation of WEE1Hu Promote Cell Cycle Progression at G2/M Transition." Molecular and Cellular Biology 25, no. 13 (July 1, 2005): 5725–37. http://dx.doi.org/10.1128/mcb.25.13.5725-5737.2005.

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ABSTRACT The serine/threonine kinase Akt is known to promote cell growth by regulating the cell cycle in G1 phase through activation of cyclin/Cdk kinases and inactivation of Cdk inhibitors. However, how the G2/M phase is regulated by Akt remains unclear. Here, we show that Akt counteracts the function of WEE1Hu. Inactivation of Akt by chemotherapeutic drugs or the phosphatidylinositide-3-OH kinase inhibitor LY294002 induced G2/M arrest together with the inhibitory phosphorylation of Cdc2. Because the increased Cdc2 phosphorylation was completely suppressed by wee1hu gene silencing, WEE1Hu was associated with G2/M arrest induced by Akt inactivation. Further analyses revealed that Akt directly bound to and phosphorylated WEE1Hu during the S to G2 phase. Serine-642 was identified as an Akt-dependent phosphorylation site. WEE1Hu kinase activity was not affected by serine-642 phosphorylation. We revealed that serine-642 phosphorylation promoted cytoplasmic localization of WEE1Hu. The nuclear-to-cytoplasmic translocation was mediated by phosphorylation-dependent WEE1Hu binding to 14-3-3θ but not 14-3-3β or -σ. These results indicate that Akt promotes G2/M cell cycle progression by inducing phosphorylation-dependent 14-3-3θ binding and cytoplasmic localization of WEE1Hu.

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28

Khalil, Md Imtiaz, Christopher Madere, Ishita Ghosh, Rosalyn M. Adam, and Arrigo De Benedetti. "Interaction of TLK1 and AKTIP as a Potential Regulator of AKT Activation in Castration-Resistant Prostate Cancer Progression." Pathophysiology 28, no. 3 (July 20, 2021): 339–54. http://dx.doi.org/10.3390/pathophysiology28030023.

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Prostate cancer (PCa) progression is characterized by the emergence of resistance to androgen deprivation therapy (ADT). AKT/PKB has been directly implicated in PCa progression, often due to the loss of PTEN and activation of PI3K>PDK1>AKT signaling. However, the regulatory network of AKT remains incompletely defined. Here, we describe the functional significance of AKTIP in PCa cell growth. AKTIP, identified in an interactome analysis as a substrate of TLK1B (that itself is elevated following ADT), enhances the association of AKT with PDK1 and its phosphorylation at T308 and S473. The interaction between TLK1 and AKTIP led to AKTIP phosphorylation at T22 and S237. The inactivation of TLK1 led to reduced AKT phosphorylation, which was potentiated with AKTIP knockdown. The TLK1 inhibitor J54 inhibited the growth of the LNCaP cells attributed to reduced AKT activation. However, LNCaP cells that expressed constitutively active, membrane-enriched Myr-AKT (which is expected to be active, even in the absence of AKTIP) were also growth-inhibited with J54. This suggested that other pathways (like TLK1>NEK1>YAP) regulating proliferation are also suppressed and can mediate growth inhibition, despite compensation by Myr-AKT. Nonetheless, further investigation of the potential role of TLK1>AKTIP>AKT in suppressing apoptosis, and conversely its reversal with J54, is warranted.

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29

Li, Fang, and Kafait U. Malik. "Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2+-dependent phospholipase A2." American Journal of Physiology-Heart and Circulatory Physiology 288, no. 5 (May 2005): H2306—H2316. http://dx.doi.org/10.1152/ajpheart.00571.2004.

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Angiotensin II (ANG II) promotes vascular smooth muscle cell (VSMC) growth, stimulates Ca2+-calmodulin (CaM)-dependent kinase II (CaMKII), and activates cytosolic Ca2+-dependent phospholipase A2 (cPLA2), which releases arachidonic acid (AA). ANG II also generates H2O2 and activates Akt, which have been implicated in ANG II actions in VSMC. This study was conducted to investigate the relationship of these signaling molecules to Akt activation in rat aortic VSMC. ANG II increased Akt activity, as measured by its phosphorylation at serine-473. ANG II (200 nM)-induced Akt phosphorylation was decreased by extracellular Ca2+ depletion and calcium chelator EGTA and inhibitors of CaM [ N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and CaMKII {(2-[ N-(2-hydroxyethyl)]- N-(4-me-thoxybenzenesulfonyl)]amino- N-(4-chlorocinnamyl)- N-methylbenzyl-amine)}. cPLA2 inhibitor pyrrolidine-1, antisense oligonucleotide, and retroviral small interfering RNA also attenuated ANG II-induced Akt phosphorylation. AA increased Akt phosphorylation, and AA metabolism inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) blocked ANG II- and AA-induced Akt phosphorylation (199.03 ± 27.91% with ANG II and 110.18 ± 22.40% with ETYA + ANG II; 405.00 ± 86.22% with AA and 153.97 ± 63.26% with ETYA + AA). Inhibitors of lipoxygenase (cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and cytochrome P-450 (ketoconazole and 17-octadecynoic acid), but not cyclooxygenase (indomethacin), attenuated ANG II- and AA-induced Akt phosphorylation. Furthermore, 5( S)-, 12( S)-, 15( S)-, and 20-hydroxyeicosatetraenoic acids and 5,6-, 11,12-, and 14,15-epoxyeicosatrienoic acids increased Akt phosphorylation. Catalase inhibited ANG II-increased H2O2 production but not Akt phosphorylation. Oleic acid, which also increased H2O2 production, did not cause Akt phosphorylation. These data suggest that ANG II-induced Akt activation in VSMC is mediated by AA metabolites, most likely generated via lipoxygenase and cytochrome P-450 consequent to AA released by CaMKII-activated cPLA2 and independent of H2O2 production.

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Arias, Edward B., Junghoon Kim, Katsuhiko Funai, and Gregory D. Cartee. "Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 292, no. 4 (April 2007): E1191—E1200. http://dx.doi.org/10.1152/ajpendo.00602.2006.

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The main purpose of this study was to determine whether the increased glucose transport (GT) found immediately postexercise (IPEX) or 4 h postexercise (4hPEX) is accompanied by increased phosphorylation of Akt substrate of 160 kDa (AS160, a protein regulator of GLUT4 translocation). Paired epitrochlearis muscles were dissected from rats (sedentary or IPEX, 2-h swim) and used to measure protein phosphorylation and insulin-independent GT. IPEX values exceeded sedentary values for GT and phosphorylations of AS160, AMP-activated protein kinase (pAMPK) and acetyl-CoA carboxylase (pACC) but not for AS160 abundance or phosphorylation of Akt serine (pSerAkt), Akt threonine (pThrAkt), or glycogen synthase kinase-3 (pGSK3). AS160 phosphorylation was significantly correlated with GT ( R = 0.801, P < 0.01) and pAMPK ( R = 0.655, P < 0.05). Muscles from other rats were studied 4hPEX along with sedentary controls. One muscle per rat was incubated without insulin, and the contralateral muscle was incubated with insulin. 4hPEX values exceeded sedentary values for insulin-stimulated GT. The elevated pAMPK and pACC found IPEX had reversed by 4hPEX. Insulin caused a significant increase in pSerAkt, pThrAkt, pGSK3, and AS160 phosphorylation with or without exercise. Exercise significantly increased AS160 phosphorylation, regardless of insulin, with unchanged AS160 abundance. Among the signaling proteins studied, insulin-stimulated GT was significantly correlated only with insulin-stimulated pThrAkt ( R = 0.720, P < 0.0005). The results are consistent with a role for increased AS160 phosphorylation in the increased insulin-independent GT IPEX, and the exercise effects on AS160 phosphorylation and/or pThrAkt at 4hPEX are potentially relevant to the increased insulin-stimulated glucose transport at this time.

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31

Korkmaz, Y., W. Bloch, D. Steinritz, M. A. Baumann, K. Addicks, K. Schneider, and W. H. M. Raab. "Bradykinin Mediates Phosphorylation of eNOS in Odontoblasts." Journal of Dental Research 85, no. 6 (June 2006): 536–41. http://dx.doi.org/10.1177/154405910608500611.

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While the activation of eNOS by Akt/PKB-dependent phosphorylation, leading to NO release, and the inhibition of enzyme activity by bradykinin (BK)-mediated phosphorylation of eNOS in endothelial cells are established, the phosphorylation of eNOS in odontoblasts is unknown. To clarify the regulation of eNOS in odontoblasts by BK, we examined the phosphorylation of eNOS, Akt/PKB, and ERK1/2 in odontoblasts of rat molars. BK (10−7 M) transiently induced the phosphorylation of eNOS at Ser1177, Akt/PKB in odontoblasts, while it induced the phosphorylation of eNOS at Thr495 throughout the entire period of BK treatment. BK receptor 2 antagonist HOE 140 (10−6 M) significantly reduced signal intensities of phosphorylated-eNOS at Ser1177, Thr495, and phosphorylated-Akt/PKB. These results suggest that BK has dual effects on the activation of eNOS in odontoblasts, the Akt/PKB-dependent up-regulation of eNOS by the transient phosphorylation at Ser1177, and the ERK1/2-independent down-regulation of eNOS by the phosphorylation at Thr495.

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32

Liu, Pengda, Zhiwei Wang, and Wenyi Wei. "Phosphorylation of Akt at the C-terminal tail triggers Akt Activation." Cell Cycle 13, no. 14 (June 16, 2014): 2162–64. http://dx.doi.org/10.4161/cc.29584.

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Khundmiri, Syed J., Vishal Amin, Jeff Henson, John Lewis, Mohamed Ameen, Madhavi J. Rane, and Nicholas A. Delamere. "Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway." American Journal of Physiology-Cell Physiology 293, no. 3 (September 2007): C1171—C1180. http://dx.doi.org/10.1152/ajpcell.00535.2006.

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Endogenous cardiotonic glycosides bind to the inhibitory binding site of the plasma membrane sodium pump (Na+/K+-ATPase). Plasma levels of endogenous cardiotonic glycosides increase in several disease states, such as essential hypertension and uremia. Low concentrations of ouabain, which do not inhibit Na+/K+-ATPase, induce cell proliferation. The mechanisms of ouabain-mediated response remain unclear. Recently, we demonstrated that in opossum kidney (OK) proximal tubular cells, low concentrations of ouabain induce cell proliferation through phosphorylation of protein kinase B (Akt) in a calcium-dependent manner. In the present study, we identified ERK as an upstream kinase regulating Akt activation in ouabain-stimulated cells. Furthermore, we provide evidence that low concentrations of ouabain stimulate Na+/K+-ATPase-mediated 86Rb uptake in an Akt-, ERK-, and Src kinase-dependent manner. Ouabain-mediated ERK phosphorylation was inhibited by blockade of intracellular calcium release, calcium entry, tyrosine kinases, and phospholipase C. Pharmacological inhibition of phosphoinositide-3 kinase and Akt failed to inhibit ouabain-stimulated ERK phosphorylation. Ouabain-mediated Akt phosphorylation was inhibited by U0126, a MEK/ERK inhibitor, suggesting that ouabain-mediated Akt phosphorylation is dependent on ERK. In an in vitro kinase assay, active recombinant ERK phosphorylated recombinant Akt on Ser473. Moreover, transient transfection with constitutively active MEK1, an upstream regulator of ERK, increased Akt phosphorylation and activation, whereas overexpression of constitutively active Akt failed to stimulate ERK phosphorylation. Ouabain at low concentrations also promoted cell proliferation in an ERK-dependent manner. These findings suggest that ouabain-stimulated ERK phosphorylation is required for Akt phosphorylation on Ser473, cell proliferation, and stimulation of Na+/K+-ATPase-mediated 86Rb uptake in OK cells.

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Liao, Hao-Yu, and Cristian O’Flaherty. "Lysophosphatidic Acid Signalling Regulates Human Sperm Viability via the Phosphoinositide 3-Kinase/AKT Pathway." Cells 12, no. 17 (September 2, 2023): 2196. http://dx.doi.org/10.3390/cells12172196.

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Lysophosphatidic acid (LPA) signalling is essential for maintaining germ cell viability during mouse spermatogenesis; however, its role in human spermatozoa is unknown. We previously demonstrated that peroxiredoxin 6 (PRDX6) calcium-independent phospholipase A2 (iPLA2) releases lysophospholipids such as LPA or arachidonic acid (AA) and that inhibiting PRDX6 iPLA2 activity impairs sperm cell viability. The exogenous addition of LPA bypassed the inhibition of PRDX6 iPLA2 activity and maintained the active phosphoinositide 3-kinase (PI3K)/AKT pathway. Here, we aimed to study PI3K/AKT pathway regulation via LPA signalling and protein kinases in maintaining sperm viability. The localization of LPARs in human spermatozoa was determined using immunocytochemistry, and P-PI3K and P-AKT substrate phosphorylations via immunoblotting. Sperm viability was determined using the hypo-osmotic swelling test. LPAR1, 3, 5 and 6 were located on the sperm plasma membrane. The inhibition of LPAR1-3 with Ki16425 promoted the impairment of sperm viability and decreased the phosphorylation of PI3K AKT substrates. Inhibitors of PKC, receptor-type PTK and PLC impaired sperm viability and the PI3K/AKT pathway. Adding 1-oleoyl-2-acetyl-snglycerol (OAG), a cell-permeable analog of diacylglycerol (DAG), prevented the loss of sperm viability and maintained the phosphorylation of PI3K. In conclusion, human sperm viability is supported by LPAR signalling and regulated by PLC, PKC and RT-PTK by maintaining phosphorylation levels of PI3K and AKT substrates.

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Bevan, H. S., J. Woolard, S. J. Harper, and D. O. Bates. "OC21 VEGF165b INHIBITS VEGF165 MEDIATED VEGF-R2 PHOSPHORYLATION AND AKT PHOSPHORYLATION." Microcirculation 11, no. 6 (September 2004): 536. http://dx.doi.org/10.1080/10739680490488319.

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36

Walther, Stefanie, Florentina Pluteanu, Susanne Renz, Yulia Nikonova, Joshua T. Maxwell, Li-Zhen Yang, Kurt Schmidt, et al. "Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177." American Journal of Physiology-Heart and Circulatory Physiology 307, no. 5 (September 1, 2014): H689—H700. http://dx.doi.org/10.1152/ajpheart.00694.2013.

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Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca2+-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.

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37

Park, Jae-Eun, and Ji-Sook Han. "HM-Chromanone, a Major Homoisoflavonoid in Portulaca oleracea L., Improves Palmitate-Induced Insulin Resistance by Regulating Phosphorylation of IRS-1 Residues in L6 Skeletal Muscle Cells." Nutrients 14, no. 18 (September 15, 2022): 3815. http://dx.doi.org/10.3390/nu14183815.

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This study investigated the effect of (E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HM-chromanone) on palmitate-induced insulin resistance and elucidated the underlying mechanism in L6 skeletal muscle cells. Glucose uptake was markedly decreased due to palmitate-induced insulin resistance in these cells; however, 10, 25, and 50 µM HM-chromanone remarkably improved glucose uptake in a concentration-dependent manner. HM-chromanone treatment downregulated protein tyrosine phosphatase 1B (PTP1B) and phosphorylation of c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), which increased because of palmitate mediating the insulin-resistance status in cells. HM-chromanone promoted insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and suppressed palmitate-induced phosphorylation of IRS-1 serine. This activated phosphoinositide 3-kinase (PI3K) and stimulated protein kinase B (AKT) phosphorylation. Phosphorylated AKT promoted the translocation of Glucose transporter type 4 to the plasma membrane and significantly enhanced glucose uptake into muscle cells. Additionally, HM-chromanone increased glycogen synthesis through phosphorylating glycogen synthase kinase 3 alpha/beta (GSK3 α/β) via AKT. Consequently, HM-chromanone may improve insulin resistance by downregulating the phosphorylation of IRS-1 serine through inhibition of negative regulators of insulin signaling and inflammation-activated protein kinases in L6 skeletal muscle cells.

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Dangelmaier, Carol, Bhanu Kanth Manne, Elizabetta Liverani, Jianguo Jin, Paul Bray, and Satya Kunapuli. "PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses." Thrombosis and Haemostasis 111, no. 03 (2014): 508–17. http://dx.doi.org/10.1160/th13-06-0484.

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Summary3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

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Trencia, Alessandra, Anna Perfetti, Angela Cassese, Giovanni Vigliotta, Claudia Miele, Francesco Oriente, Stefania Santopietro, et al. "Protein Kinase B/Akt Binds and Phosphorylates PED/PEA-15, Stabilizing Its Antiapoptotic Action." Molecular and Cellular Biology 23, no. 13 (July 1, 2003): 4511–21. http://dx.doi.org/10.1128/mcb.23.13.4511-4521.2003.

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ABSTRACT The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser116. In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser116 PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser116. In addition, a mutant of PED/PEA-15 featuring the substitution of Ser116→Gly (PEDS116→G) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also induced phosphorylation of PED/PEA-15 at Ser116. Based on pull-down and coprecipitation assays, PED/PEA-15 specifically bound Akt, independently of Akt activity. Serum activation of Akt as well as BAD phosphorylation by Akt showed no difference in 293 cells transfected with PED/PEA-15 and in untransfected cells (which express no endogenous PED/PEA-15). However, the antiapoptotic action of PED/PEA-15 was almost twofold reduced in PEDS116→G compared to that in PED/PEA-15WT cells. PED/PEA-15 stability closely paralleled Akt activation by serum in 293 cells. In these cells, the nonphosphorylatable PEDS116→G mutant exhibited a degradation rate threefold greater than that observed with wild-type PED/PEA-15. In the U373MG glioma cells, blocking Akt also reduced PED/PEA-15 levels and induced sensitivity to tumor necrosis factor-related apoptosis-inducing ligand apoptosis. Thus, phosphorylation by Akt regulates the antiapoptotic function of PED/PEA-15 at least in part by controlling the stability of PED/PEA-15. In part, Akt survival signaling may be mediated by PED/PEA-15.

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40

Hung, Ming-Jui, Ming-Yow Hung, Wen-Jin Cherng, and Li-Fu Li. "Increased cardiac microvascular permeability and activation of cardiac endothelial nitric oxide synthase in high tidal volume ventilation-induced lung injury." Asian Biomedicine 4, no. 1 (February 1, 2010): 27–36. http://dx.doi.org/10.2478/abm-2010-0004.

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Abstract Background: Positive pressure ventilation with large tidal volumes has been shown to cause lung injury via the serine/threonine kinase-protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS)-pathways. However, the effects of high tidal volume (VT) ventilation on the heart are unclear. Objectives: Evaluate the effect of VT ventilation on the cardiac vascular permeability and intracellular Akt and eNOS signaling pathway. Methods: C57BL/6 and Akt knock-out (heterozygotes, +/−) mice were exposed to high VT (30 mL/kg) mechanical ventilation with room air for one and/or five hours. Results: High VT ventilation increased cardiac microvascular permeability and eNOS phosphorylation in a timedependent manner. Serum cardiac troponin I was increased after one hour of high VT ventilation. Cardiac Akt phosphorylation was accentuated after one hour and attenuated after five hours of high VT ventilation. Pharmacological inhibition of Akt with LY294002 and high VT ventilation of Akt+/− mice attenuated cardiac Akt phosphorylation, but not eNOS phosphorylation. Conclusion: High VT ventilation increased cardiac myocardial injury, microvascular permeability, and eNOS phosphorylation. Involvement of cardiac Akt in high VT ventilation was transient.

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Rane, Madhavi J., Ye Song, Shunying Jin, Michelle T. Barati, Rui Wu, Hina Kausar, Yi Tan, et al. "Interplay between Akt and p38 MAPK pathways in the regulation of renal tubular cell apoptosis associated with diabetic nephropathy." American Journal of Physiology-Renal Physiology 298, no. 1 (January 2010): F49—F61. http://dx.doi.org/10.1152/ajprenal.00032.2009.

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Hyperglycemia induces p38 MAPK-mediated renal proximal tubular cell (RPTC) apoptosis. The current study hypothesized that alteration of the Akt signaling pathway by hyperglycemia may contribute to p38 MAPK activation and development of diabetic nephropathy. Immunoblot analysis demonstrated a hyperglycemia-induced increase in Akt phosphorylation in diabetic kidneys at 1 mo, peaking at 3 mo, and dropping back to baseline by 6 mo. Immunohistochemical staining with anti-pAkt antisera localized Akt phosphorylation to renal tubules. Maximal p38 MAPK phosphorylation was detected concomitant with increase in terminal uridine deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and caspase-3 activity in 6-mo diabetic kidneys. Exposure of cultured RPTCs to high glucose (HG; 22.5 mM) significantly increased Akt phosphorylation at 3, 6, and 9 h, and decreased thereafter. In contrast, p38 MAPK phosphorylation was detected between 9 and 48 h of HG treatment. Increased p38 MAPK activation at 24 and 48 h coincided with increased apoptosis, demonstrated by increased caspase-3 activity at 24 h and increased TUNEL-positive cells at 48 h of HG exposure. Blockade of p38 cascade with SB203850 inhibited HG-induced caspase-3 activation and TUNEL-positive cells. Overexpression of constitutively active Akt abrogated HG-induced p38 MAPK phosphorylation and RPTC apoptosis. In addition, blockade of the phosphatidylinositol-3 kinase/Akt pathway with LY294002 and silencing of Akt expression with Akt small interfering RNA induced p38 MAPK phosphorylation in the absence of HG. These results collectively suggest that downregulation of Akt activation during long-term hyperglycemia contributes to enhanced p38 MAPK activation and RPTC apoptosis. Mechanism of downregulation of Akt activation in 6-mo streptozotocin diabetic kidneys was attributed to decreased Akt-heat shock protein (Hsp) 25, Akt-p38 interaction, and decreased PTEN activity. Thus PTEN or Hsp25 could serve as potential therapeutic targets to modulate Akt activation and control p38 MAPK-mediated diabetic complications.

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Lynn, Matthew A., Heidi L. Rupnow, Dean J. Kleinhenz, William A. Kanner, Samuel C. Dudley, and C. Michael Hart. "Fatty Acids Differentially Modulate Insulin-Stimulated Endothelial Nitric Oxide Production by an Akt-lndependent Pathway." Journal of Investigative Medicine 52, no. 2 (March 2004): 129–36. http://dx.doi.org/10.1177/108155890405200222.

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Background Insulin increases endothelial nitric oxide (NO) production by activating endothelial nitric oxide synthase (eNOS) through protein kinase B (Akt)-mediated phosphorylation of serine residue 1179 (p-eNOS serine 1179). Because fatty acids modulate insulin-stimulated Akt signaling cascades in smooth muscle cells, we hypothesized that fatty acids would differentially regulate endothelial Akt signaling, eNOS phosphorylation, and NO production. Methods Porcine pulmonary artery endothelial cells (PAECs) were treated for 3 hours with 100 μM oleic (18:1) or eicosapentaenoic (20:5) acids or with an equivalent volume of ethanol vehicle (0.1%). PAECs were then treated with graded concentrations (109–10-5 M) of insulin or incubated overnight (24 hours) in culture medium without fatty acids before insulin treatment. Activation and phosphorylation of Akt and eNOS were determined by immunoblotting. NO production was measured with a chemiluminescence NO analyzer or with a NO-selective carbon fiber microelectrode. Results Insulin-stimulated Akt phosphorylation, eNOS phosphorylation, and NO production. The phosphatidylinositol-3 kinase inhibitor wortmannin attenuated insulin-stimulated Akt activation and NO production. Treatment with the co-3 fatty acid 20:5, but not 18:1, enhanced insulin-stimulated NO production but failed to alter insulin-stimulated Akt activation or eNOS serine 1179 phosphorylation. Conclusion Individual fatty acyl species have distinct effects on insulin-stimulated endothelial NO production. Although fatty acids alter Akt signaling in muscle cells, the current results indicate that fatty acids do not modulate endothelial NO production through alterations in insulin-stimulated, Akt-mediated eNOS phosphorylation.

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Jacques-Silva, Maria C., Richard Rodnight, Guido Lenz, Zhongji Liao, Qiongman Kong, Minh Tran, Yuan Kang, Fernando A. Gonzalez, Gary A. Weisman, and Joseph T. Neary. "P2X7 receptors stimulate AKT phosphorylation in astrocytes." British Journal of Pharmacology 141, no. 7 (April 2004): 1106–17. http://dx.doi.org/10.1038/sj.bjp.0705685.

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Salani, Barbara, Silvia Ravera, Adriana Amaro, Annalisa Salis, Mario Passalacqua, Enrico Millo, Gianluca Damonte, et al. "IGF1 regulates PKM2 function through Akt phosphorylation." Cell Cycle 14, no. 10 (May 19, 2015): 1559–67. http://dx.doi.org/10.1080/15384101.2015.1026490.

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45

Cinar, Ozgur, Yasemin Seval, Yesim H. Uz, Hakan Cakmak, Murat Ulukus, Umit A. Kayisli, and Aydin Arici. "Differential regulation of Akt phosphorylation in endometriosis." Reproductive BioMedicine Online 19, no. 6 (December 2009): 864–71. http://dx.doi.org/10.1016/j.rbmo.2009.10.001.

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46

Wang, Shouye, and Marc D. Basson. "Akt directly regulates focal adhesion kinase through association and serine phosphorylation: implication for pressure-induced colon cancer metastasis." American Journal of Physiology-Cell Physiology 300, no. 3 (March 2011): C657—C670. http://dx.doi.org/10.1152/ajpcell.00377.2010.

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Although focal adhesion kinase (FAK) is typically considered upstream of Akt, extracellular pressure stimulates cancer cell adhesion via Akt-dependent FAK activation. How Akt regulates FAK is unknown. We studied Akt-FAK interaction in colon cancer cells under 15 mmHg increased extracellular pressure. Pressure enhanced Akt-FAK association, blocked by inhibiting FAK or silencing Akt1 but not Akt2, and stimulated FAK serine phosphorylation in Caco-2 and human colon cancer cells from surgical specimens Akt1-dependently. FAK includes three serine (S517/601/695) and one threonine (T600)-containing consensus sequences for Akt phosphorylation. Studying S–>A nonphosphorylatable point mutants suggests that these sites coordinately upregulate FAK Y397 tyrosine phosphorylation, which conventionally initiates FAK activation, and mediate pressure-induced cancer cell adhesion. FAK(T600A) mutation did not prevent pressure-induced FAK(Y397) phosphorylation or adhesion. Akt1 appeared to directly bind FAK, and this binding did not depend on the FAK autophosphorylation site (Y397). In addition, our results demonstrated that Akt phosphorylated FAK at three novel serine phosphorylation sites, which were also not required for FAK-Akt binding. This novel interaction suggests that FAK and Akt may be dual kinase targets to prevent cancer cell adhesion and metastasis.

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47

García-Martínez, Juan M., Jennifer Moran, Rosemary G. Clarke, Alex Gray, Sabina C. Cosulich, Christine M. Chresta, and Dario R. Alessi. "Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)." Biochemical Journal 421, no. 1 (June 12, 2009): 29–42. http://dx.doi.org/10.1042/bj20090489.

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mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of ∼10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.

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48

Gold, Michael R., Michael P. Scheid, Lorna Santos, May Dang-Lawson, Richard A. Roth, Linda Matsuuchi, Vincent Duronio, and Danielle L. Krebs. "The B Cell Antigen Receptor Activates the Akt (Protein Kinase B)/Glycogen Synthase Kinase-3 Signaling Pathway Via Phosphatidylinositol 3-Kinase." Journal of Immunology 163, no. 4 (August 15, 1999): 1894–905. http://dx.doi.org/10.4049/jimmunol.163.4.1894.

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Abstract We have previously shown that the B cell Ag receptor (BCR) activates phosphatidylinositol (PI) 3-kinase. We now show that a serine/threonine kinase called Akt or protein kinase B is a downstream target of PI 3-kinase in B cells. Akt has been shown to promote cell survival as well as the transcription and translation of proteins involved in cell cycle progression. Using an Ab that specifically recognizes the activated form of Akt that is phosphorylated on serine 473, we show that BCR engagement activates Akt in a PI 3-kinase-dependent manner. These results were confirmed using in vitro kinase assays. Moreover, BCR ligation also induced phosphorylation of Akt of threonine 308, another modification that is required for activation of Akt. In the DT40 chicken B cell line, phosphorylation of Akt on serine 473 was completely dependent on the Lyn tyrosine kinase, while the Syk tyrosine kinase was required for sustained phosphorylation of Akt. Complementary experiments in BCR-expressing AtT20 endocrine cells confirmed that Src kinases are sufficient for BCR-induced Akt phosphorylation, but that Syk is required for sustained phosphorylation of Akt on both serine 473 and threonine 308. In insulin-responsive cells, Akt phosphorylates and inactivates the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). Inactivation of GSK-3 may promote nuclear accumulation of several transcription factors, including NF-ATc. We found that BCR engagement induced GSK-3 phosphorylation and decreased GSK-3 enzyme activity. Thus, BCR ligation initiates a PI 3-kinase/Akt/GSK-3 signaling pathway.

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49

Cersosimo, Eugenio, Xiaojing Xu, and Nicolas Musi. "Potential role of insulin signaling on vascular smooth muscle cell migration, proliferation, and inflammation pathways." American Journal of Physiology-Cell Physiology 302, no. 4 (February 15, 2012): C652—C657. http://dx.doi.org/10.1152/ajpcell.00022.2011.

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To investigate the role of insulin signaling pathways in migration, proliferation, and inflammation of vascular smooth muscle cells (VSMCs), we examined the expression of active components of the phosphatidyl inositol 3 (PI-3) kinase (p-Akt) and mitogen-activated protein kinase (MAPK) (p-Erk) in primary cultures of VSMCs from human coronary arteries. VSMCs were treated in a dose-response manner with insulin (0, 1, 10, and 100 nM) for 20 min, and Akt and Erk phosphorylation were measured by Western blot analysis. In separate experiments, we evaluated the effect of 200 μM palmitate, in the presence and absence of 8 μM pioglitazone, on insulin-stimulated (100 nM for 20 min) Akt and Erk phosphorylation. The phosphorylation of Akt and Erk in VSMCs exhibited a dose dependency with a three- to fourfold increase, respectively, at the highest dose (100 nM). In the presence of palmitate, insulin-induced Akt phosphorylation was completely abolished, and there was a threefold increase in p-Erk. With addition of pioglitazone, the phosphorylation of Akt by insulin remained unchanged, whereas insulin-stimulated Erk phosphorylation was reduced by pioglitazone. These data in VSMCs indicate that high palmitate decreases insulin-stimulated Akt phosphorylation and stimulates MAPK, whereas preexposure peroxisome proliferator-activated receptor-γ agonist pioglitazone preserves Akt phosphorylation and simultaneously attenuates MAPK signaling. Our results suggest that metabolic and mitogenic insulin signals have different sensitivity, are independently regulated, and may play a role in arterial smooth muscle cells migration, proliferation, and inflammation in conditions of acute hyperinsulinemia.

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Yu, Yongjun, and James C. Alwine. "Interaction between Simian Virus 40 Large T Antigen and Insulin Receptor Substrate 1 Is Disrupted by the K1 Mutation, Resulting in the Loss of Large T Antigen-Mediated Phosphorylation of Akt." Journal of Virology 82, no. 9 (February 27, 2008): 4521–26. http://dx.doi.org/10.1128/jvi.02365-07.

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ABSTRACT The cellular kinase Akt is a key controller of cellular metabolism, growth, and proliferation. Many viruses activate Akt due to its beneficial effects on viral replication. We previously showed that wild-type (WT) simian virus 40 (SV40) large T antigen (TAg) inhibits apoptosis via the activation of PI3K/Akt signaling. Here we show that WT TAg expressed from recombinant adenoviruses in U2OS cells induced the phosphorylation of Akt at both T308 and S473. In contrast, Akt phosphorylation was eliminated by the K1 mutation (E107K) within the retinoblastoma protein (Rb) binding motif of TAg. This suggested that Akt phosphorylation may depend on TAg binding to Rb or one of its family members. However, in Rb-negative SAOS2 cells depleted of p107 and p130 by using small hairpin RNAs (shRNAs), WT TAg still mediated Akt phosphorylation. These results suggested that the K1 mutation affects another TAg function. WT-TAg-mediated phosphorylation of Akt was inhibited by a PI3K inhibitor, suggesting that the effects of TAg originated upstream of PI3K; thus, we examined the requirement for insulin receptor substrate 1 (IRS1), which binds and activates PI3K. Depletion of IRS1 by shRNAs abolished the WT-TAg-mediated phosphorylation of Akt. Immunoprecipitation studies showed that the known interaction between TAg and IRS1 is significantly weakened by the K1 mutation. These data indicate that the K1 mutation disrupts not only Rb binding but also IRS1 binding, contributing to the loss of activation of PI3K/Akt signaling.

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