The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf

Cross, Darren A.E.; Alessi, Dario R.; Vandenheede, Jackie R.; McDowell, H E; Hundal, Harinder S.; Cohen, Philip

doi:10.1042/bj3030021

Cited by 454 publications

(228 citation statements)

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“…Distinct experimental approaches have led to the conclusion that PI 3-kinase is necessary for insulin-stimulated GLUT4 translocation [38][39][40][41][42][43][44]. Evidence from other sources has also demonstrated a correlation between PI 3-kinase activity and glycogen metabolism [45,46]. Thus, it is reasonable to speculate that the IRS-1/PI 3-kinase pathway may be linked to the activation of glucose transport in muscle and glycogen synthesis in liver and muscle, and that a reduction in this association in pregnancy may have a role in the insulin resistance in liver and muscle of pregnant rats.…”

Section: Discussionmentioning

confidence: 99%

Defects in insulin signal transduction in liver and muscle of pregnant rats

et al. 1997

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Insulin resistance during pregnancy has been reported both in women and in experimental animals [1][2][3][4][5][6][7], but the mechanism of this resistance remains unclear. In vivo studies have shown that pregnant rats become progressively resistant to insulin after day 16 of gestation [5]. In these animals, the insulin resistance of skeletal muscle has been clearly demonstrated in vitro, while the insulin resistance in vivo in peripheral tissues and liver has been substantiated in studies using the euglycaemic-hyperinsulinaemic clamp technique [6,7].Insulin initiates its metabolic and growth-promoting effects by binding to the a subunit of its tetrameric receptor, thereby activating the kinase in the b subunit [8]. This interaction catalyses the intramolecular autophosphorylation of specific tyrosine residues of the b subunit which further enhances the tyrosine kinase activity of the receptor toward other protein substrates [8]. In most cells, this primary event leads to the subsequent tyrosyl phosphorylation of a cytoplasmic protein with an apparent molecular weight of 160-185 kDa, called insulin receptor substrate 1 (IRS-1) [9][10][11]. Considerable evidence indicates that insulin receptor tyrosine Diabetologia (1997) 40: 179-186 Defects in insulin signal transduction in liver and muscle of pregnant rats Summary Pregnancy is known to induce insulin resistance, but the exact molecular mechanism involved is unknown. In the present study, we have examined the levels and phosphorylation state of the insulin receptor and of insulin receptor substrate 1(IRS-1), as well as the association between IRS-1 and phosphatidylinositol 3-kinase (PI 3-kinase) in the liver and muscle of pregnant rats (day 20 of gestation) by immunoprecipitation and immunoblotting with anti-insulin receptor, anti-IRS-1, anti-PI 3-kinase and antiphosphotyrosine antibodies. There were no changes in the insulin receptor concentration in the liver and muscle of pregnant rats. However, insulin stimulation of receptor autophosphorylation, as determined by immunoblotting with antiphosphotyrosine antibody, was reduced by 30 ± 6 % (p < 0.02) in muscle and 36 ± 5 % (p < 0.01) in liver at day 20 of gestation. IRS-1 protein levels decreased by 45 ± 6 % (p < 0.002) in liver and by 56 ± 9 % (p < 0.002) in muscle of pregnant rats. In samples previously immunoprecipitated with anti-IRS-1 antibody and blotted with antiphosphotyrosine antibody, the insulin-stimulated IRS-1 phosphorylation levels in the muscle and liver of pregnant rats decreased by 70 ± 9 % (p < 0.01) and 75 ± 8 % (p < 0.01), respectively. The insulin-stimulated IRS-1 association with PI 3-kinase decreased by 81 ± 6 % in muscle (p < 0.01) and 79 ± 11 % (p < 0.01) in the liver during pregnancy. These data suggest that changes in the early steps of insulin signal transduction may have a role in the insulin resistance observed in pregnancy. [Diabetologia (1997) 40: 179-186]

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Section: Discussionmentioning

confidence: 99%

Defects in insulin signal transduction in liver and muscle of pregnant rats

et al. 1997

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“…However, SB 203580 does not induce any activation of MKK1 or MAPK2 (Figure 2). Moreover, incubation of Swiss 3T3 cells with PD 98059 (50 mM), which prevents the activation of MAPK2 by EGF (Figure 2 The activation of c-Raf by di erent agonists can result from the stimulation of several signalling pathways, including the activation of protein kinase C (PKC) (Morrison and Cutler, 1997) or phosphatidylinositide (PtdIns) 3-kinase (Cross et al, 1994). However, c-Raf activation is una ected by Ro 318220 (Figure 4), an inhibitor of PKC and some other protein kinases (Alessi, 1997), under conditions where the PMA-induced activation of MAPK2 measured in the same cells was decreased by 85%.…”

Section: Mechanism Of Activation Of C-raf By Sb 203580mentioning

confidence: 99%

Effect of SB 203580 on the activity of c-Raf in vitro and in vivo

et al. 1999

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The inhibition of SAPK2a/p38 (a mitogen activated protein (MAP) kinase family member) by SB 203580 depends on the presence of threonine at residue 106. Nearly all other protein kinases are insensitive to this drug because a more bulky residue occupies this site (Eyers et al., 1998). Raf is one of the few protein kinases that possesses threonine at this position, and we show that SB 203580 inhibits c-Raf with an IC 50 of 2 mM in vitro. However, SB 203580 does not suppress either growth factor or phorbol ester-induced activation of the classical MAP kinase cascade in mammalian cells. One of the reasons for this is that SB 203580 also triggers a remarkable activation of c-Raf in vivo (when measured in the absence of the drug). The SB 203580-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras, is not prevented by inhibitors of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase, and is not triggered by the binding of this drug to SAPK2a/p38. The paradoxical activation of cRaf by SB 203580 (and by another structurally unrelated c-Raf inhibitor) suggests that inhibitors of the kinase activity of c-Raf may not be e ective as anti-cancer drugs.

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“…However, it is now clear that Ras also regulates the activity of a number of other signaling pathways through activation of proteins such as phosphatidylinositol 3-kinase (Pl3-K) and RalGDS (4,55). There is much interest in the cross talk that exists between these different, parallel Ras pathways, and in some but not all cell types, inhibition of Pl3-K that leads to suppression of ERK has been shown (8,16,24,56). In different studies, Pl3-K has been shown to regulate ERK activation at the level of Ras or at the level of Raf-1 (7,8,27,56).…”

mentioning

confidence: 99%

“…There is much interest in the cross talk that exists between these different, parallel Ras pathways, and in some but not all cell types, inhibition of Pl3-K that leads to suppression of ERK has been shown (8,16,24,56). In different studies, Pl3-K has been shown to regulate ERK activation at the level of Ras or at the level of Raf-1 (7,8,27,56). More recently, it was suggested that Pl3-K regulates Raf-1 by activating protein kinases that can phosphorylate Raf-1 directly.…”

mentioning

confidence: 99%

S338 Phosphorylation of Raf-1 Is Independent of Phosphatidylinositol 3-Kinase and Pak3

Chiloeches

Mason

Marais

2001

Molecular and Cellular Biology

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The Raf-1 serine/threonine protein kinase requires phosphorylation of the serine at position 338 (S338) for activation. Ras is required to recruit Raf-1 to the plasma membrane, which is where S338 phosphorylation occurs. The recent suggestion that Pak3 could stimulate Raf-1 activity by directly phosphorylating S338 through a Ras/phosphatidylinositol 3-kinase (Pl3-K)/-Cdc42-dependent pathway has attracted much attention. Using a phospho-specific antibody to S338, we have reexamined this model. Using LY294002 and wortmannin, inhibitors of Pl3-K, we find that growth factor-mediated S338 phosphorylation still occurs, even when Pl3-K activity is completely blocked. Although high concentrations of LY294002 and wortmannin did suppress S338 phosphorylation, they also suppressed Ras activation. Additionally, we show that Pak3 is not activated under conditions where S338 is phosphorylated, but when Pak3 is strongly activated, by coexpression with V12Cdc42 or by mutations that make it independent of Cdc42, it did stimulate S338 phosphorylation. However, this occurred in the cytosol and did not stimulate Raf-1 kinase activity. The inability of Pak3 to activate Raf-1 was not due to an inability to stimulate phosphorylation of the tyrosine at position 341 but may be due to its inability to recruit Raf-1 to the plasma membrane. Taken together, our data show that growth factorstimulated Raf-1 activity is independent of Pl3-K activity and argue against Pak3 being a physiological mediator of S338 phosphorylation in growth factor-stimulated cells.The Raf-1 serine/threonine-specific protein kinase is the first component of a three-tiered protein kinase cascade that regulates many biological events such as cell growth, differentiation, and apoptosis (for reviews, see references 12, 39, and 44). Raf-1 phosphorylates and activates the dual-specificity mitogen-activated protein kinase (MAPK) kinases MEK1 and MEK2, which in turn activate the MAPKs ERK1 and ERK2. The ERKs phosphorylate and regulate the activity of transcription factors, cytoskeletal proteins, metabolic enzymes, and other protein kinases to modulate cellular responses to extracellular signals. Raf-1 regulation is highly complex. It is cytosolic in unstimulated cells, but following activation of the small G-protein Ras, it translocates to the plasma membrane, where activation takes place (for reviews, see references 23, 38, and 41). Interaction with Ras alone is not sufficient to activate Raf-1 and other membrane-localized events such as oligomerization, interaction with other proteins, and interactions with lipids all appear to play a role.Phosphorylation also plays a key role in Raf-1 activation, and both positive and negative regulatory sites have been mapped (23,38,41). Two sites whose phosphorylation has been shown to be necessary for activation are the serine located at position 338 (S338) and the tyrosine located at position 341 (Y341) (3,15,18,36,40,42). These amino acids are located 10 to 15 amino acids N terminal to the glycine-rich loop of the ATP-binding domain...

show abstract

The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf

Cited by 454 publications

References 43 publications

Defects in insulin signal transduction in liver and muscle of pregnant rats

Defects in insulin signal transduction in liver and muscle of pregnant rats

Effect of SB 203580 on the activity of c-Raf in vitro and in vivo

S338 Phosphorylation of Raf-1 Is Independent of Phosphatidylinositol 3-Kinase and Pak3

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