Expression of a constitutively active, membrane-associated Akt-1 (PKB ␣) construct in 3T3L1 adipocytes was shown to induce glucose uptake in the absence of insulin by stimulating Glut4 translocation to the plasma membrane (Kohn, A. D., Summers, S. A., Birnbaum, M. J., and Roth, R. A. (1996) J. Biol. Chem. 271, 31372-31378). However, in rat fat cell the vast majority of Akt-1 is cytosolic and shows no re-distribution to the plasma membrane in response to insulin. On the other hand, little work has been done with other Akt family members such as Akt-2 (PKB ) or Akt-3 (PKB ␥). In this report, an analysis of the subcellular distribution of Akt-2 in rat adipocytes shows that Akt-2 is present in significant amounts in various membrane compartments, as well as in the cytosol, and the former include the light microsomes where Glut4 is present in the basal state. The distribution of Akt-2 in resting adipocytes was found to substantially overlap with that of Glut4 when light microsomes were subfractionated by a sucrose velocity gradient indicating possible co-localization. We confirmed co-localization of Akt-2 and Glut4 in the basal state by immunopurification of Glut4 vesicles, which exhibited a 5.5-fold increase in Akt-2 in response to insulin relative to the amount of Glut4. These results are consistent with the possibility that Akt-2 may be involved in Glut4 vesicle translocation.
It is now well-recognized that the mitogen-activated protein (MAP) kinase cascade facilitates signaling from an activated tyrosine kinase receptor to the nucleus. In fact, an increasing number of extracellular effectors have been reported to activate the MAP kinase cascade, with a significant number of cellular responses attributed to this activation. We set out to explore how two extracellular effectors, basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF-1), which have both been reported to activate MAP kinase, generate quite distinct cellular responses in C2C12 myoblasts. We demonstrate here that bFGF, which is both a potent mitogen and inhibitor of myogenic differentiation, is a strong MAP kinase agonist. By contrast, IGF-1, which is equally mitogenic for C2C12 cells but ultimately enhances the differentiated phenotype, is a weak activator of the MAP kinase cascade. We further demonstrate that IGF-1 is a potent activator of both insulin receptor substrate IRS-1 tyrosyl phosphorylation and association of IRS-1 with activated phosphatidylinositol 3-kinase (PI 3-kinase). Finally, use of the specific MAP kinase kinase inhibitor, PD098059, and wortmannin, a PI 3-kinase inhibitor, suggests the existence of an IGF-1-induced, MAP kinase-independent signaling event which contributes to the mitogenic response of this factor, whereas bFGF-induced mitogenesis appears to strongly correlate with activation of the MAP kinase cascade.The binding of hormones or growth factors to their cognate cell surface receptor/tyrosine kinases initiates an intricate array of phosphorylation events that have been implicated in the control of cellular growth, differentiation, and metabolism. The mitogen-activated protein (MAP) kinase phosphorylation cascade is one mechanism by which a signal generated by ligand binding at the cell membrane is transduced to the nucleus. The MAP kinase signaling cascade is triggered by hormone or growth factor binding to a receptor/tyrosine kinase followed by receptor autophosphorylation and recruitment of the Src homology 2 (SH2)-domain-containing proteins to phosphorylated tyrosine residues on the receptor (7, 48). Subsequent to these receptor-proximal events, a protein phosphorylation cascade that involves association of GTP-bound Ras with Raf followed by activation of MAP kinase kinase (MEK) and MAP kinase (23, 43) is stimulated. In turn, activated MAP kinases translocate to the nucleus, where they phosphorylate appropriate transcription factors and ultimately exert their effects on cellular growth and differentiation (9, 13, 37). A paradox exists, however, because activation of the MAP kinase signaling pathway by various extracellular effectors appears to be initiated by a common cascade of phosphorylation events, whereas the downstream cellular responses which have been attributed to activation of this pathway can vary dramatically. For instance, in neuronal cells, MAP kinase activity results from stimulation with nerve growth factor, basic fibroblast growth factor (bFGF), insul...
Protein-tyrosine phosphatases (PTPases) play a key role in maintaining the steady-state tyrosine phosphorylation of the insulin receptor (IR) and its substrate proteins such as insulin receptor substrate 1 (IRS-1). However, the PTPase(s) that inactivate IR and IRS-1 under physiological conditions remain unidentified. Here, we analyze the subcellular distribution in rat adipocytes of several PTPases thought to be involved in the counterregulation of insulin signaling. We found that the transmembrane enzymes, protein-tyrosine phosphatase (PTP)-␣ and leukocyte common antigen-related (LAR), were detected predominantly in the plasma membrane and to a lesser extent in the heavy microsomes, a distribution similar to that of insulin receptor. PTP-1B and IRS-1 were present in light microsomes and cytosol, whereas SHPTP2/Syp was exclusively cytosolic. Insulin induced a redistribution of PTP-␣ from the plasma membrane to the heavy microsomes in a parallel fashion with the receptor. The distribution of PTP-1B in the light microsomes from resting adipocytes was similar to that of IRS-1 as determined by sucrose velocity gradient fractionation. Analysis of the catalytic activity of partially purified rat adipocyte PTP-␣ and LAR and recombinant PTP-1B showed that all three PTPases dephosphorylate IR. When a mix of IR/IRS-1 was used as a substrate, PTP-1B was particularly effective in dephosphorylating IRS-1. Considering that IR and IRS-1 can be dephosphorylated in internal membrane compartments from rat adipocytes (Kublaoui, B., Lee, J., and Pilch, P.F. (1995) J. Biol. Chem. 270, 59 -65) and that PTP-␣ and PTP-1B are the respective PTPases in these fractions, we conclude that these PTPases are responsible for the counterregulation of insulin signaling there, whereas both LAR and PTP-␣ may act upon cell surface insulin receptors.The initial steps in insulin signaling require protein tyrosine phosphorylation. The interaction of insulin with its plasma membrane receptor elicits a rapid autophosphorylation on specific tyrosine residues in several domains of the receptor's cytoplasmic -subunit followed by activation of the receptor's exogenous tyrosine kinase activity as reflected by enhanced substrate phosphorylation (reviewed in Refs. 1 and 2). The principle insulin receptor substrates (IRSs) 1 involved in metabolic regulation are IRS-1 and IRS-2, and there is abundant biochemical evidence to support this (reviewed in Ref.3). Genetic evidence from animals devoid of IRS-1 (4, 5) and IRS-2 (6) further suggests that these molecules are intimately involved in the signaling to metabolic pathways regulated by insulin. Two additional members of the IRS family, IRS-3 and IRS-4, have been described, but they are unlikely to be involved in insulin-dependent metabolic regulation because the former is not expressed in the appropriate tissues (7) and a knockout of the latter has no obvious metabolic phenotype (8). IRS-1 and IRS-2 contain numerous tyrosine residues that, when phosphorylated, act as docking sites for a certain set of Src homolo...
Connexin43 is a major component of the gap junctions between pigmented and non-pigmented cells of the double-layered epithelium in the ciliary body of the eye. We directly tested the hypothesis that gap junctions play a crucial role in the production of the aqueous humor by inactivating the GJA1 (connexin43) gene in the pigmented epithelium with cre-loxP technology. To accomplish this, we crossed a line expressing cre recombinase driven by the nestin promoter and a line with floxed connexin43 alleles. Resultant lines exhibited loss of connexin43 from the pigmented epithelium, iris, retinal pigment epithelium and the lens. We observed plasma proteins in the aqueous humor and pathological changes consistent with a loss of intraocular pressure. As the ciliary body is responsible for aqueous humor production, these data support the hypothesis that the gap junctions between pigmented and non-pigmented epithelium are necessary for production of the aqueous humor that is in turn required for the generation of normal intraocular pressure and nourishment of the postnatal lens. The loss of connexin43 expression in the iris correlated with a separation of the posterior pigmented epithelium from the anterior myoepithelium and with meiosis, possibly resulting from a loss of function of the dilator pupillae.
The allelopathic potential ofI. tricolor, used in traditional agriculture as a weed controller, has been demonstrated by measuring the inhibitory activity of aqueous lixiviates and organic extracts of the plant material on seedling growth ofAmaranthus leucocarpus andEchinochloa crusgalli. Bioactivity-guided fractionation of the most active organic extract led to the isolation of the allelopathic principles, which turned out to be a mixture of glycosides, having jalapinolic acid as the aglycone portion glycosidically linked in the 11 position to an oligosaccharide composed of glucose, rhamnose, and fucose, which also combines with the carboxyl group of the aglycone to form a macrocyclic ester.
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