Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398

Protein synthesis consumes a high proportion of the metabolic energy of mammalian cells, and most of this is used by peptide chain elongation. An important regulator of energy supply and demand in eukaryotic cells is the AMP-activated protein kinase (AMPK). The rate of peptide chain elongation can be modulated through the phosphorylation of eukaryotic elongation factor (eEF) 2, which inhibits its activity and is catalyzed by a specific calcium/calmodulin-dependent protein kinase termed eEF2 kinase. Here we show that AMPK directly phosphorylates eEF2 kinase, and we identify the major site of phosphorylation as Ser-398 in a regulatory domain of eEF2 kinase. AMPK also phosphorylates two other sites (Ser-78 and Ser-366) in eEF2 kinase in vitro. We develop appropriate phosphospecific antisera and show that phosphorylation of Ser-398 in eEF2 kinase is enhanced in intact cells under a range of conditions that activate AMPK and increase the phosphorylation of eEF2. Ser-78 and Ser-366 do not appear to be phosphorylated by AMPK within cells. Although cardiomyocytes appear to contain a distinct isoform of eEF2 kinase, it also contains a site corresponding to Ser-398 that is phosphorylated by AMPK in vitro. Stimuli that activate AMPK and increase eEF2 phosphorylation within cells increase the activity of eEF2 kinase. Thus, AMPK and eEF2 kinase may provide a key link between cellular energy status and the inhibition of protein synthesis, a major consumer of metabolic energy.     

Akt mediates insulin-stimulated phosphorylation of Ndrg2: evidence for cross-talk with protein kinase C theta

The protein kinase Akt mediates several metabolic and mitogenic effects of insulin, whereas activation of protein kinase C (PKC) isoforms has been implicated in the inhibition of insulin action. We have previously shown that both PKC and PKCepsilon are activated in skeletal muscle of insulin-resistant high fat-fed rats, and to identify potential substrates for these kinases, we incubated recombinant PKC isoforms with rat muscle fractions in vitro. PKC specifically phosphorylated a 48-kDa protein that was subsequently identified by mass spectrometry as Ndrg2. Ndrg2 is highly related to N-Myc downstream-regulated protein 1, which has been linked to stress responses, cell proliferation, and differentiation, although Ndrg2 itself is not repressed by N-Myc. Ndrg2 contains several potential phosphorylation sites, including three Akt consensus sequences. Ndrg2 phosphorylation was enhanced in [32P]orthophosphate-labeled C2C12 muscle cells co-overexpressing either PKC or Akt. Phosphorylation of Ndrg2 was examined further using a phospho (Ser/Thr) Akt substrate antibody. Insulin increased Ndrg2 phosphorylation in C2C12 cells in a wortmannin- and palmitate-inhibitable manner, whereas rapamycin, PD98059, and bisindoylmaleimide I had no effect, supporting a direct role for Akt. Mutation of Ndrg2 indicated that Thr-348 is the major phosphorylation site detected by the antibody and that Akt stimulates phosphorylation of this site, whereas PKC phosphorylates Ser-332. PKC overexpression, however, diminished the effect of insulin on Thr-348 phosphorylation without reducing Akt activation, suggesting that this is mediated through phosphorylation of Ndrg2 at Ser-332. Our data identify Ndrg2 as a novel insulin-dependent phosphoprotein and suggest that PKC may inhibit insulin action in part by reducing its phosphorylation by Akt.     

Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302

Inhibitory serine phosphorylation is a potential molecular mechanism for insulin resistance. We have developed a new variant of the yeast two-hybrid method, referred to as disruptive yeast tri-hybrid (Y3H), to identify inhibitory kinases and sites of phosphorylation in insulin receptors (IR) and IR substrates, IRS-1. Using IR and IRS-1 as bait and prey, respectively, and c-Jun NH(2)-terminal kinase (JNK1) as the disruptor, we now show that phosphorylation of IRS-1 Ser-307, a previously identified site, is necessary but not sufficient for JNK1-mediated disruption of IR/IRS-1 binding. We further identify a new phosphorylation site, Ser-302, and show that this too is necessary for JNK1-mediated disruption. Seven additional kinases potentially linked to insulin resistance similarly block IR/IRS-1 binding in the disruptive Y3H, but through distinct Ser-302- and Ser-307-independent mechanisms. Phosphospecific antibodies that recognize sequences surrounding Ser(P)-302 or Ser(P)-307 were used to determine whether the sites were phosphorylated under relevant conditions. Phosphorylation was promoted at both sites in Fao hepatoma cells by reagents known to promote Ser/Thr phosphorylation, including the phorbol ester phorbol 12-myristate 13-acetate, anisomycin, calyculin A, and insulin. The antibodies further showed that Ser(P)-302 and Ser(P)-307 are increased in animal models of obesity and insulin resistance, including genetically obese ob/ob mice, diet-induced obesity, and upon induction of hyperinsulinemia. These findings demonstrate that phosphorylation at both Ser-302 and Ser-307 is necessary for JNK1-mediated inhibition of the IR/IRS-1 interaction and that Ser-302 and Ser-307 are phosphorylated in parallel in cultured cells and in vivo under conditions that lead to insulin resistance.     

Nicotiana tabacum osmotic stress-activated kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop

NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress. Our previous results as well as data presented by others indicate that phosphorylation is involved in activation of SnRK2 kinases. Here, we have mapped the regulatory phosphorylation sites of NtOSAK by mass spectrometry with collision-induced peptide fragmentation. We show that active NtOSAK, isolated from NaCl-treated tobacco BY-2 cells, is phosphorylated on Ser-154 and Ser-158 in the kinase activation loop. Prediction of the NtOSAK three-dimensional structure indicates that phosphorylation of Ser-154 and Ser-158 triggers changes in enzyme conformation resulting in its activation. The involvement of Ser-154 and Ser-158 phosphorylation in regulation of NtOSAK activity was confirmed by site-directed mutagenesis of NtOSAK expressed in bacteria and in maize protoplasts. Our data reveal that phosphorylation of Ser-158 is essential for NtOSAK activation, whereas phosphorylation of Ser-154 most probably facilitates Ser-158 phosphorylation. The time course of NtOSAK phosphorylation on Ser-154 and Ser-158 in BY-2 cells subjected to osmotic stress correlates with NtOSAK activity, indicating that NtOSAK is regulated by reversible phosphorylation of these residues in vivo. Importantly, Ser-154 and Ser-158 are conserved in all SnRK2 subfamily members, suggesting that phosphorylation at these sites may be a general mechanism for SnRK2 activation.     

Shp2 is required for protein kinase C-dependent phosphorylation of serine 307 in insulin receptor substrate-1

The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms. However, in an in vitro system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), we detected only Ser-318, but not Ser-307 phosphorylation, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. As we have observed recently that the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, we studied the role of Shp2 in this context. We found that phorbol ester-induced Ser-307 phosphorylation is reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2-/-) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depended on the presence of Shp2. Furthermore, Shp2-dependent phorbol ester effects on Ser-307 were blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. This suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1.     

Identification of Akt-independent Regulation of Hepatic Lipogenesis by Mammalian Target of Rapamycin (mTOR) Complex 2

Mammalian target of rapamycin complex 2 (mTORC2) is a key activator of protein kinases that act downstream of insulin and growth factor signaling. Here we report that mice lacking the essential mTORC2 component rictor in liver (Lrictor(KO)) are unable to respond normally to insulin. In response to insulin, Lrictor(KO) mice failed to inhibit hepatic glucose output. Lrictor(KO) mice also fail to develop hepatic steatosis on a high fat diet and manifest half-normal serum cholesterol levels. This is accompanied by lower levels of expression of SREBP-1c and SREBP-2 and genes of fatty acid and cholesterol biosynthesis. Lrictor(KO) mice had defects in insulin-stimulated Akt Ser-473 and Thr-308 phosphorylation, leading to decreased phosphorylation of Akt substrates FoxO, GSK-3β, PRAS40, AS160, and Tsc2. Lrictor(KO) mice also manifest defects in insulin-activated mTORC1 activity, evidenced by decreased S6 kinase and Lipin1 phosphorylation. Glucose intolerance and insulin resistance of Lrictor(KO) mice could be fully rescued by hepatic expression of activated Akt2 or dominant negative FoxO1. However, in the absence of mTORC2, forced Akt2 activation was unable to drive hepatic lipogenesis. Thus, we have identified an Akt-independent relay from mTORC2 to hepatic lipogenesis that separates the effects of insulin on glucose and lipid metabolism.     

Multisite phosphorylation regulates Bim stability and apoptotic activity

The proapoptotic BH3-only protein Bim is established to be an important mediator of signaling pathways that induce cell death. Multisite phosphorylation of Bim by several members of the MAP kinase group is implicated as a regulatory mechanism that controls the apoptotic activity of Bim. To test the role of Bim phosphorylation in vivo, we constructed mice with a series of mutant alleles that express phosphorylation-defective Bim proteins. We show that mutation of the phosphorylation site Thr-112 causes decreased binding of Bim to the antiapoptotic protein Bcl2 and can increase cell survival. In contrast, mutation of the phosphorylation sites Ser-55, Ser-65, and Ser-73 can cause increased apoptosis because of reduced proteasomal degradation of Bim. Together, these data indicate that phosphorylation can regulate Bim by multiple mechanisms and that the phosphorylation of Bim on different sites can contribute to the sensitivity of cellular apoptotic responses.     

Interplay and effects of temporal changes in the phosphorylation state of serine-302, -307, and -318 of insulin receptor substrate-1 on insulin action in skeletal muscle cells

Transduction of the insulin signal is mediated by multisite Tyr and Ser/Thr phosphorylation of the insulin receptor substrates (IRSs). Previous studies on the function of single-site phosphorylation, particularly phosphorylation of Ser-302, -307, and -318 of IRS-1, showed attenuating as well as enhancing effects on insulin action. In this study we investigated a possible cross talk of these opposedly acting serine residues in insulin-stimulated skeletal muscle cells by monitoring phosphorylation kinetics, and applying loss of function, gain of function, and combination mutants of IRS-1. The phosphorylation at Ser-302 was rapid and transient, followed first by Ser-318 phosphorylation and later by phosphorylation of Ser-307, which remained elevated for 120 min. Mutation of Ser-302 to alanine clearly reduced the subsequent protein kinase C-zeta-mediated Ser-318 phosphorylation. The Ser-307 phosphorylation was independent of Ser-302 and/or Ser-318 phosphorylation status. The functional consequences of these phosphorylation patterns were studied by the expression of IRS-1 mutants. The E302A307E318 mutant simulating the early phosphorylation pattern resulted in a significant increase in Akt and glycogen synthase kinase 3 phosphorylation. Furthermore, glucose uptake was enhanced. Because the down-regulation of the insulin signal was not affected, this phosphorylation pattern seems to be involved in the enhancement but not in the termination of the insulin signal. This enhancing effect was completely absent when Ser-302 was unphosphorylated and Ser-307 was phosphorylated as simulated by the A302E307E318 mutant. Phospho-Ser-318, sequentially phosphorylated at least by protein kinase C-zeta and a mammalian target of rapamycin/raptor-dependent kinase, was part of the positive as well as of the subsequent negative phosphorylation pattern. Thus we conclude that insulin stimulation temporally generates different phosphorylation statuses of the same residues that exert different functions in insulin signaling.     

Common inhibitory serine sites phosphorylated by IRS-1 kinases, triggered by insulin and inducers of insulin resistance

The Insulin Receptor Substrate (IRS) proteins are key players in insulin signal transduction and are the best studied targets of the insulin receptor. Ser/Thr phosphorylation of IRS proteins negatively modulates insulin signaling; therefore, the identification of IRS kinases and their target Ser phosphorylation sites is of physiological importance. Here we show that in Fao rat hepatoma cells, the IkappaB kinase beta (IKKbeta) is an IRS-1 kinase activated by selected inducers of insulin resistance, including sphingomyelinase, ceramide, and free fatty acids. Moreover, IKKbeta shares a repertoire of seven potential target sites on IRS-1 with protein kinase C zeta (PKCzeta), an IRS-1 kinase activated both by insulin and by inducers of insulin resistance. We further show that mutation of these seven sites (Ser-265, Ser-302, Ser-325, Ser-336, Ser-358, Ser-407, and Ser-408) confers protection from the action of IKKbeta and PKCzeta when they are overexpressed in Fao cells or primary hepatocytes. This enables the mutated IRS proteins to better propagate insulin signaling. These findings suggest that insulin-stimulated IRS kinases such as PKCzeta overlap with IRS kinases triggered by inducers of insulin resistance, such as IKKbeta, to phosphorylate IRS-1 on common Ser sites.     

Phosphorylation of Dopamine Transporter Serine 7 Modulates Cocaine Analog Binding

As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (−)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.     

S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-(alpha) signaling through IKK2

S6K1 (p70S6K) is a serine kinase downstream from Akt in the insulin signaling pathway that is involved in negative feedback regulation of insulin action. S6K1 is also activated by TNF-alpha, a pro-inflammatory cytokine. However, its role remains to be characterized. In the current study, we elucidated a mechanism for S6K1 to mediate TNF-alpha-induced insulin resistance in adipocytes and hepatocytes. S6K1 was phosphorylated at Thr-389 in response to TNF-alpha. This led to phosphorylation of IRS-1 by S6K1 at multiple serine residues including Ser-270, Ser-307, Ser-636, and Ser-1101 in human IRS-1 (Ser-265, Ser-302, Ser-632, and Ser-1097, in rodent IRS-1). Direct phosphorylation of these sites by S6K1 was observed in an in vitro kinase assay using purified IRS-1 and S6K1. Phosphorylation of all these serines was increased in the adipose tissue of obese mice. RNAi knockdown demonstrated an important role for S6K1 in mediating TNF-alpha-induced IRS-1 inhibition that led to impaired insulin-stimulated glucose uptake in adipocytes. A point mutant of IRS-1 (S270A) impaired association of IRS-1 with S6K1 resulting in diminished phosphorylation of IRS-1 at three other S6K1 phosphorylation sites (Ser-307, Ser-636, and Ser-1101). Expression of a dominant negative S6K1 mutant prevented TNF-induced Ser-270 phosphorylation and IRS-1 protein degradation. Moreover, in IKK2 (but not IKK1)-null cells, TNF-alpha treatment did not result in Thr-389 phosphorylation of S6K1. We present a new mechanism for TNF-alpha to induce insulin resistance that involves activation of S6K by an IKK2-dependent pathway. S6K directly phosphorylates IRS-1 on multiple serine residues to inhibit insulin signaling.     

Ser-64 and Ser-111 in PHAS-I are dispensable for insulin-stimulated dissociation from eIF4E

Insulin stimulates phosphorylation of multiple sites in the eIF4E-binding protein, PHAS-I, leading to dissociation of the PHAS-I.eIF4E complex and to an increase in cap-dependent translation. The Ser-64 and Ser-111 sites have been proposed to have key roles in controlling the association of PHAS-I and eIF4E. To determine whether the effects of insulin require these sites, we assessed the control of PHAS-I proteins having Ala-64 or Ala-111 mutations. The results indicate that phosphorylation of neither site is required for insulin to promote release of PHAS-I from eIF4E. Also, the mutation of Ser-111, which has been proposed to serve as a necessary priming site for the phosphorylation of other sites in PHAS-I, did not affect the phosphorylation of Thr-36/45, Ser-64, or Thr-69. Insulin promoted the release of eIF4E from PHAS-II, a PHAS isoform that lacks the Ser-111 site, but it was without effect on the amount of eIF4E bound to the third isoform, PHAS-III. The results demonstrate that contrary to widely accepted models, Ser-64 and Ser-111 are not required for the control of PHAS-I binding to eIF4E in cells, implicating phosphorylation of the Thr sites in dissociation of the PHAS-I.eIF4E complex. The findings also indicate that PHAS-II, but not PHAS-III, contributes to the control of protein synthesis by insulin.     

Insulin-stimulated protein kinase B phosphorylation on Ser-473 is independent of its activity and occurs through a staurosporine-insensitive kinase

Full activation of protein kinase B (PKB, also called Akt) requires phosphorylation on two regulatory sites, Thr-308 in the activation loop and Ser-473 in the hydrophobic C-terminal regulatory domain (numbering for PKB alpha/Akt-1). Although 3'-phosphoinositide-dependent protein kinase 1 (PDK1) has now been identified as the Thr-308 kinase, the mechanism of the Ser-473 phosphorylation remains controversial. As a step to further characterize the Ser-473 kinase, we examined the effects of a range of protein kinase inhibitors on the activation and phosphorylation of PKB. We found that staurosporine, a broad-specificity kinase inhibitor and inducer of cell apoptosis, attenuated PKB activation exclusively through the inhibition of Thr-308 phosphorylation, with Ser-473 phosphorylation unaffected. The increase in Thr-308 phosphorylation because of overexpression of PDK1 was also inhibited by staurosporine. We further show that staurosporine (CGP 39360) potently inhibited PDK1 activity in vitro with an IC(50) of approximately 0.22 microm. These data indicate that agonist-induced phosphorylation of Ser-473 of PKB is independent of PDK1 or PKB activity and occurs through a distinct Ser-473 kinase that is not inhibited by staurosporine. Moreover, our results suggest that inhibition of PKB signaling is involved in the proapoptotic action of staurosporine.     

cAMP-dependent protein kinase (PKA) inhibits T cell activation by phosphorylating ser-43 of raf-1 in the MAPK/ERK pathway

cAMP-dependent protein kinase (PKA) has been suggested to interfere with T-cell activation by inhibiting interleukin (IL-2) receptor alpha-chain (CD25) expression and IL-2 production. The Ras/MAP kinase pathway has been found to be necessary for induction of the IL-2 production. In this study, we have scrutinized the Ras/MAP kinase pathway in Jurkat T-cells to attempt to identify any sites for PKA-mediated regulatory phosphorylations. Here we unambiguously demonstrate that PKA directly inhibits anti-CD3-induced MAP kinase activation. In vitro phosphorylation experiments showed that Raf-1 was extensively phosphorylated by PKA, while ERK2 and MEK were not. Phosphopeptide mapping identified Ser-43 of Raf-1 as the only site phosphorylated by PKA in the Ras/MAPK pathway. Transient transfection experiments demonstrated that mutations of Ser-43 of the Raf-1 kinase were rendered insensitive to cAMP-mediated inhibition.     

Multiple phosphorylations of cytochrome c oxidase and their functions

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial electron transport chain, is regulated by isozyme expression, allosteric effectors such as the ATP/ADP ratio, and reversible phosphorylation. Of particular interest is the "allosteric ATP-inhibition," which has been hypothesized to keep the mitochondrial membrane potential at low healthy values (<140 mV), thus preventing the formation of superoxide radical anions, which have been implicated in multiple degenerative diseases. It has been proposed that the "allosteric ATP-inhibition" is switched on by the protein kinase A-dependent phosphorylation of COX. The goal of this study was to identify the phosphorylation site(s) involved in the "allosteric ATP-inhibition" of COX. We report the mass spectrometric identification of four new phosphorylation sites in bovine heart COX. The identified phosphorylation sites include Tyr-218 in subunit II, Ser-1 in subunit Va, Ser-2 in subunit Vb, and Ser-1 in subunit VIIc. With the exception of Ser-2 in subunit Vb, the identified phosphorylation sites were found in enzyme samples with and without "allosteric ATP inhibition," making Ser-2 of subunit Vb a candidate site enabling allosteric regulation. We therefore hypothesize that additional phosphorylation(s) may be required for the "allosteric ATP-inhibition," and that these sites may be easily dephosphorylated or difficult to identify by mass spectrometry.