Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.

The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.     

Dual regulation of glycogen synthase kinase-3beta by the alpha1A-adrenergic receptor

Catecholamines, acting through adrenergic receptors, play an important role in modulating the effects of insulin on glucose metabolism. Insulin activation of glycogen synthesis is mediated in part by the inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3). In this study, catecholamine regulation of GSK-3beta was investigated in Rat-1 fibroblasts stably expressing the alpha1A-adrenergic receptor. Treatment of these cells with either insulin or phenylephrine (PE), an alpha1-adrenergic receptor agonist, induced Ser-9 phosphorylation of GSK-3beta and inhibited GSK-3beta activity. Insulin-induced GSK-3beta phosphorylation is mediated by the phosphatidylinositol 3-kinase/Akt signaling pathway. PE treatment does not activate phosphatidylinositol 3-kinase or Akt (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z. (2000) J. Biol. Chem. 275, 4803-4809), but instead inhibits insulin-induced Akt activation and GSK-3beta phosphorylation. Experiments using protein kinase C (PKC) inhibitors suggest that phorbol ester-sensitive novel PKC and G? 6983-sensitive atypical PKC isoforms are involved in the PE-induced phosphorylation of GSK-3beta. Indeed, PE treatment of Rat-1 cells increased the activity of atypical PKCzeta, and expression of PKCzeta in COS-7 cells stimulated GSK-3beta Ser-9 phosphorylation. In addition, PE-induced GSK-3beta phosphorylation was reduced in Rat-1 cells treated with a cell-permeable PKCzeta pseudosubstrate peptide inhibitor. These results suggest that the alpha1A-adrenergic receptor regulates GSK-3beta through two signaling pathways. One pathway inhibits insulin-induced GSK-3beta phosphorylation by blocking insulin activation of Akt. The second pathway stimulates Ser-9 phosphorylation of GSK-3beta, probably via PKC.     

Akt/protein kinase B isoforms are differentially regulated by epidermal growth factor stimulation

Overexpression of epidermal growth factor receptor (EGFR) in certain cancers is well established. There is growing evidence that epidermal growth factor (EGF) activates Akt/protein kinase B (PKB) in a phosphoinositide 3-OH kinase (PI3K)-dependent manner, but it is not yet clear which Akt isoforms are involved in this signal transduction pathway. We investigated the functional regulation of three Akt isoforms, Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma, in esophageal cancer cells where EGFR is frequently overexpressed. Upon EGF simulation, phosphorylation of Akt1 at the Ser-473 residue was remarkably induced. This result was corroborated by in vitro Akt kinase assays using glycogen synthase kinase 3beta as the substrate. PI3K inhibitors, wortmannin or LY294002, significantly blocked the Akt kinase activity induced by EGF. Akt2 activity was evaluated by electrophoretic mobility shift assays. Robust activation of Akt2 by EGF was observed in some cell lines in a PI3K-dependent manner. EGF-induced Akt3 activation was demonstrated by Ser-472 phosphorylation of Akt3 but in a restrictive fashion. In aggregate, EGF-mediated activation of Akt isoforms is overlapping and distinctive. The mechanism by which EGFR recruits the PI3K/Akt pathway was in part differentially regulated at the level of Ras but independent of heterodimerization of EGFR with either ErbB2 or ErbB3 based upon functional dissection of pathways in esophageal cancer cell lines.     

Exercise does not alter subcellular localization, but increases phosphorylation of insulin-signaling proteins in human skeletal muscle

The subcellular localization of insulin signaling proteins is altered by various stimuli such as insulin, insulin-like growth factor I, and oxidative stress and is thought to be an important mechanism that can influence intracellular signal transduction and cellular function. This study examined the possibility that exercise may also alter the subcellular localization of insulin signaling proteins in human skeletal muscle. Nine untrained males performed 60 min of cycling exercise (approximately 67% peak pulmonary O2 uptake). Muscle biopsies were sampled at rest, immediately after exercise, and 3 h postexercise. Muscle was fractionated by centrifugation into the following crude fractions: cytosolic, nuclear, and a high-speed pellet containing membrane and cytoskeletal components. Fractions were analyzed for protein content of insulin receptor, insulin receptor substrate (IRS)-1 and -2, p85 subunit of phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3). There was no significant change in the protein content of the insulin signaling proteins in any of the crude fractions after exercise or 3 h postexercise. Exercise had no significant effect on the phosphorylation of IRS-1 Tyr612 in any of the fractions. In contrast, exercise increased (P < 0.05) the phosphorylation of Akt Ser473 and GSK-3alpha/beta Ser9/21 in the cytosolic fraction only. In conclusion, exercise can increase phosphorylation of downstream insulin signaling proteins specifically in the cytosolic fraction but does not result in changes in the subcellular localization of insulin signaling proteins in human skeletal muscle. Change in the subcellular protein localization is therefore an unlikely mechanism to influence signal transduction pathways and cellular function in skeletal muscle after exercise.     

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.     

Dual phospholipase C/diacylglycerol requirement for protein kinase D1 activation in lymphocytes

The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.     

Reconstitution of phosphoinositide 3-kinase-dependent insulin signaling in a cell-free system

Early insulin signaling events were examined in a novel cell-free assay utilizing subcellular fractions derived from 3T3-L1 adipocytes. The following cellular processes were observed in vitro in a manner dependent on insulin, time of incubation, and exogenous ATP: 1) autophosphorylation and activation of the insulin receptor; 2) tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1); 3) association of tyrosine-phosphorylated IRS-1 with phosphoinositide 3-kinase; 4) activation of the kinase Akt via its phosphorylation on Thr-308 and Ser-473; and 5) phosphorylation of glycogen synthase kinase-3 by activated Akt. The activation of Akt in vitro was abolished in the presence of the phosphoinositide 3-kinase inhibitor, wortmannin, thus recapitulating the most notable regulatory feature of Akt observed in vivo. Evidence is presented indicating that the critical spatial compartmentalization of signaling molecules necessary for efficient signal transduction is likely to be preserved in the cell-free system. Additionally, data are provided demonstrating that full Akt activation in this system is dependent on plasma membrane-associated IRS-1, cannot be mediated by robust cytosol-specific tyrosine phosphorylation of IRS-1, and occurs in the complete absence of detectable IRS-2 phosphorylation in the cytosol and plasma membrane.     

Altered profile of gene expression in rat hearts induced by chronic nicotine consumption

Using a cDNA microarray technique, we analyzed the expression profile of 1081 genes in the whole heart tissue of rats. The expressions of three classes of genes encoding cellular energy metabolism enzymes, transmembrane receptors, and intracellular kinase network members were reduced by more than 2.5-fold in cardiac tissues from the rats fed with nicotine (3mg/kg/day) for 3 months. The down-regulated 11 genes included mitochondrial ATP synthase beta subunit, mitochondrial H(+) transporting ATP synthase F1 complex alpha subunit isoform 1, liver mitochondrial aldehyde dehydrogenase 2, glutathione-S-transferase mu type 2, corticotropin-releasing factor receptor 2, metabotropic glutamate receptor 2, N-methyl-D-aspartate receptor subtype 2B, muscarinic acetylcholine receptor M3, transmembrane receptor Unc5H1, glycogen synthase kinase 3alpha, and Ca(2+)/calmodulin-dependent protein kinase II beta subunit. It appears that chronic nicotine treatment affects cardiac function by modulating the expressions of genes involved in energy metabolism and signal transduction.     

The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiating signal transduction in NIH3T3 cells.

The ability of the receptor for the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to function in non-hematopoietic cells is unknown. NIH3T3 fibroblasts were transfected with cDNAs encoding the alpha and beta subunit of the human GM-CSF receptor and a series of stable transformants were isolated that bound GM-CSF with either low (KD = 860 - > 1000 pM) or high affinity (KD = 20-80 pM). Low affinity receptors were not functional. However, the reconstituted high affinity receptors were found to be capable of activating a number of signal transduction pathways, including tyrosine kinase activity, phosphorylation of Raf-1, and the transient induction of c-fos and c-myc mRNAs. The activation of protein tyrosine phosphorylation by GM-CSF in NIH3T3 cells was rapid (< 1 min) and transient (peaking at 5-20 min) and resulted in the phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58-60 kDa. Some of these proteins co-migrated with proteins from myeloid cells that were phosphorylated on tyrosine residues in response to GM-CSF. In particular, p42 and p44 were identified as mitogen-activated protein kinases (MAP kinases), and the phosphorylation on tyrosine residues of p42 and p44 MAP kinases occurred at the same time as the phosphorylation of Raf-1. However, despite evidence for activation of many mitogenic signal transduction molecules, GM-CSF did not induce significant proliferation of transfected NIH3T3 cells. These results suggest that murine fibroblasts contain signal transducing molecules that can effectively interact with the human GM-CSF receptor, and that are sufficient to activate at least some of the same signal transduction pathways this receptor activates in myeloid cells, including activation of one or more tyrosine kinase(s). However, the level of activation of signal transduction is either below a threshold of necessary activity or at least one mitogenic signal necessary for proliferation is missing.     

Role of Akt2 in contraction-stimulated cell signaling and glucose uptake in skeletal muscle

The serine/threonine kinase Akt/PKB plays diverse roles in cells, and genetic studies have indicated distinct roles for the three Akt isoforms expressed in mammalian cells and tissues. Akt2 is a key signaling intermediate for insulin-stimulated glucose uptake and glycogen synthesis in skeletal muscle. Akt2 has also been shown to be activated by exercise and muscle contraction in both rodents and humans. In this study, we used Akt2 knockout mice to explore the role of Akt2 in exercise-stimulated glucose uptake and glycogen synthesis as well as intracellular signaling pathways that regulate glycogen metabolism in skeletal muscle. We found that Akt2 deficiency does not affect basal or exercise-stimulated glucose uptake or intracellular glycogen content in the soleus muscle. In addition, lack of Akt2 did not result in alterations in basal Akt Thr(308) or basal and contraction-stimulated glycogen synthase kinase-3beta (GSK-3beta) Ser(9) phosphorylation, glycogen synthase phosphorylation, or glycogen synthase activity. In contrast, in situ contraction failed to elicit normal increases in Akt T-loop Thr(308) phosphorylation and GSK-3alpha Ser(21) phosphorylation in tibialis anterior muscles from Akt2-deficient animals. Our data establish a key role for Akt2 in the regulation of GSK-3alpha Ser(21) phosphorylation with contraction and add genetic evidence to support the separation of the intracellular pathways regulated by insulin and exercise that converge on glucose uptake and glycogen synthesis in skeletal muscle.     

Mechanistic analysis of the amplification and diversification events induced by Vav proteins in B-lymphocytes

Vav proteins participate in the assembly of a multibranched signal transduction pathway in lymphocytes, including the stimulation of the phosphatidylinositol 3-kinase/protein kinase B and the phospholipase C-gamma/Ras GDP-releasing protein/Ras/Erk routes. In the present work, we used a genetic approach in chicken DT40 B-cell lines to investigate additional elements of the Vav route, the synergisms existing among the different Vav signaling branches, and the activities exerted by wild-type and oncogenic Vav proteins in B-lymphocytes. We show here that the Vav pathway is ramified in B-lymphocytes in additional diacylglycerol-dependent signaling branches such as those involving protein kinase C, protein kinase D, and phospholipase D. By using side-by-side comparisons of the activation levels of those signal transduction pathways in inhibitor-treated and knockout DT40 cells, we show that B-cells have different requirements regarding Vav proteins for the activation of antigen receptor downstream elements. Furthermore, we have detected interpathway cross-talk at the level of the most proximal elements but not among the most distal effector molecules of the Vav route. Finally, we show that the oncogenic versions of Vav1 and RhoA can activate alternative routes that could contribute to signal amplification and diversification events in transformed lymphocytes.     

Signal Transduction Cross Talk Mediated by Jun N-Terminal Kinase-Interacting Protein and Insulin Receptor Substrate Scaffold Protein Complexes

Scaffold proteins have been established as important mediators of signal transduction specificity. The insulin receptor substrate (IRS) proteins represent a critical group of scaffold proteins that are required for signal transduction by the insulin receptor, including the activation of phosphatidylinositol 3 kinase. The c-Jun NH₂-terminal kinase (JNK)-interacting proteins (JIPs) represent a different group of scaffold molecules that are implicated in the regulation of the JNK. These two signaling pathways are functionally linked because JNK can phosphorylate IRS1 on the negative regulatory site Ser-307. Here we demonstrate the physical association of these signaling pathways using a proteomic approach that identified insulin-regulated complexes of JIPs together with IRS scaffold proteins. Studies using mice with JIP scaffold protein defects confirm that the JIP1 and JIP2 proteins are required for normal glucose homeostasis. Together, these observations demonstrate that JIP proteins can influence insulin-stimulated signal transduction mediated by IRS proteins.The c-Jun NH₂-terminal kinase (JNK)-interacting proteins (JIPs) are implicated in the regulation of the JNK signal transduction pathway (8, 28). The JIP1 and JIP2 proteins are structurally related with similar modular domains (SH3 and PTB) and binding sites for the mixed-lineage protein kinase (MLK) group of mitogen-activated protein kinase (MAPK) kinase kinases, the MAPK kinase MKK7, and JNK (19). These JIP proteins also interact with the microtubule motor protein kinesin, several guanine nucleotide exchange factors, the phosphatase MKP7, Src-related protein kinases, and AKT to form multifunctional protein complexes (19).One potential physiological role of JIP scaffold proteins is the response to metabolic stress, insulin resistance, and diabetes. Several lines of evidence support this hypothesis. First, JIP1 is required for metabolic stress-induced activation of JNK in white adipose tissue (12). Second, MLKs that interact with JIP proteins are implicated as essential components of a signaling pathway that mediates the effects of metabolic stress on JNK activation (13). Third, studies have demonstrated that the human Jip1 gene may contribute to the development of type 2 diabetes, because a Jip1 missense mutation was found to segregate with type 2 diabetes (26). Collectively, these data suggest that JIP proteins play a role in the cellular response to metabolic stress and the regulation of insulin resistance.It is established that the insulin receptor substrate (IRS) group of scaffold proteins plays a central role in insulin signaling (27). Treatment of cells with insulin causes tyrosine phosphorylation of the insulin receptor, the recruitment of IRS proteins to the insulin receptor, and the subsequent tyrosine phosphorylation of IRS proteins on multiple residues that act as docking sites for insulin-regulated signaling molecules, including phosphatidylinositol 3 kinase (27). Negative regulation of IRS proteins is implicated as a mechanism of insulin resistance and can be mediated by multiple pathways, including IRS protein phosphorylation and degradation. Thus, the mTOR/p70^S6K (21, 22, 24) and the SOCS-1/3 (20) signaling pathways can regulate IRS protein degradation. Multisite phosphorylation on Ser/Thr residues can also regulate IRS protein function, including JNK phosphorylation of IRS1 on the inhibitory site Ser-307 that prevents recruitment of IRS1 to the activated insulin receptor (2).The IRS and JIP groups of scaffold proteins may function independently to regulate JNK-dependent and insulin-dependent signal transduction. However, functional connections between these scaffold proteins have been identified. Thus, studies using Jip1^−/− mice demonstrate that JIP1 is required for high-fat-diet-induced JNK activation in white adipose tissue, IRS1 phosphorylation on the inhibitory site Ser-307, and insulin resistance (12). These data suggest that JIP scaffold proteins function cooperatively with IRS proteins to regulate signal transduction by the insulin receptor. The purpose of this study was to examine cross talk between the JIP and IRS scaffold complexes. We demonstrate that the JIP and IRS scaffold complexes physically interact in an insulin-dependent manner and confirm that JIP proteins influence normal glucose homeostasis.     

Glycogen Synthase Kinase-3β Is Involved in Electroacupuncture Pretreatment via the Cannabinoid CB1 Receptor in Ischemic Stroke

We have previously shown that electroacupuncture (EA) pretreatment produces neuroprotective effects, which were mediated through an endocannabinoid signal transduction mechanism. Herein, we have studied the possible contribution of the phosphorylated form of glycogen synthase kinase-3β (GSK-3β) in EA pretreatment-induced neuroprotection via the cannabinoid CB1 receptor (CB1R). Focal transient cerebral ischemia was induced by middle cerebral artery occlusion in rats. Phosphorylation of GSK-3β at Ser-9 [p-GSK-3β (Ser-9)] was evaluated in the penumbra tissue following reperfusion. Infarct size and neurological score were assessed in the presence of either PI3K inhibitors or a GSK-3β inhibitor 72 h after reperfusion. Cellular apoptosis was evidenced by TUNEL staining and determination of the Bax/Bcl-2 ratio 24 h after reperfusion. The present study showed that EA pretreatment increased p-GSK-3β(Ser-9) 2 h after reperfusion in the ipsilateral penumbra. Augmented phosphorylation of GSK-3β induced similar neuroprotective effects as did EA pretreatment. By contrast, inhibition of PI3K dampened the levels of p-GSK-3β(Ser-9), and reversed not only the neuroprotective effect but also the anti-apoptotic effect following EA pretreatment. Regulation of GSK-3β by EA pretreatment was abolished following treatment with a CB1R antagonist and CB1R knockdown, whereas two CB1R agonists enhanced the phosphorylation of GSK-3β. Therefore we conclude that EA pretreatment protects against cerebral ischemia/reperfusion injury through CB1R-mediated phosphorylation of GSK-3β.     

Cross-talk between the two divergent insulin signaling pathways is revealed by the protein kinase B (Akt)-mediated phosphorylation of adapter protein APS on serine 588

The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.     

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.