The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor

Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRβ) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRβ′s tyrosine kinase activity and directly promotes IRβ interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

Structural basis for phosphotyrosine recognition by the Src homology-2 domains of the adapter proteins SH2-B and APS

SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 A resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS.     

Binding specificity of SH2 domains: insight from free energy simulations

Cellular signal transduction pathways are controlled by specific protein-protein interactions mediated by the binding of short peptides to small modular interaction domains. To gain insights into the specificity of these interactions, the association of phosphotyrosine-containing peptides to Src Homology 2 (SH2) domains is characterized using computations. Molecular dynamics simulations based on high-resolution crystal structures complemented by homology models are used to calculate the absolute binding free energies for 25 SH2-peptides pairs. The calculations are carried out using a potential of mean force free energy simulations method with restraining potentials that was developed previously (Woo and Roux, Proc Natl Acad Sci USA 2005;102:6825-6830). The method is utilized in conjunction with an implicit solvent representation to reduce the computational cost to characterize the association of five SH2 domains and five peptides. Specificity is ascertained by directly comparing the affinities of a given SH2 domain binding for any of the different peptides. For three of the five SH2 domains, the computational results rank the native peptides, as the most preferred binding motif. For the remaining two SH2 domains, high affinity binding motifs other than the native peptides are identified. This study illustrates how free energy computations can complement experiments in trying to elucidate complex protein-protein interactions networks.     

Substrates and signalling complexes: the tortured path to insulin action.

In the last few years several potential substrates of the insulin receptor tyrosine kinase have been identified, purified, and their cDNAs isolated. These putative substrates include: 1) pp15, a fatty acid-binding protein; 2) pp120, a plasma membrane ecto-ATPase; 3) pp42, a MAP serine/threonine kinase; 4) pp85, a subunit of the Type 1 phosphatidylinositol kinase; and 5) pp185, a phosphatidylinositol kinase binding protein. Although the tyrosine phosphorylation of several of these substrates correlates with the signalling capabilities of various mutant receptors, the role of these substrates in mediating any one of insulin's many biological responses is still unknown. In addition, recent data indicate that the tyrosine phosphorylation of pp42 may in fact be due to autophosphorylation, thereby removing it from the list of putative substrates of the insulin receptor kinase. Finally, the present review discusses the question of whether signalling occurs as a result of the tyrosine phosphorylation of substrates or via the formation of signalling complexes.     

Transient ischemia enhances tyrosine phosphorylation and binding of the NMDA receptor to the Src homology 2 domain of phosphatidylinositol 3-kinase in the rat hippocampus

Tyrosine phosphorylation of the NMDA receptor has been implicated in the regulation of the receptor channel. We investigated the effects of transient (15 min) global ischemia on tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B, and the interaction of NR2 subunits with the SH2 domain of phosphatidylinositol 3-kinase (PI3-kinase) in vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Transient ischemia induced a marked increase in the tyrosine phosphorylation of NR2A in both regions. The tyrosine phosphorylation of NR2B in CA3/dentate gyrus after transient ischemia was sustained and greater than that in CA1. PI3-kinase p85 was co-precipitated with NR2B after transient global ischemia. The SH2 domain of the p85 subunit of PI3-kinase bound to NR2B, but not to NR2A. Binding to NR2B was increased following ischemia and the increase in binding in CA3/dentate gyrus (4.5-fold relative to sham) was greater than in CA1 (1.7-fold relative to sham) at 10 min of reperfusion. Prior incubation of proteins with an exogenous protein tyrosine phosphatase or with a phosphorylated peptide (pYAHM) prevented binding. The results suggest that sustained increases in tyrosine phosphorylation and increased interaction of NR2B with the SH2 domain of PI3-kinase may contribute to altered signal transduction in the CA3/dentate gyrus after transient ischemia.     

Phosphorylation of glycolytic and gluconeogenic enzymes by the insulin receptor kinase

Various glycolytic and gluconeogenic enzymes were tested as substrates for the insulin receptor kinase. Phosphofructokinase and phosphoglycerate mutase were found to be the best substrates. Phosphorylation of these enzymes was rapid, stimulated 2- to 6-fold by 10(-7) M insulin and occurred exclusively on tyrosine residues. Enolase, fructose 1,6-bisphosphatase, lactate dehydrogenases in decreasing order, were also subject to insulin-stimulated phosphorylation but to a smaller extent than that for phosphofructokinase or phosphoglycerate mutase. The phosphorylation of phosphofructokinase was studied most extensively since phosphofructokinase is known to catalyze a rate-limiting step in glycolysis. The apparent Km of the insulin receptor for phosphofructokinase was 0.1 microM, which is within the physiologic range of concentration of this enzyme in most cells. Tyrosine phosphorylation of phosphofructokinase paralleled autophosphorylation of the beta-subunit of the insulin receptor with respect to time course, insulin dose response (half maximal effect between 10(-9) and 10(-8) M insulin), and cation requirement (Mn2+ greater than Mg2+ much greater than Ca2+). Further study will be required to determine whether the tyrosine phosphorylation of phosphofructokinase plays a role in insulin-stimulated increases in glycolytic flux.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins. J. Cell. Biochem. 68:139–150, 1998. © 1998 Wiley-Liss, Inc.     

Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.     

Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.     

Regulation of the insulin signalling pathway by cellular protein-tyrosine phosphatases

Protein-tyrosine phosphatases (PTPases) have been implicated in the physiological regulation of the insulin signalling pathway. In cellular and molecular studies, the transmembrane, receptor-type PTPase LAR and the intracellular, non-receptor enzyme PTP1B have been shown to have a direct impact on insulin action in intact cell models. Since insulin signalling can be enhanced by reducing the abundance or activity of specific PTPases, pharmaceutical agents directed at blocking the interaction between individual PTPases and the insulin receptor may have potential clinical relevance to the treatment of insulin-resistant states such as obesity and Type II diabetes mellitus.     

基于SH2超亲体的微量样品酪氨酸磷酸化蛋白质组学技术研究进展及应用

酪氨酸磷酸化是生物体内一种重要的蛋白质磷酸化修饰类型,参与细胞信号转导、细胞迁移、凋亡等众多的生命活动过程.在磷酸化蛋白质组学研究中,由于酪氨酸磷酸化蛋白丰度低且有时起始样品量有限,传统的磷酸化蛋白质组富集方法用于酪氨酸磷酸化肽富集效率较低.微量样品的制备技术以及SH2超亲体的引入正在改变酪氨酸磷酸化蛋白质组研究的现状...     

Proline Isomerization Preorganizes the Itk SH2 Domain for Binding to the Itk SH3 Domain

We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires.     

In vivo activation of insulin receptor tyrosine kinase by melatonin in the rat hypothalamus

Melatonin is the pineal hormone that acts via a pertussis toxin-sensitive G-protein to inhibit adenylate cyclase. However, the intracellular signalling effects of melatonin are not completely understood. Melatonin receptors are mainly present in the suprachiasmatic nucleus (SCN) and pars tuberalis of both humans and rats. The SCN directly controls, amongst other mechanisms, the circadian rhythm of plasma glucose concentration. In this study, using immunoprecipitation and immunoblotting, we show that melatonin induces rapid tyrosine phosphorylation and activation of the insulin receptor beta-subunit tyrosine kinase (IR) in the rat hypothalamic suprachiasmatic region. Upon IR activation, tyrosine phosphorylation of IRS-1 was detected. In addition, melatonin induced IRS-1/PI3-kinase and IRS-1/SHP-2 associations and downstream AKT serine phosphorylation and MAPK (mitogen-activated protein kinase) phosphorylation, respectively. These results not only indicate a new signal transduction pathway for melatonin, but also a potential cross-talk between melatonin and insulin.     

Phosphorylation of the insulin receptor by casein kinase I

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.     

High‐resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase

Fes and Fes‐related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N‐terminal Fer/CIP4 homology‐Bin/Amphiphysin/Rvs (F‐BAR) domain, a central Src homology 2 (SH2) domain, and a C‐terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine‐containing ligands to the SH2 domain. Here, the X‐ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D‐E‐pY‐E‐N‐V‐D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer‐phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I β‐turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.     

Thy-1 associated pp85--90 is a potential docking site for SH2 domain-containing signal transduction molecules

Thy-1, a glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed at high levels on thymocytes, has been implicated in positive and negative signal transduction. We show that Thy-1 associates with a protein of 85--90 kDa, which is prominently phosphorylated in vitro as well as in vivo following the stimulation of thymocytes with pervanadate. pp85--90 is not identical to known proteins that are phosphorylated following T cell activation. The SH2 domains of fyn, csk, phosphatidylinositol 3'-kinase, rasGAP, vav and lck bind to pp85--90 with varying affinities. The SH2 domains of ZAP70, SHP-1 and PLC gamma 1 and the SH3 domains of lck, vav and HS1 did not bind to pp85--90. The molecular weight, iso-electric point, efficient phosphorylation by fyn and lck and preferential binding to the SH2 domain of fyn compared to that of lck indicate that Thy-1-associated pp85-90 may be identical to a recently cloned, fyn-associated transmembrane adaptor protein, PAG-85.     

Role of binding proteins to IRS-1 in insulin signalling

Insulin elicits its divergent metabolic and mitogenic effects by binding to its specific receptor, which belongs to the family of receptor tyrosine kinases. The activated insulin receptor phosphorylates the intracellular substrate IRS-1, which then binds various signalling molecules that contain SRC homology 2 domains, thereby propagating the insulin signal. Among these IRS-1-binding proteins, the Grb2-Sos complex and the protein tyrosine phosphatase SHP-2 transmit mitogenic signals through the activation of Ras, and phosphoinositide 3-kinase is implicated in the major metabolic actions of insulin. Although substantial evidence indicates the importance of IRS-1 in insulin signal transduction, the generation of IRS-1-deficient mice has revealed the existence of redundant signalling pathways.