Detectable Tyrosine Phosphorylation Research Articles

Label-Free SERS Sensors for Real-Time Monitoring of Tyrosine Phosphorylation.

Dysregulation of receptor tyrosine kinases (RTKs) has been shown to correlate with cancer cell proliferation and drug resistance. Thus, monitoring the activity of RTKs at a chemical level could provide new biomedical insights and methods to assess the drug efficacy. However, direct monitoring of kinase activity is challenging and most commonly relies on in vitro techniques such as Western blotting and ELISAs. Herein, we report the development of a gold nanoparticle-based surface-enhanced Raman scattering (SERS) sensor, which allows the real-time monitoring of tyrosine phosphorylation of a reporter peptide (Axltide) by the Axl enzyme. We demonstrate that our sensor shows strong signal localization, and we are able to detect tyrosine phosphorylation of the reporter peptide through chemical phosphorylation and enzymatically with similar peak changes to those observed in the spontaneous Raman spectra. Through monitoring the SERS spectrum, we can observe changes in phosphorylation in real time.

Network pharmacology and experiment indicated that medicinal food homologous components play important roles in insomnia

AbstractInsomnia refers to a subjective experience where insufficient sleep duration and quality affect daytime social functioning. This study aims to screen bioactive components with medicinal food homologous (MFH), such as quercetin, beta‐sitosterol, kaempferol, stigmasterol, and capsaicin, which can treat insomnia and demonstrate that quercetin acts as a direct inhibitor of insomnia‐related glucosylceramidase and as an insulin receptor tyrosine phosphatase inhibitor in the upstream insulin signaling pathway, affecting insomnia targets in the downstream pathway for the treatment of insomnia, using network pharmacology and cell experiment approaches. The results showed that 703 components in 44 MFH were enriched in 139 metabolic pathways and acted on 210 potential targets of insomnia. The protein–protein interaction network is analyzed by cytoNCA, according to the topological analysis results, quercetin, beta‐sitosterol, kaempferol, stigmasterol, and capsaicin of betweenness ranked in the top 10. Quercetin and glucosylceramidase were shown to directly inhibit insomnia proteins through reverse molecular docking. Quercetin was used to culture and stimulate HepG2 cells to detect tyrosine phosphorylation of key cellular proteins in the insulin signaling pathway, concluding that quercetin inhibits the activity of tyrosine phosphatase in a dose‐dependent manner, activating the insulin signaling pathway, triggering a cascade reaction, and affecting the expression of insomnia targets in the downstream pathway. The molecular docking of tyrosine phosphatase and quercetin revealed that quercetin interacted with it near the catalytic site, thereby inhibiting its interaction with the substrate. Overall, this study facilitates the development of medicinal food homologous, which exerts good pharmacological effects in the treatment of insomnia.

Novel Tyrosine Phosphorylation Signals in the Nucleus and on Mitotic Spindle Fibers and Lysosomes Revealed by Strong Inhibition of Tyrosine Dephosphorylation

Protein-tyrosine phosphorylation is one of the posttranslational modifications and plays critical roles in regulating a wide variety of cellular processes, such as cell proliferation, differentiation, adhesion, migration, survival, and apoptosis. Protein-tyrosine phosphorylation is reversibly regulated by protein-tyrosine kinases and protein-tyrosine phosphatases. Strong inhibition of protein-tyrosine phosphatase activities is required to undoubtedly detect tyrosine phosphorylation. Our extremely careful usage of Na3VO4, a potent protein-tyrosine phosphatase inhibitor, has revealed not only the different intracellular trafficking pathways of Src-family tyrosine kinase members but also novel tyrosine phosphorylation signals in the nucleus and on mitotic spindle fibers and lysosomes. Furthermore, despite that the first identified oncogene product v-Src is generally believed to induce transformation through continuous stimulation of proliferation signaling by its strong tyrosine kinase activity, v-Src-driven transformation was found to be caused not by continuous proliferation signaling but by v-Src tyrosine kinase activity-dependent stochastic genome alterations. Here, I summarize our findings regarding novel tyrosine phosphorylation signaling in a spatiotemporal sense and highlight the significance of the roles of tyrosine phosphorylation in transcriptional regulation inside the nucleus and chromosome dynamics.

Engineered proteins with sensing and activating modules for automated reprogramming of cellular functions

Protein-based biosensors or activators have been engineered to visualize molecular signals or manipulate cellular functions. Here we integrate these two functionalities into one protein molecule, an integrated sensing and activating protein (iSNAP). A prototype that can detect tyrosine phosphorylation and immediately activate auto-inhibited Shp2 phosphatase, Shp2-iSNAP, is designed through modular assembly. When Shp2-iSNAP is fused to the SIRPα receptor which typically transduces anti-phagocytic signals from the ‘don’t eat me’ CD47 ligand through negative Shp1 signaling, the engineered macrophages not only allow visualization of SIRPα phosphorylation upon CD47 engagement but also rewire the CD47-SIRPα axis into the positive Shp2 signaling, which enhances phagocytosis of opsonized tumor cells. A second SIRPα Syk-iSNAP with redesigned sensor and activator modules can likewise rewire the CD47-SIRPα axis to the pro-phagocytic Syk kinase activation. Thus, our approach can be extended to execute a broad range of sensing and automated reprogramming actions for directed therapeutics.

Tyrosine 601 of Bacillus subtilis DnaK Undergoes Phosphorylation and Is Crucial for Chaperone Activity and Heat Shock Survival.

In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple Stable Isotope Labeling by Amino acids in Cell culture-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type (WT), were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the WT dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

Click Conjugation of a Binuclear Terbium(III) Complex for Real-Time Detection of Tyrosine Phosphorylation

Phosphorylation of proteins is closely associated with various diseases, and, therefore, its detection is vitally important in molecular biology and drug discovery. Previously, we developed a binuclear Tb(III) complex, which emits notable luminescence only in the presence of phosphotyrosine. In this study, we conjugated a newly synthesized binuclear Tb(III) complex to substrate peptides by using click chemistry. Using these conjugates, we were able to detect tyrosine phosphorylation in real time. These conjugates were superior to nonconjugated Tb(III) complexes for the detection of tyrosine phosphorylation, especially when the substrate peptides used were positively charged. Luminescence intensity upon phosphorylation was enhanced 10-fold, making the luminescence intensity of this system one of the largest among lanthanide luminescence-based systems. We also determined Michaelis-Menten parameters for the phosphorylation of various kinase/peptide combinations and quantitatively analyzed the effects of mutations in the peptide substrates. Furthermore, we successfully monitored the inhibition of enzymatic phosphorylation by inhibitors in real time. Advantageously, this system detects only the phosphorylation of tyrosine (phosphorylated serine and threonine are virtually silent) and is applicable to versatile peptide substrates. Our study thus demonstrates the applicability of this system for the analysis of kinase activity, which could lead to drug discovery.

Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

PTEN Is a Key Regulator of Collagen-Induced Platelet Activation and Is Involved in αIIbβ3 -Mediated Outside-in Signaling,

Abstract 3252PTEN (Phosphatase and tensin homologue deleted in chromosome 10) is a well-known tumor suppressor. PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2 and thereby negatively regulates PIP3-downstream signaling. According to present studies, PTEN is also a protein tyrosine phosphatase. In 2010 (Blood. 2010;116(14):2579–81), we reported that hematopoietic specific PTEN deficiency causes an increased platelet count, a shortened tail vein bleeding time, and enhanced platelet activation in response to stimulation by collagen and other agonists along with augmented collagen-induced phosphorylation of platelet Akt at Ser473. The PI3K specific inhibitor LY294002 almost completely blocked collagen-induced WT platelet aggregation, but had less effect on PTEN-deficient platelets. This result implies that PTEN may regulate collagen-induced platelet activation via both PI3K/Akt signaling and an yet to be identified pathway. In this study, we investigated the mechanism underlying PTEN regulation of collagen-induced platelet activation.It is well-known that the GPVI receptor signaling cascade is similar to that of T- and B-cell immune receptors. This signaling cascade relies on the formation of a signalosome composed of the transmembrane adapter LAT (linker for activation of T cells), the two cytosolic adapters SLP-76 and Gads, and the activation of PLCγ2. Accordingly, we investigated the phosphorylation of LAT, SLP-76 and PLCγ2 in PTEN-deficient and WT platelets in response to low level collagen (2μg/ml), with and without LY294002 pre-incubation. The platelet lysate was immunoprecipitated with 4G10, a specific anti-phosphotyrosine monoclonal antibody, to detect tyrosine phosphorylation. Interestingly, we found that in the presence of LY294002, the phosphorylation levels of LAT, SLP-76 and PLCγ2 were very high in PTEN-deficient platelets while the phosphorylation of these molecules was hardly detectable in WT platelets, all in response to low level collagen. These results suggested that PTEN may also regulate platelet activation through LAT, SLP-76 and PLCγ2. This hypothesis was supported by co-immunopricipitation experiments demonstrating that PTEN interacts with LAT in platelets upon collagen stimulation. Moreover, we also found that PTEN Ser380 phosphorylation was correlated with collagen-induced WT platelet activation without LY294002, and PTEN Ser380 phosphorylation could be inhibited by CK2 inhibitor DMAT and PDK1 inhibitor II, but not by Akt inhibitor SH6 or the GSK3β inhibitor LiCl. In contrast, the PDK1 inhibitor II blocked collagen-induced activation of CK2. Radioactivity assays indicated that collagen-induced enzyme activity activation of CK2 was blocked by the PDK1 inhibitor II. Ser380 phosphorylation is thought to reflect the loss of PTEN phosphatase function. So, because inhibition of PI3K, and PDK1 do not result in phosphorylation of PTEN S380 and the resulting loss of PTEN phosphatase function, it may be true that PDK1 inactivates PTEN. Therefore, we conclude that the mechanism of PTEN involvement in collagen-induced platelet activation is complicated, and PTEN plays a key role in GPVI mediated platelet activation probably through down-regulating both the PI3K/Akt and PI3K-PDK1-LAT pathways.We also investigated PTEN’s function in αIIbβ3 mediated platelet activation. Platelet spreading and clot retraction experiments were performed. We found that PTEN deficiency significantly promoted clot retraction, but had no effect on platelet spreading on immobilized fibrinogen. Moreover, U46619 and thrombin induced PTEN ser380 phosphorylation was inhibited in β3 deficient platelet. These results indicate that PTEN is also involved in αIIbβ3 mediated outside-in signaling. Although the molecular mechanism of PTEN modulation of αIIbβ3 mediated outside-in signaling is unclear, our results may partially explain why PTEN deficiency also enhances platelet aggregation and secretion in response to agonists other than collagen. Disclosures:No relevant conflicts of interest to declare.

The Neuroblastoma ALK(I1250T) Mutation Is a Kinase-Dead RTK In Vitro and In Vivo

Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) have recently been shown to be an important determinant in the genetics of the childhood tumor neuroblastoma. Here we discuss an in-depth analysis of one of the reported gain-of-function ALK mutations—ALKI1250T—identified in the germ line DNA of one patient. Our analyses were performed in cell culture-based systems and subsequently confirmed in a Drosophila model. The results presented here indicate that the germ line ALKI1250T mutation is most probably not a determinant for tumor initiation or progression and, in contrast, seems to generate a kinase-dead mutation in the ALK receptor tyrosine kinase (RTK). Consistent with this, stimulation with agonist ALK antibodies fails to lead to stimulation of ALKI1250T and we were unable to detect tyrosine phosphorylation under any circumstances. In agreement, ALKI1250T is unable to activate downstream signaling pathways or to mediate neurite outgrowth, in contrast to the activated wild-type ALK receptor or the activating ALKF1174S mutant. Identical results were obtained when the ALKI1250T mutant was expressed in a Drosophila model, confirming the lack of activity of this mutant ALK RTK. We suggest that the ALKI1250T mutation leads to a kinase-dead ALK RTK, in stark contrast to assumed gain-of-function status, with significant implications for patients reported to carry this particular ALK mutation.

Detecting tyrosine-phosphorylated proteins by Western blot analysis.

The development of monoclonal antibodies (mAbs) that recognize nearly all of the phosphorylated tyrosine residues, irrespective of the surrounding sequences, enables researchers to detect the phosphorylation state of proteins through the use of anti-phosphotyrosine western blotting. The availability of this simple, reliable, nonradioactive and yet sensitive method created a boom in signal transduction research. While the methodology of how to perform an anti-phosphotyrosine western blot remains unchanged since the procedure became widely used in the early part of 1990s, steady improvements in reagents and detection technologies have allowed researchers to detect tyrosine phosphorylation quantitatively, at unprecedented sensitivity. In addition to the improvements in the western blot-based systems, powerful new phosphotyrosine detection platforms, based on proteomic technologies, are emerging rapidly. This unit will describe in detail the steps needed to perform the standard anti-phosphotyrosine western blot analysis.

Enhancement of ABL Kinase Catalytic Efficiency by a Direct Binding Regulator Is Independent of Other Regulatory Mechanisms

ABL family tyrosine kinases are tightly regulated by autoinhibition and phosphorylation mechanisms. These kinases maintain an inactive conformation through intramolecular interactions involving SH3 and SH2 domains. RIN1, a downstream effector of RAS, binds to the ABL SH3 and SH2 domains and stimulates ABL tyrosine kinase activity. RIN1 binding to the ABL2 kinase resulted in a large decrease in Km and a small increase in Vmax toward an ABL consensus substrate peptide. The enzyme efficiency (k(cat)/Km) was increased more than 5-fold by RIN1. In addition, RIN1 strongly enhanced ABL-mediated phosphorylation of CRK, PSTPIP1, and DOK1, all established ABL substrates but with unique protein structures and distinct target sequences. Importantly RIN1-mediated stimulation of ABL kinase activity was independent of activation by SRC-mediated phosphorylation. RIN1 increased the kinase activity of both ABL1 and ABL2, and this occurred in the presence or absence of ABL regulatory domains outside the SH3-SH2-tyrosine kinase domain core. We further demonstrate that a catalytic site mutation associated with broad drug resistance, ABL1T315I, remains responsive to stimulation by RIN1. These findings are consistent with an allosteric kinase activation mechanism by which RIN1 binding promotes a more accessible ABL catalytic site through relief of autoinhibition. Direct disruption of RIN1 binding may therefore be a useful strategy to suppress the activity of normal and oncogenic ABL, including inhibitor-resistant mutants that confound current therapeutic strategies. Stimulation through derepression may be applicable to many other tyrosine kinases autoinhibited by coupled SH3 and SH2 domains.

Regulation of STAT pathways and IRF1 during human dendritic cell maturation by TNF-α and PGE2

Maturation of dendritic cells (DCs) by TLR ligands induces expression of IFN-beta and autocrine activation of IFN-inducible Stat1-dependent genes important for DC function. In this study, we analyzed the regulation of STAT signaling during maturation of human DCs by TNF-alpha and PGE2, which induced maturation of human DCs comparably with LPS but did not induce detectable IFN-beta production or Stat1 tyrosine phosphorylation. Consistent with these results, TNF-alpha and PGE2 did not induce Stat1 DNA binding to a standard Stat1-binding oligonucleotide. Instead, TNF-alpha and PGE2 increased Stat1 serine phosphorylation and Stat4 tyrosine phosphorylation and activated expression of the NF-kappaB and Stat1 target gene IFN regulatory factor 1 (IRF1), which contributes to IFN responses. TNF-alpha and PGE2 induced a complex that bound an oligonucleotide derived from the IRF1 promoter that contains a STAT-binding sequence embedded in a larger palindromic sequence, and this complex was recognized by Stat1 antibodies. These results suggest that TNF-alpha and PGE2 activate STAT-mediated components of human DC maturation by alternative pathways to the IFN-beta-mediated autocrine loop used by TLRs.

Prostaglandin E2 Activates HPK1 Kinase Activity via a PKA-dependent Pathway

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 serine/threonine kinase super family. We recently reported that the immunosuppressive eicosanoid, prostaglandin E(2) (PGE(2)), is capable of activating HPK1 in T cells. In this report, we demonstrate that unlike the TCR-induced activation of HPK1 kinase activity, the induction of HPK1 catalytic activity by PGE(2) does not require the presence of phosphotyrosine-based signaling molecules such as Lck, ZAP-70, SLP-76, and Lat. Nor does the PGE(2)-induced HPK1 activation require the intermolecular interaction between its proline-rich regions and the SH3 domain-containing adaptor proteins, as required by the signaling from the TCR to HPK1. Instead, our study reveals that PGE(2) signal to HPK1 via a 3' -5 '-cyclic adenosine monophosphate-regulated, PKA-dependent pathway. Consistent with this observation, changing the serine 171 residue that forms the optimal PKA phosphorylation site within the "activation loop" of HPK1 to alanine completely prevents this mutant from responding to PGE(2)-generated stimulation signals. Moreover, the inability of HPK1 to respond to PGE(2) stimulation in PKA-deficient S49 cells further supports the importance of PKA in this signaling pathway. We speculate that this unique signaling pathway enables PGE(2) signals to engage a proven negative regulator of TCR signal transduction pathway and uses it to inhibit T cell activation.

Protein-tyrosine Phosphatase 1B Deficiency Reduces Insulin Resistance and the Diabetic Phenotype in Mice with Polygenic Insulin Resistance

Mice heterozygous for insulin receptor (IR) and IR substrate (IRS)-1 deficiency provide a model of polygenic type 2 diabetes in which early-onset, genetically programmed insulin resistance leads to diabetes. Protein-tyrosine phosphatase 1B (PTP1B) dephosphorylates tyrosine residues in IR and possibly IRS proteins, thereby inhibiting insulin signaling. Mice lacking PTP1B are lean and have increased insulin sensitivity. To determine whether PTP1B can modify polygenic insulin resistance, we crossed PTP1B-/- mice with mice with a double heterozygous deficiency of IR and IRS-1 alleles (DHet). DHet mice weighed slightly less than wild-type mice and exhibited severe insulin resistance and hyperglycemia, with approximately 35% of DHet males developing diabetes by 9-10 weeks of age. Body weight in DHet mice with PTP1B deficiency was similar to that in DHet mice. However, absence of PTP1B in DHet mice markedly improved glucose tolerance and insulin sensitivity at 10-11 weeks of age and reduced the incidence of diabetes and hyperplastic pancreatic islets at 6 months of age. Insulin-stimulated phosphorylation of IR, IRS proteins, Akt/protein kinase B, glycogen synthase kinase 3beta, and p70(S6K) was impaired in DHet mouse muscle and liver and was differentially improved by PTP1B deficiency. In addition, increased phosphoenolpyruvate carboxykinase expression in DHet mouse liver was reversed by PTP1B deficiency. In summary, PTP1B deficiency reduces insulin resistance and hyperglycemia without altering body weight in a model of polygenic type 2 diabetes. Thus, even in the setting of high genetic risk for diabetes, reducing PTP1B is partially protective, further demonstrating its attractiveness as a target for prevention and treatment of type 2 diabetes.

Multiple seropathotypes of verotoxin-producing Escherichia coli (VTEC) disrupt interferon- γ-induced tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1

Verotoxin-producing Escherichia coli (VTEC) O157:H7 inhibits interferon- γ-stimulated tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1 in epithelial cells, independent of Verotoxins and the locus of enterocyte effacement pathogenicity island. Although E. coli O157:H7 is the major cause of disease in humans, non-O157:H7 VTEC also cause human disease. However, the virulence properties of non-O157:H7 VTEC are less well characterized. The aims of this study were to define the ability of VTEC strains of differing seropathotypes (classified as A–E) to inhibit interferon- γ stimulated Stat1-phosphorylation and to further characterize the bacterial-derived inhibitory factor. Confluent T84 and HEp-2 cells were infected with VTEC strains (MOI 100:1, 6 h, 37 °C), and then stimulated with interferon- γ (50 ng/mL) for 0.5 h at 37 °C. Whole-cell protein extracts of infected cells were collected and prepared for immunoblotting to detect tyrosine phosphorylation of Stat1. The effects of E. coli O55 strains, the evolutionary precursors of VTEC, on Stat1-tyrosine phosphorylation were also determined. The effects of isogenic mutants of O-islands 47 and 122 were tested to determine the role of genes encoded on these putative pathogenicity islands in mediating VTEC inhibition of the interferon- γ-Stat1 signaling cascade. To evaluate potential mechanism(s) of inhibition, VTEC O157:H7-infected cells were treated with pharmacological inhibitors, including, wortmannin and LY294002. Relative to uninfected cells, Stat1-tyrosine phosphorylation was significantly reduced after 6 h infection of both T84 and HEp-2 cells by VTEC strains of all five seropathotypes. E. coli O55 strains, but not enteropathogenic E. coli (EPEC), also caused inhibition of Stat1-tyrosine phosphorylation, suggesting that this effect was acquired early in the evolution of VTEC. Stat1-activation did not recover in epithelial cells infected with isogenic mutants of O-islands 47 and 122, indicating that the inhibitory factor was not contained in these genomic regions. Stat1-phosphorylation remained intact when VTEC-infected cells were treated with wortmannin (0–100 nM), but not by treatment with the more specific PI3-kinase inhibitor, LY294002. Inhibition of interferon- γ stimulated Stat1-tyrosine phosphorylation by VTEC of multiple seropathotypes indicates the presence of a common inhibitory factor that is independent of bacterial virulence in humans. The results of treatment with wortmannin suggest that the bacterial-derived inhibitory factor employs host cell signal transduction to mediate inhibition of Stat1-activation.

Human Tyrosine Kinase 2 Deficiency Reveals Its Requisite Roles in Multiple Cytokine Signals Involved in Innate and Acquired Immunity

Tyrosine kinase 2 (Tyk2) is a nonreceptor tyrosine kinase that belongs to the Janus kinase (Jak) family. Here we identified a homozygous Tyk2 mutation in a patient who had been clinically diagnosed with hyper-IgE syndrome. This patient showed unusual susceptibility to various microorganisms including virus, fungi, and mycobacteria and suffered from atopic dermatitis with elevated serum IgE. The patient's cells displayed defects in multiple cytokine signaling pathways including those for type I interferon (IFN), interleukin (IL)-6, IL-10, IL-12, and IL-23. The cytokine signals were successfully restored by transducing the intact Tyk2 gene. Thus, the Tyk2 deficiency is likely to account for the patient's complex clinical manifestations, including the phenotype of impaired T helper 1 (Th1) differentiation and accelerated Th2 differentiation. This study identifies human Tyk2 deficiency and demonstrates that Tyk2 plays obligatory roles in multiple cytokine signals involved in innate and acquired immunity of humans, which differs substantially from Tyk2 function in mice.

A Growth-suppressive Function for the c-Fes Protein-Tyrosine Kinase in Colorectal Cancer

The human c-fes locus encodes a non-receptor protein-tyrosine kinase implicated in myeloid, vascular endothelial, and neuronal cell differentiation. A recent analysis of the tyrosine kinome in colorectal cancer identified c-fes as one of only seven genes with consistent kinase domain mutations. Although four mutations were identified (M704V, R706Q, V743M, S759F), the consequences of these mutations on Fes kinase activity were not explored. To address this issue, Fes mutants with these substitutions were co-expressed with STAT3 in human 293T cells. Surprisingly, the M704V, R706Q, and V743M mutations substantially reduced Fes autophosphorylation and STAT3 Tyr-705 phosphorylation compared with wild-type Fes, whereas S759F had little effect. These mutations had a similar impact on Fes kinase activity in a yeast expression system, suggesting that they inhibit Fes by affecting kinase domain structure. We have also demonstrated for the first time that endogenous Fes is strongly expressed at the base of colonic crypts where it co-localizes with epithelial cells positive for the progenitor cell marker Musashi-1. In contrast to normal colonic epithelium, Fes expression was reduced or absent in colon tumor sections from most individuals. Fes protein levels were also low or absent in a panel of human colorectal cancer cell lines, including HT-29 and HCT 116 cells. Introduction of Fes into these lines with a recombinant retrovirus suppressed their growth in soft agar. Together, our findings strongly implicate the c-Fes protein-tyrosine kinase as a tumor suppressor rather than a dominant oncogene in colorectal cancer.

Inducible phosphorylation of cortactin is not necessary for cortactin-mediated actin polymerisation

Cortactin is an SH3 domain-containing protein that contributes to the formation of dynamic cortical actin-associated structures, such as lamellipodia and membrane ruffles. It was originally identified as a substrate for the protein kinase Src; however, the role of tyrosine phosphorylation in the translocation of cortactin to the cell periphery and in the subsequent actin polymerisation is still unclear. Recently, two serine/threonine kinases, Pak1 and Erk, have been implicated in the regulation of cortactin. Therefore, we systematically investigated whether phosphorylation on either tyrosine or serine/threonine residues is necessary for cortactin function. In COS7 cells over-expressing Vav2 or treated with EGF, we could not detect tyrosine phosphorylation, although cortactin was translocated to cell periphery and induced membrane ruffle formation. In addition, the selective MEK inhibitor, PD98059, did not influence in vivo the ability of cortactin to bind to and induce membrane ruffles upon Vav2 over-expression or short-term EGF treatment. Finally, using a constitutively active Pak1 mutant, Pak1 T423E, we showed that Pak1 is not capable of phosphorylating cortactin either in vitro or in COS7 cells. These results suggest that cortactin-mediated actin polymerisation at cell periphery requires only Rac activation but neither tyrosine nor serine/threonine phosphorylation.

Phosphotyrosine Proteomic Study of Interferon α Signaling Pathway Using a Combination of Immunoprecipitation and Immobilized Metal Affinity Chromatography

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

Activation of the IκB kinase complex is sufficient for neuronal differentiation of PC12 cells

We examined the role of the IkappaB kinase complex in nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. We showed that neurite outgrowth is accompanied by an activation of the IKK complex and a delayed elevation of NF-kappaB-dependent transcription. Ectopic expression of a constitutively active form of IKK2 but not of IKK1 promoted neurite outgrowth in the absence of NGF. In addition, increased expression of Bcl-2 and Bcl-xL and resistance to apoptosis upon serum withdrawal were found. The IKK2-driven neurite outgrowth was not blocked by MEK1/2 and PI3K inhibitors but was repressed by the SN50 peptide suggesting that NF-kappaB activation is critical for this differentiation process. Transdominant mutants of IkappaBalpha (32/36-SS/AA) and IKK1 only marginally reduced NGF-driven neuritogenesis. However, a dominant negative mutant of IKK2 or an IkappaBalpha protein lacking the complete N-terminus was able to repress neuritogenesis. We also detected tyrosine phosphorylation of IkappaBalpha during differentiation. Consequently, PC12 cells expressing mutant IkappaBalpha (Y42F) show an impaired neuritogenesis. Furthermore, PC12 cells ectopically expressing p65 show almost no signs of neurite outgrowth which is, however, found to some extent in c-Rel-expressing cells. Our data suggest that NGF-induced PC12 differentiation includes activation of IKK2 which may promote the release of c-Rel-containing dimers.