Anti-phosphotyrosine Immunoblotting Research Articles

Effect of Sintering Temperature of Bioactive Glass Nanoceramics on the Hemolytic Activity and Oxidative Stress Biomarkers in Erythrocytes.

The nature of the surface is critical in determining the biological activity of silica powders. A novel correlation between toxicity and surface properties of bioactive glass ceramics (BGCs) synthesized via the sol-gel method was attempted in this study. The behavior of BGCs after their attachment to the surface of red blood cells (RBCs) was evaluated and their toxic effects were determined based on hemolysis, membrane injury via anti-phosphotyrosine immunoblot of Band 3, lipid peroxidation, potential to generate reactive oxygen species, and antioxidant enzyme production. In particular, three BGCs were synthesized and treated at three sintering temperatures (T1 = 835°C, T2 = 1000°C and T3 = 1100°C) to investigate possible relation between surface charge or structure and hemolytic potential. Their toxicity based on hemolysis was dose dependent, while BGC-T2 had the best hemocompatibility in compare with the other BGCs.No BGCs in dosages lower than 0.125mg/mL could damage erythrocytes. On the other hand, all BGCs promoted the production of reactive oxygen species in certain concentrations, with the BGC-T2 producing the lowest ROS and increasing the glutathione levels in RBCs protecting their damage. The results suggest that various factors such as size, a probable different proportion of surface silanols, a balanced mechanism between calcium and magnesium cellular uptake or the different crystalline nature may have contributed to this finding; however, future research is needed to clarify the underlying mechanisms.

IL-7-induced phosphorylation of the adaptor Crk-like and other targets

IL-7 is required for T cell differentiation and mature T cell homeostasis and promotes pro-B cell proliferation and survival. Tyrosine phosphorylation plays a central role in IL-7 signaling. We identified by two-dimensional electrophoresis followed by anti-phosphotyrosine immunoblotting and mass spectrometry sixteen tyrosine phosphorylated proteins from the IL-7-dependent cell line D1. IL-7 stimulation induced the phosphorylation of the proteins STI1, ATIC and hnRNPH, involved in pathways related to survival, proliferation and gene expression, respectively, and increased the phosphorylation of CrkL, a member of a family of adaptors including the highly homologous Crk isoforms CrkII and CrkI, important in multiple signaling pathways. We observed an increased phosphorylation of CrkL in murine pro-B cells and in murine and human T cells. In addition, IL-7 increased the association of CrkL with the transcription factor Stat5, essential for IL-7 pro-survival activity. The selective tyrosine kinase inhibitor Imatinib. counteracted the IL-7 pro-survival effect in D1 cells and decreased CrkL phosphorylation. These data suggested that CrkL could play a pro-survival role in IL-7-mediated signaling. We observed that pro-B cells also expressed, in addition to CrkL, the Crk isoforms CrkII and CrkI and therefore utilized pro-B cells conditionally deficient in all three to evaluate the role of these proteins. The observation that the IL-7 pro-survival effect was reduced in Crk/CrkL conditionally-deficient pro-B cells further pointed to a pro-survival role of these adaptors. To further evaluate the role of these proteins, gene expression studies were performed in Crk/CrkL conditionally-deficient pro-B cells. IL-7 decreased the transcription of the receptor LAIR1, which inhibits B cell proliferation, in a Crk/CrkL-dependent manner, suggesting that the Crk family of proteins may promote pro-B cell proliferation. Our data contribute to the understanding of IL-7 signaling and suggest the involvement of Crk family proteins in pathways promoting survival and proliferation.

Abstract A25: Dual inhibition of Flt3 and Fes tyrosine kinases potently blocks proliferation of AML cells expressing an active Flt3 mutant.

Abstract Acute myelogenous leukemia (AML) is a hematologic cancer associated with a relatively poor prognosis, especially in the elderly. Although the diverse genetic changes associated with AML make treatment a challenge, about one-third of cases involve activating mutations in the receptor tyrosine kinase, Flt3. While many Flt3 tyrosine kinase inhibitors have been tested in the clinic, most have performed poorly as single agents indicating that additional signaling pathways contribute to the transformed phenotype. Previous studies have linked the Fes protein-tyrosine kinase to Flt3 in AML, and RNAi-knockdown studies suggest that Fes may be required for Flt3 function. In the present study, we tested the phenoxyquinazoline Fes kinase inhibitor, TL02-59, for its growth-suppressive activity against MV4-11 myeloid leukemia cells. These cells have an activating mutation in Flt3 (internal tandem duplication or ITD) that causes constitutive Fes activation downstream. TL02-59 blocked MV4-11 cell proliferation with an IC50 value of 25 nM, rivaling the potency of the clinical Flt3 inhibitors lestaurtinib and tandutinib in this cell line. In contrast, TL02-59 had no effect on the growth of THP-1 cells, which are wild-type for Flt3. We next explored the inhibitory activity of TL02-59 against Fes using an in vitro kinase assay. Surprisingly, TL02-59 inhibited recombinant Fes with an IC50 value of only 1.5 µM, suggesting that it may have additional kinase targets in AML cells. In vitro kinase assays with recombinant Flt3 support this idea, where TL02-59 also blocked Flt3 kinase activity with an IC50 value of 0.6 µM. This observation suggests that the potency of TL02-59 in MV4-11 cells may be the result of dual inhibition of both Fes and Flt3-ITD kinase activities, a possibility confirmed by anti-phosphotyrosine immunoblotting of Fes and Flt3 immunoprecipitates from TL02-59-treated cells. We next investigated downstream targets linked uniquely to Fes by engineering mutants of Flt3-ITD that are resistant to TL02-59 based on clinical cases of acquired drug resistance to other Flt3 inhibitors. These mutants were used to transform human TF-1 myeloid cells to cytokine independence, resulting in constitutive activation of endogenous Fes and sensitization to growth suppression by TL02-59 (IC50 = 24 nM). Interestingly, TF-1 cells transformed with TL02-59-resistant mutants of Flt3-ITD were less sensitive to the growth-suppressive actions of the inhibitor, consistent with its dual specificity. Using these cell lines, we will be able to identify downstream phosphorylation targets unique to Fes, because TL02-59 treatment will only affect Fes and not Flt3-ITD directly. Preliminary experiments performed in TF-1 cells transformed with one TL02-59-resistant mutant of Flt3-ITD, show inhibitor treatment still results in inhibition of STAT5 phosphorylation. These results support a critical role for Fes in downstream signaling from Flt3-ITD+ AML, and suggest that dual inhibition of both Flt3-ITD and Fes may provide a therapeutic advantage for the treatment of this subtype of AML. Citation Format: Mark Weir, Sabine Hellwig, Nathanael S. Gray, Thomas E. Smithgall. Dual inhibition of Flt3 and Fes tyrosine kinases potently blocks proliferation of AML cells expressing an active Flt3 mutant. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A25.

Differential Modulation of Fast Inactivation in Cardiac Sodium Channel Splice Variants by Fyn Tyrosine Kinase

Background/Aims: Post-translational modifications such as phosphorylation and dephosphorylation can finely tune the function of ion channels. Na_v1.5 is the main sodium channel in human hearts and alternative splicing of the transcript generates two major splice variants, characterized by the presence (Q-pre) or absence (Q-del) of glutamine at position 1077. In the heart, both the Na_v1.5 channel and Fyn tyrosine kinase are colocalized at adherens junctions. This study aimed to investigate the modulation of the aforementioned splice variants by Fyn tyrosine kinase. Methods and Results: Q-del and Q-pre were transiently expressed alone, with catalytically active Fyn kinase (FynKa) or with a catalytically dead Fyn kinase (FynKd). Co-expression of Na_v1.5 channel splice variants and Fyn kinase was confirmed by Western blotting and their Interaction was established by co-immunoprecipitation experiments. The enzymatic activity of Fyn kinase and phosphorylation of Na_v1.5 channel were ascertained by immunoprecipitation and anti-phosphotyrosine immunoblotting. Whole-cell ionic currents were recorded in patch clamp experiments to examine the modulation of Na_v1.5 channel variants by Fyn kinase, which indicated a hyperpolarizing shift of 9.68 mV in fast inactivation of Q-del. In contrast, a depolarizing shift of 8.77 mV in fast inactivation was observed in the case of Q-pre, while activation curves remained unaltered for both splice variants. This differential modulation in fast inactivation was further assessed by mutating tyrosine 1495 to phenylalanine in the inactivation loop, which completely removed the modulatory effect of Fyn kinase in Q-pre splice variant, while in Q-del variant hyperpolarizing shift in fast inactivation was reduced to 4.74 mV. Finally, the modulatory effect of Fyn kinase was compensated at a mid-value of 94.63 ± 0.34, when both splice variants were co-expressed at a normal physiological ratio. Conclusion: Q-del and Q-pre were differentially modulated by Fyn kinase, and this fine modification resulted in smooth electrical activity in the heart.

Frank-ter Haar Syndrome Protein Tks4 Regulates Epidermal Growth Factor-dependent Cell Migration

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.

Activation of Src and transformation by an RPTPα splice mutant found in human tumours

Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ∼30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.

Acetylome and phosphoproteome modifications in imatinib resistant chronic myeloid leukaemia cells treated with valproic acid

Chronic myeloid leukaemia has a specific therapy: BCR/ABL inhibitor imatinib. Resistance due to BCR/ABL dependent and independent mechanisms is partially reversible by histone deacetylase inhibitors. We analysed by 2D-electrophoresis and anti-pan-acetylated and anti-phosphotyrosine immunoblots, followed by spot-matching and MALDI-TOF mass spectrometry, which proteome modifications would parallel restoration of sensitivity to imatinib by valproic acid (VPA). VPA plus imatinib significantly increased acetylation of HSP90 and hnRNP L and decreased phosphorylation of HSPs and hnRNPs in imatinib resistant cells. VPA was able to modify profoundly acetylome and phosphoproteome of CML cells, while reverting resistance to imatinib.

Control of T Lymphocyte Signaling by Ly108, a Signaling Lymphocytic Activation Molecule Family Receptor Implicated in Autoimmunity

The signaling lymphocytic activation molecule family of receptors has been implicated in the pathophysiology of autoimmunity in humans and mice. One member of the family, Ly108, was strongly linked to lupus susceptibility in mice. High expression of a Ly108 isoform, Ly108-1, was observed in lymphocytes of lupus-prone mice. Herein, we examined the molecular basis for the influence of Ly108 on lupus susceptibility by studying Ly108 signal transduction in T cells. We observed that Ly108 was able to mediate a tyrosine phosphorylation signal implicating Ly108, Vav-1, and c-Cbl in a manner strictly dependent on engagement of the extracellular domain of Ly108 and co-expression of the Src homology 2 (SH2) domain-containing adaptor signaling lymphocytic activation molecule (SLAM)-associated protein (SAP). Evaluation of T cells from mice carrying mutations in the SAP-FynT pathway indicated that Ly108-triggered protein tyrosine phosphorylation was due to the capacity of SAP to recruit FynT. Importantly, Ly108-1 was more apt at triggering tyrosine phosphorylation signals in T cells when compared with the predominant Ly108 isoform found in non-lupus-prone mice, Ly108-2. This difference was due in part to the presence in Ly108-1 of a unique intra-cytoplasmic tyrosine-based motif that promoted Ly108 signal transduction. Together these data provided a molecular explanation for the involvement of Ly108 in lupus susceptibility in mice.

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.

C-Abl-mediated Phosphorylation of WAVE3 Is Required for Lamellipodia Formation and Cell Migration

The activity of the Wiskott-Aldrich syndrome-related WAVE3 protein is critical for the regulation of the Arp2/3-dependent cytoskeleton organization downstream of Rac-GTPase. The Ableson (Abl) non-receptor tyrosine kinase is also involved in the remolding of actin cytoskeleton in response to extracellular stimuli. Here we show that platelet-derived growth factor stimulation of cultured cells results in WAVE3-Abl interaction and localization to the cell periphery. WAVE3-Abl interaction promotes the tyrosine phosphorylation of WAVE3 by Abl, and STI-571, a specific inhibitor of Abl kinase activity, abrogates the Abl-mediated phosphorylation of WAVE3. We have also shown that Abl targets and phosphorylates four tyrosine residues in WAVE3 and that the Abl-dependent phosphorylation of WAVE3 is critical for the stimulation of lamellipodia formation and cell migration. Our results show that the activation of WAVE3 to promote actin remodeling is enhanced by the c-Abl-mediated tyrosine phosphorylation of WAVE3.

SLP-65 Signal Transduction Requires Src Homology 2 domain-mediated Membrane Anchoring and a Kinase-independent Adaptor Function of Syk

The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.

Focal adhesion signaling is required for myometrial ERK activation and contractile phenotype switch before labor

In late pregnancy rapidly increasing fetal growth dramatically increases uterine wall tension. This process has been implicated in the activation of the myometrium for labor, but the mechanisms involved are unclear. Here, we tested, using a rat model, the hypothesis that gestation-dependent stretch, via activation of focal adhesion signaling, contributes to the published activation of myometrial ERK at the end of pregnancy. Consistent with this hypothesis, we show here that ERK is targeted to adhesion plaques during late pregnancy. Furthermore, myometrial stretch triggers a dramatic increase in myometrial contractility and ERK and caldesmon phosphorylation, confirming the presence of stretch sensitive myometrial signaling element. Screening by anti-phosphotyrosine immunoblotting for focal adhesion signaling in response to stretch reveals a significant increase in the tyrosine phosphorylated bands identified as focal adhesion kinase (FAK), A-Raf, paxillin, and Src. Pretreatment with PP2, a Src inhibitor, significantly suppresses the stretch-induced increases in FAK, paxillin, Src, ERK and caldesmon phosphorylation and myometrial contractility. Thus, focal adhesion-Src signaling contributes to ERK activation and promotes contraction in late pregnancy. These results point to focal adhesion signaling molecules as potential targets in the modulation of the myometrial contractility and the onset of labor.

Genome-Wide Analysis and Experimentation of Plant Serine/ Threonine/Tyrosine-Specific Protein Kinases

Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.

Phosphorylation of Neurogenin2 Specifies the Migration Properties and the Dendritic Morphology of Pyramidal Neurons in the Neocortex

The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 (Ngn2) is both necessary and sufficient for specifying the dendritic morphology of pyramidal neurons in vivo by specifying the polarity of its leading process during the initiation of radial migration. The ability of Ngn2 to promote a polarized leading process outgrowth requires the phosphorylation of a single tyrosine residue at position 241, an event that is neither involved in Ngn2 direct transactivation properties nor its proneural function. Interestingly, the migration defect observed in the Ngn2 knockout mouse and in progenitors expressing the Ngn2(Y241F) mutation can be rescued by inhibiting the activity of the small-GTPase RhoA in cortical progenitors. Our results demonstrate that Ngn2 coordinates the acquisition of the radial migration properties and the unipolar dendritic morphology characterizing pyramidal neurons through molecular mechanisms distinct from those mediating its proneural activity.

93 CALCIUM REMOVAL INCREASES THE PROTECTIVE EFFECTS OF β-CYCLODEXTRIN PLUS CHOLESTEROL ON PORCINE SPERM DURING COLD SHOCK

Porcine sperm are extremely sensitive to the damaging effects of cold shock and cryopreservation. Cholesterol-binding molecules, such as 2-hydroxypropyl-�-cyclodextrin (HBCD), improve post-thaw and post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. Depending upon the environmental cholesterol content, HBCD can act either as a cholesterol shuttle or sink to increase or decrease, respectively, sperm plasma membrane cholesterol content. Increasing the sperm cholesterol to phospholipid ratio reduces cold shock sensitivity whereas decreasing the ratio initiates the process of sperm capacitation. An increase in protein tyrosine phosphorylation correlates with sperm capacitation and has been shown to be dependent upon the presence of extracellular calcium. Sperm intracellular calcium also increases during cold shock. The objective of this study was to determine the combined effects of extracellular calcium and membrane cholesterol manipulation on porcine sperm viability and protein tyrosine phosphorylation following cold shock (10�C for 10 min). Viability was assessed using CFDA/propidium iodide staining. Protein tyrosine phosphorylation, previously shown to correlate with porcine sperm capacitation, was evaluated via antiphosphotyrosine (clone 4G10) immunoblots. We report here that following cold shock, porcine sperm incubated in defined medium containing both 0.8 mM HBCD and 0.5 mM cholesterol 3-sulfate (ChS) incubated in the absence of added extracellular calcium and the presence of 6 mM EGTA have significantly improved viability (90.5 � 6.3%, n = 3) when compared with cold-shocked sperm incubated in either the same medium with calcium (46.1 � 3.8%), without HBCD or ChS (26.5 � 7.4% with calcium; 46.5 � 13.1% without calcium), or with HBCD alone (17.0 � 7.4% with calcium, 36.8 � 7.5% without calcium). As we have found previously, treatment with 0.8 mM HBCD plus 0.5 mM ChS completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment. Although protein tyrosine phosphorylation correlates with porcine sperm capacitation, the ability of cold shock treatment to induce the same phosphorylation pattern indicates that other processes or pathways may contribute to its appearance. Removing extracellular calcium consistently decreased, but did not completely eliminate, the protein tyrosine phosphorylation induced by cold shock. These results indicate that cold shock-induced protein tyrosine phosphorylation is not dependent upon, but can be modulated by, extracellular calcium. The combined effects of calcium, HBCD and ChS on viability suggest that porcine sperm viability following cold shock is best maintained by removing extracellular calcium and increasing membrane cholesterol content via the cholesterol shuttle activity of HBCD. This work was supported by grants from PA Dept. Ag. (ME 443291) and the NIH (5-K08-HD041430).

HIV-1 Nef Alleles Show Differential Activation of Hematopoietic Cell Kinase in a Yeast Model System.

Hematopoietic cell kinase (Hck) is a member of the Src family of non-receptor protein-tyrosine kinases that is expressed strongly in macrophages, an important target cell type for human immunodeficiency virus (HIV). The HIV Nef protein, a critical AIDS progression factor, binds Hck with unusually high affinity and induces Hck activation. Nef-mediated Hck activation has been proposed to trigger signaling pathways important for the establishment of HIV infection and subsequent progression to AIDS. Previous studies have utilized sequence alignments and binding assays to predict the functionality of Hck-Nef interactions, but these studies do not directly investigate the ability of Nef alleles to activate Hck. To better address this issue, we have created a model system in Saccharomyces cerevisiae based on previous findings that ectopic expression of c-Src arrests yeast growth in a kinase-dependent fashion. To establish the utility of this system for Hck and Nef, we first created a Hck variant containing a C-terminal sequence modified to encode the high-affinity SH2-domain binding sequence Tyr-Glu-Glu-Ile (HckYEEI). This modified sequence allows for autophosphorylation of the new tail and the subsequent intramolecular binding to the Hck SH2 domain. The resulting HckYEEI molecule models the physiologically down-regulated (inactive) form of Hck in structure and function. Unlike wild-type Hck, HckYEEI showed little kinase activity and failed to induce growth suppression in yeast. Importantly, co-expression with the Nef-SF2 allele activated HckYEEI, resulting in growth arrest. This effect was dependent on the conserved Nef proline-rich motif essential for Hck binding through its SH3 domain. However, Nef-SF2 did not activate FynYEEI, consistent with our previous data that the Fyn tyrosine kinase is not activated by Nef in mammalian cells. We then tested several additional laboratory HIV-1 Nef alleles for their ability to activate HckYEEI. Consensus, Lai, NL4.3, SF2, and YU-2 Nef alleles activated HckYEEI to varying degrees, as reflected by yeast growth inhibition. In contrast, Nef-Eli failed to suppress growth in the presence of HckYEEI, consistent with its inability to bind the Hck SH3 domain in vitro. Growth inhibition paralleled tyrosine phosphorylation of yeast proteins on anti-phosphotyrosine immunoblots. The differential effects of these Nef alleles on Hck activation correlated closely with previous results in mammalian cells. Taken together, these studies validate the use of yeast to reconstitute Nef-Hck interactions in a system amenable to screens of clinical Nef isolates, which may have predictive value in terms of AIDS progression.

Generation and Verification of a Three-Dimensional Model of the ALK Kinase Domain: ALK - a “Supermutant” of Abl.

Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase that plays a key role in oncogenesis in a subset of non-Hodgkin's lymphomas, through the generation of fusion proteins containing its kinase domain (KD), such as NPM/ALK (N/A). A three-dimensional (3D) model of the ALK KD was generated by homology modelling, using the crystal structures of the inactive conformations of Abl and insulin receptor kinase as templates. Sequence alignment of ALK and Abl, which share 34% identity and 61% homology in their KDs indicated that sequences diverged at positions known to be critical for imatinib binding to Abl.ALK sequence differs from Abl at 13 of the 18 amino acids known to be mutated in the Abl KD of imatinib resistant CML patients, with 3 of these ALK amino acids being identical to mutations observed in patients (Y253F, F317L and V379I). Hence, ALK could be considered as a ‘supermutant' of Abl that should be resistant to Abl inhibitors. Mutation of ALK to an Abl-like sequence should be able to ‘restore' sensitivity of ALK to Abl inhibitors. In Bcr/Abl, a key determinant in imatinib sensitivity is threonine315 (T315), which is often mutated to a bulky isoleucine residue in relapsed CML patients. In ALK, the corresponding amino acid is a leucine (L256 of N/A), which similarly to isoleucine, causes a steric hindrance in the ATP binding pocket and eliminates the possibility of hydrogen bond formation. Therefore, we hypothesised that a L256T mutation in N/A might render this kinase sensitive to Abl inhibitors. Molecular docking of Abl inhibitors to ALK wild type (WT) and L256T 3D models predicted that WT-ALK should be insensitive to three Abl inhibitors (compound 583, compound 683, imatinib), while L256T-ALK should be inhibited by compound 583 and 683, but not imatinib, which would require further mutagenization. To test this hypothesis we generated BaF3 cell lines stably transfected with WT- and L256T-N/A. We observed that L256T-BaF3 cells were 5 times more sensitive to growth arrest induced by compound 583 than WT-BaF3 cells as determined by 3[H]-thymidine uptake (IC50: 0.2±0.02 v 1.0±0.07 μM [mean ± sem]), and 7 and 3.5 times more sensitive to cell death induced by compounds 583 (70 v 10% after 48h) and 683 (70 v 20% after 16h) respectively, as determined by trypan blue exclusion. No difference in sensitivity to imatinib was observed in the 2 cell lines.In addition, autophosphorylation activity of L256T-N/A, assessed by anti-phosphotyrosine immunoblotting and in vitro radioactive kinase assays, was inhibited by compounds 583 and 683 with IC50s of approximately 0.2 and 3 μM respectively, whereas autophosphorylation activity of WT-N/A was not affected by either inhibitor. Neither L256T- nor WT-N/A kinase activities were inhibited by imatinib. Summarizing, L256T-N/A was significantly more sensitive to compounds 583 and 683 compared with WT-N/A. These data provide experimental evidence supporting our modelling predictions, thereby verifying the reliability of the ALK 3D model. The sensitisation of ALK to compounds 583 and 683, but not imatinib, by the L256T mutation is in agreement with data describing the activities of these inhibitors on Abl mutants, excluding the T315I, and supports the ‘supermutant' hypothesis.

Selective tyrosine hyperphosphorylation of cytoskeletal and stress proteins in primary human breast cancers: implications for adjuvant use of kinase-inhibitory drugs.

Small-molecule growth factor receptor inhibitors block cell growth in vitro and downstream signaling in vivo, but controlled trials in patients with advanced solid tumors have yielded disappointing response rates. To clarify this discrepancy, we compared the patterns of tyrosine phosphoprotein expression in human cancer cells and primary tumors. Immunoaffinity chromatography, two-dimensional electrophoresis, and antiphosphotyrosine immunoblotting were combined with mass spectrometry to determine the phosphoproteomic signatures of 40 matched normal and malignant tissues from patients with breast or liver cancer. The identities and abundance of the detected tyrosine phosphoproteins were compared with those of ligand-responsive A431 cells. Patterns of tyrosine-phosphorylated proteins are similar among normal tissues of the same origin but vary markedly between different tissues. Primary breast tumors exhibit a strikingly homogeneous tyrosine phosphorylation profile, whereas liver cancers display greater phosphoproteomic diversity. The main breast-tumor-specific tyrosine phosphoproteins are cytoskeletal molecules (actin, tubulin, and vimentin) and molecular chaperones (Hsp70, Hsc71, and Grp75). In contrast, control studies in ligand-stimulated A431 human cancer cells revealed an additional phosphorylated subset of promitogenic phosphoproteins (Grb2, Shc, Jnk2, phospholipase C-gamma, and phosphatidylinositol 3'-kinase). Identification of cytoskeletal and stress proteins as the most abundant tyrosine phosphoproteins in breast tumors implicates these molecules, rather than promitogenic effectors, as the prime stoichiometric substrates for kinase-inhibitory anticancer drugs in vivo. Because phosphorylated cytoskeletal proteins and chaperones mediate cell motility and apoptotic resistance, respectively, these data raise the intriguing possibility that small-molecule tyrosine kinase inhibitors may be of greatest value either as adjuvant antimetastatic/-invasive drugs or as chemo-/radiosensitizers.

The Aryl Hydrocarbon Receptor (AhR) Tyrosine 9, a Residue That Is Essential for AhR DNA Binding Activity, Is Not a Phosphoresidue but Augments AhR Phosphorylation

We delineate a mechanism by which dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD)-mediated formation of the aryl hydrocarbon receptor (AhR) DNA binding complex is disrupted by a single mutation at the conserved AhR tyrosine 9. Replacement of tyrosine 9 with the structurally conservative phenylalanine (AhRY9F) abolished binding to dioxin response element (DRE) D, E, and A and abrogated DRE-driven gene induction mediated by the AhR with no effect on TCDD binding, TCDD-induced nuclear localization, or ARNT heterodimerization. The speculated role for phosphorylation at tyrosine 9 was also examined. Anti-phosphotyrosine immunoblotting could not detect a major difference between the AhRY9F mutant and wild-type AhR, but a basic isoelectric point shift was detected by two-dimensional gel electrophoresis of AhRY9F. However, an antibody raised to recognize only phosphorylated tyrosine 9 (anti-AhRpY9) confirmed that AhR tyrosine 9 is not a phosphorylated residue required for DRE binding. Kinase assays using synthetic peptides corresponding to the wild-type and mutant AhR residues 1-23 demonstrated that a tyrosine at position 9 is important for substrate recognition at serine(s)/threonine(s) within this sequence by purified protein kinase C (PKC). Also, compared with AhRY9F, immunopurified full-length wild-type receptor was more rapidly phosphorylated by PKC. Furthermore, co-treatment of AhR-deficient cells that expressed AhRY9F and a DRE-driven luciferase construct with phorbol 12-myristate 13-acetate and TCDD resulted in a 30% increase in luciferase activity compared with AhRY9F treated with TCDD alone. Overall, AhR tyrosine 9, which is not a phosphorylated residue itself but is required for DNA binding, appears to play a crucial role in AhR activity by permitting proper phosphorylation of the AhR.

Tyrosyl Phosphorylation of Shp2 Is Required for Normal ERK Activation in Response to Some, but Not All, Growth Factors

The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542F- or Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the "Grb2-adapter" model of Shp2 action.