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Unveiling of cancer genomes is unleashing new therapeutic strategies for cancer. With cancer parts lists in hand, new approaches to personalized medicine can be developed by understanding the assembly of cancer machines using modular domains in proteins and their associated networks. Using the Src-homology-2 (SH2) domain as an example, new profiling approaches can discern global patterns of tyrosine phosphorylation in cancer cells that can enable molecular cancer medicine. Identifying and quantifying protein–protein interactions also has the potential to subtype tumors and guide clinical decision making. These approaches should extend the impact of genomics through viewing the architecture of cancer systems and improve predictions of patient outcome and therapeutic response, as well as guide combination therapy approaches that attack cancer systems.

The initial steps of heat stress in A431 cells were previously characterized by ligand-independent EGFR transactivation via an unknown mechanism and concomitant secretion of Hsp70. In this work we demonstrate that the depletion of Hsp70 from the conditioned medium of heated cells abolishes EGFR transactivation indicating that secreted Hsp70 is essential for EGFR transactivation during heat shock. This notion is supported by the findings that purified Hsp70 can induce EGFR transactivation and the activation of EGFR-dependent signaling pathways. Both heat stress and pure Hsp70 stimulate activation of TLR2/4 and their association with EGFR. These results suggest that the secreted Hsp70 mediates the cross-communication of TLR and EGFR signaling systems in A431 cells.

The G protein-coupled inwardly rectifying GIRK5 and Delta5GIRK5 splicing variants do not express functional potassium channels. In contrast, Delta25GIRK5 forms functional homomultimers in Xenopus laevis oocytes. A tyrosine is present at the N-term of the non-functional isoforms. We studied the effect of endogenous tyrosine phosphorylation on the GIRK5 surface and functional expression. Unlike wild type channels, GIRK5Y16A and Delta5GIRK5Y16A mutants displayed inwardly rectifying currents and inhibitors of Src tyrosine kinase promoted the traffiking of GIRK5 to the cell surface. This is the first evidence that endogenous phosphorylation of a tyrosine residue in a GIRK channel inhibits its surface expression.

Low-molecular-weight organic chromium complexes such as chromium picolinate are often used as dietary supplements to improve insulin sensitivity and to correct dyslipidemia. However, toxicity associated with such chromium compounds has compromised their therapeutic value. The aim of this study was to evaluate the impact of a newly synthesized complex of chromium with phenylalanine, Cr(pa)3 on insulin-signaling and glucose tolerance. Cr(pa)3 was synthesized by chelating chromium(III) with d-phenylalanine ligand in aqueous solution. In mouse 3T3-adipocytes, Cr(pa)3 augmented insulin-stimulated glucose-uptake as assessed by a radioactive-glucose uptake assay. At the molecular level, Cr(pa)3 enhanced insulin-stimulated phosphorylation of Akt in a time- and concentration-dependent manner without altering the phosphorylation of insulin receptor. Oral treatment with Cr(pa)3 (150μg/kg/d, for six weeks) in ob/ob(+/+) obese mice significantly alleviated glucose tolerance compared with untreated obese mice. Unlike chromium picolinate, Cr(pa)3 does not cleave DNA under physiological reducing conditions. Collectively, these data suggest that Cr(pa)3 may represent a novel, less-toxic chromium supplement with potential therapeutic value to improve insulin sensitivity and glycemic control in type II diabetes.

Glutathione-S-transferase (GST)-pp60c-Src (GST-Src) expressed in Escherichia coli is as catalytically active as purified, activated pp60c-Src protein derived from human platelets. We utilized the bacterially expressed enzyme, together with information about the structures of Src family kinases in complex with their inhibitors PP1 and PP2, to modify PP1 in a quest for improved inhibitors. Despite the detailed structural information on Hck-PP1 and Lck-PP2 complexes, which shows that PP1 and PP2 bind to the adenosine triphosphate (ATP) pocket, we were unable to improve the affinity between modified PP1 and Src. Puzzled, we examined in detail the mechanism by which PP1 inhibits the kinase activity of Src. Here we report that PP1 is non-competitive with ATP for the inhibition of Src, at variance with what is currently accepted, and is a ‘mixed competitive inhibitor’ vis-à-vis the substrate. These findings shed new light on the mechanism whereby PP1-like molecules inhibit Src. Examination of the homology between the kinase domain of Src and those of Hck and Lck reveals significant differences outside the ATP binding pocket, whereas they are identical within the ATP binding domain. These results suggest that PP1 may be a leading compound for ATP non-competitive inhibitors of Src family kinases. Since Src in its active form is the hallmark of numerous cancers, understanding how PP1 inhibits activated Src will aid in the discovery of potent and selective Src kinase inhibitors.

Previous studies from this laboratory have shown that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (HCRI) causes apoptotic cell death of a muscle cell-derived cell line, L6 myoblasts, by involving the phosphatidylinositol-3 (PI-3) kinase pathway and tyrosine phosphorylation of several cellular proteins, although the relationship between PI-3 kinase pathway and tyrosine phosphorylation responses remained to be elucidated. Here, we show that HCRI induces tyrosine phosphorylation of catalytic subunit p110 of PI-3 kinase as early as 5 min after addition of HCRI into culture medium. We could not detect the tyrosine phosphorylation of the regulatory subunit p85 of PI-3 kinase under the present experimental conditions. Concomitantly, the kinase activity toward PI in p110 immunoprecipitates was decreased with a similar time course. Furthermore, both herbimycin A and genistein, potent inhibitors of tyrosine kinase activity, inhibited HCRI-induced inhibition of PI-3 kinase activity as well as HCRI-induced apoptotic cell death. Once the catalytic subunit p110 becomes tyrosine-phosphorylated, the regulatory subunit p85 appears to be dissociated from the catalytic subunit, because we observed a decreasing amount of p85 regulatory subunits in p110 immunoprecipitates in response to HCRI treatment. These results strongly suggest the novel function of tyrosine phosphorylation of catalytic subunit p110 of PI-3 kinase in the regulation of its kinase activity. The tyrosine phosphorylation of these catalytic subunits may play an important role in the intracellular signal transduction of apoptotic cell death. To our knowledge, this is the first report that tyrosine phosphorylation of p110 catalytic subunit acts as a negative regulator of its kinase activity.

p97/Gab2 is a recently characterized member of a large family of scaffold proteins that play essential roles in signal transduction. Gab2 becomes tyrosine-phosphorylated in response to a variety of growth factors and forms multimolecular complexes with SH2 domain-containing signaling molecules such as the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K), the tyrosine phosphatase SHP-2 and the adapter protein CrkL. To characterize the interactions between Gab2 and its SH2-containing binding partners, we designed a modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed in a regulated manner in yeast. Using this assay, we demonstrated that p97/Gab2 specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL. Interaction with p85-PI3K is mediated by tyrosine residues Y452, Y476 and Y584 of Gab2, while interaction with SHP-2 depends exclusively on tyrosine Y614. CrkL interaction is mediated by its SH2 domain recognizing Y266 and Y293, despite the latter being in a non-consensus (YTFK) environment.

We examined the role of the mitogen-activated protein (MAP) kinase pathway in tissue inhibitor of metalloproteinases-1 (TIMP-1)-mediated cellular effects in a human erythroleukemic cell line UT-7. We show that TIMP-1 induced both UT-7 cell erythroid differentiation and proliferation and tyrosine phosphorylation of many intracellular proteins. Using a panel of phosphospecific antibodies, we also demonstrate that phosphorylation of the p38 and c-Jun N-terminal kinases is increased by TIMP-1 whereas phosphorylation of extracellular signal-regulated kinase 1/2 is not induced. Moreover, inhibition of the p38 activity by SB203580 significantly reduces erythroid differentiation induced by TIMP-1, suggesting that the p38 MAP kinase pathway is involved in TIMP-1-induced erythroid differentiation.

Desmoplastic small round cell tumor (DSRCT) is a malignant human cancer that is associated with a specific t(11;22) chromosome translocation, where 265 amino acids from the EWS amino-terminus are fused to the DNA binding domain of the WT1 tumor suppressor gene. We have noticed the presence of several SH3 interacting domains within the amino-terminus of EWS and have assessed the potential of EWS/WT1 to interact with such motifs. We find that EWS/WT1 can associate with the SH3 domain of several proteins, including v-Src. Ectopic expression of v-Src phosphorylates EWS/WT1 in vivo, as well as enhances the transactivation ability of the EWS amino-terminal domain. Structural alteration of the v-Src SH2 or SH3 domains produced mutants that could not interact with EWS/WT1 nor augment the transcriptional properties of EWS. Taken together, our results suggest the possibility that some transcriptional properties of EWS/WT1 may be regulated by a cytoplasmic signaling pathway.

Hic-5 is a CAKbeta-binding protein localized at focal adhesions. Here we show that overexpression of CAKbeta or Fyn, but not FAK, enhanced the tyrosine phosphorylation of coexpressed Hic-5 in COS-7 cells. These phosphorylations were further augmented by stimulating cells with osmotic stress. The Y60F mutant of Hic-5 was not phosphorylated, and Hic-5 phosphorylated on tyrosine 60 was bound specifically to the SH2 domain of Csk. Coexpression experiments revealed that the phosphorylation of Hic-5 by CAKbeta required the kinase activation of CAKbeta and binding of Hic-5 by CAKbeta. Specific phosphorylation of Hic-5 by CAKbeta and Fyn may activate a signaling pathway mediated by Hic-5.

The nerve growth factor receptor, TrkA, is essential for the survival and differentiation of neurons in the central and peripheral nervous systems. To understand the molecular principles underlying this differentiation step, we employed a yeast two-hybrid screening protocol using human TrkA as bait. We isolated c-Abl as a TrkA-interacting protein, in addition to known proteins such as phospholipase Cγ and SH2-B. This interaction was confirmed by an in vitro binding assay using glutathione S-tranferase–Abl fusion protein. Furthermore, we show here that c-Abl binds to phosphotyrosine residue(s) in the kinase activation loop of TrkA.

Vav functions as a specific GDP/GTP nucleotide exchange factor which is regulated by tyrosine phosphorylation in the hematopoietic system. Loss of the amino-terminus sequences of Vav was sufficient to control its transforming potential and its function in T cells. We report here the identification of the hematopoietic GDP dissociation inhibitor protein, Ly-GDI, as a protein that interacts with the amino-terminus of Vav. Further analysis confirmed that Vav and Ly-GDI interact both in in vitro and in in vivo assays. This association is maximal only when the amino region of Vav is intact and requires an intact carboxy-terminus of Ly-GDI. The interaction between Vav and Ly-GDI is not dependent on the tyrosine phosphorylation status of Vav. In addition, Rho-GDI, the highly homologous protein to Ly-GDI, associates with Vav as well. The contribution of the interaction between Vav and GDIs, proteins that are involved in the GDP/GTP exchange processes, to the biological function of Vav is further discussed.

We previously have shown that the overexpression of phospholipase C-gamma1 (PLC-gamma1) in rat 3Y1 fibroblasts results in malignant transformation (Chang, J.-S., Noh, D.Y., Park, I.A., Kim, M;.J., Song, H., Ryu, S.H. and Suh, P.-G. (1997) Cancer Res. 57, 5465-5468). The transformed cells, which initially are in an elongated and flat form after seeding in plastic dishes, become rounded during continued culture. We found that tyrosine dephosphorylation of paxillin accompanies this morphological change of the transformed cells and that PLC-gamma1 co-immunoprecipitates together with paxillin and vice versa, but not after the cells have become round. Transformed cells growing on fibronectin-pre-coated dishes regain their flat morphology and this is accompanied by paxillin tyrosine phosphorylation. Furthermore, immunoprecipitation analysis showed that paxillin forms a heteromeric complex with PLC-gamma1 in cells grown on fibronectin. These results suggest that a complex formation between paxillin and PLC-gamma1 may play a role in cell-substrate adhesion.

Recent studies have shown that, in addition to its role as an adhesion receptor, platelet endothelial cell adhesion molecule 1/CD31 becomes phosphorylated on tyrosine residues Y663 and Y686 and associates with protein tyrosine phosphatases SHP-1 and SHP-2. In this study, we screened for additional proteins which associate with phosphorylated platelet endothelial cell adhesion molecule 1, using surface plasmon resonance. We found that, besides SHP-1 and SHP-2, platelet endothelial cell adhesion molecule 1 binds the cytoplasmic signalling proteins SHIP and PLC-gamma1 via their Src homology 2 domains. Using two phosphopeptides, NSDVQpY663TEVQV and DTETVpY686SEVRK, we demonstrate differential binding of SHP-1, SHP-2, SHIP and PLC-gamma1. All four cytoplasmic signalling proteins directly associate with cellular platelet endothelial cell adhesion molecule 1, immunoprecipitated from pervanadate-stimulated THP-1 cells. These results suggest that overlapping immunoreceptor tyrosine-based inhibition motif/immunoreceptor tyrosine-based activation motif-like motifs within platelet endothelial cell adhesion molecule 1 mediate differential interactions between the Src homology 2 containing signalling proteins SHP-1, SHP-2, SHIP and PLC-gamma1.

In this study we show that platelet-derived growth factor (PDGF)-induced DNA binding as well as transcriptional activation of Stat5b are markedly increased by inhibition of the MAP (mitogen-activated protein) kinase kinase MEK. In addition to the previously demonstrated tyrosine phosphorylation, we show that serine and threonine phosphorylation of Stat5b is increased in response to PDGF stimulation. However, inhibition of MEK had no effect on the phosphorylation level of Stat5b or on the nuclear translocation of Stat5b. These observations indicate that MEK is a negative modulator of PDGF-induced Stat5b activation through a mechanism not involving direct phosphorylation of Stat5b.

Tyrosine phosphorylation has been associated with several aspects of the regulation of cholinergic synaptic function, including nicotinic acetylcholine receptor (AChR) desensitization as well as the synthesis and clustering of synaptic components. While some progress has been made in elucidating the molecular events initiating such signals, the downstream targets of these tyrosine kinase pathways have yet to be characterized. In this paper we have used molecular cloning techniques to identify proteins which are tyrosine phosphorylated at the cholinergic synapse. Phosphotyrosine containing proteins (PYCPs) were isolated from the electric organ of Torpedo californica by anti-phosphotyrosine immunoaffinity chromatography. Peptide sequencing and expression cloning then identified the isolated proteins. The proteins identified included heat shock protein 90, type III intermediate filament from Torpedo electric organ, α-fodrin, β-tubulin, actin and rapsyn. These tyrosine phosphorylated proteins may play a role in the regulation of synaptic function by tyrosine kinases.

Covalent complexes of nucleic acids and proteins are widespread among viruses. Covalent complexes of RNA and proteins are proposed to exist in eukaryotic cells. The goal of this work was to obtain specific antibodies to the covalent linkage unit (CLU) between virus RNA and protein to search cellular RNA-protein complexes. Antibodies were generated by direct immunization of a rabbit with the BSA-coupled EMC virus RNA-VPg complex. By a dot-blot immunoassay and immunofluorescent microscopy it was found that the antibodies specifically recognize both EMC virus RNA-VPg and synthetic CLU-containing compounds. Thus, a fraction of the antibodies was directed to CLU.

Oxidative stress-induced tyrosine phosphorylation has been ascribed to activation of phosphotyrosine kinase or to inhibition of phosphotyrosine phosphatase (PTP). We have previously identified a PTP associated with band 3 in the human erythrocyte membrane, a PTP that is normally highly active and prevents the appearance of band 3 phosphotyrosine. Here we show that treatment of erythrocytes with the thiol-oxidizing agent diamide leads to the formation of PTP disulfides (PTP-band 3 mixed disulfides) and inhibition of dephosphorylation, allowing the accumulation of band 3 phosphotyrosine. Upon reduction of the disulfides, the band 3 phosphotyrosine is dephosphorylated. Erythrocyte thiol alkylation by N-ethylmaleimide results in irreversible PTP inhibition and irreversible phosphorylation. The results are consistent with the notion that alterations in cellular thiol status affect the cell phosphotyrosine status and that oxidative stress-induced tyrosine phosphorylation involves inhibition of PTP.

Oncogenic transformation by v-Src is accompanied by marked morphological changes and cytoskeletal reorganization. Yet, the cytoskeleton-associated proteins with which v-Src interacts are largely unknown. We have studied the binding of v-Src-SH3 domain to cellular proteins utilizing a blot overlay procedure with a GST-v-Src-SH3 fusion protein as probe. A major 62–64 kDa v-Src-SH3-binding protein, present in detergent-insoluble cellular fractions, was identified as p21ras-GTPase-activating protein-associated p62 (GAPA62). In non-transformed cells, including NIH 3T3 cells, GAPA62 was present in both the RIPA-soluble and RIPA-insoluble fractions, but only the latter form was tyrosine-phosphorylated. In contrast, in polyoma middle T antigen-transformed 3T3 cells, GAPA62 was present only in the RIPA-insoluble fraction, where it was highly phosphorylated. It is suggested that tyrosine phosphorylation of GAPA62 may be an important determinant of its cellular localization and its possible function as a mediator of v-Src actions.© 1997 Federation of European Biochemical Societies.

Gastrin/CCKB G protein-coupled receptors have been shown to mediate proliferative effects of their endogenous ligands. In the present study, we examined the signal transduction mechanisms linked to the G/CCKB receptor occupancy. We report here that gastrin stimulates MAP kinase activation in a dose- and time-dependent manner, a pathway known to play a key role in cell proliferation. We also characterized the molecular events, upstream of p21-Ras, that may link the MAP kinase pathway to G/CCKB receptors. Gastrin induced a rapid and transient increase in tyrosine phosphorylation of several proteins including the 2 isoforms (46 and 52 kDa) of the adaptor protein She. Phosphorylated Shc subsequently associated with a complex that includes Grb2 and the p21-Ras activator, Sos. Our results also indicate that Sos becomes phosphorylated in response to gastrin as shown by a reduction in electrophoretic mobility of the protein. Tyrosine phosphorylation of Shc and subsequent complex formation with Grb2 and Sos appear to be a common mechanism by which tyrosine kinase receptors and the G/CCKB G protein-coupled receptor stimulate the Ras-dependent MAP kinase pathway.

The growth and differentiation of megakaryocytes are regulated by thrombopoietin (TPO), a recently characterized cytokine which exerts its effects via a member of the hematopoietin receptor superfamily, c-Mpl. Since many cytokines which bind hematopoietin receptors activate the STAT family of transcription factors, we investigated whether STAT proteins were activated by TPO. TPO induced the formation of a DNA-binding complex recognizing a known STAT binding sequence. STAT5 was a major component of this DNA-binding complex, and STAT5 was tyrosine phosphorylated in response to TPO. Additionally, TPO-induced the tyrosine phosphorylation and DNA-binding activity of STAT3. Together with the recent demonstration of JAK2 activation in response to TPO, the data presented here define a rapid signaling pathway likely to be important in TPO-induced gene regulation.

The cytokine interleukin-6 (IL-6) rapidly activates a latent cytoplasmic transcription factor, acute-phase response factor (APRF), by tyrosine phosphorylation. Activation and DNA binding of APRF are inhibited by inhibitors of protein tyrosine kinases but not serine/threonine kinases. However, immediate-early gene induction by IL-6 and, as we show here, stimulation of the promoters of the genes for α2-macroglobulin, Jun-B, and intercellular adhesion molecule-1 (ICAM-1) are blocked by the serine/threonine kinase inhibitor H7. We now show that IL-6 triggers a delayed phosphorylation of APRF at serine resudues which can be reversed in vitro by protein phosphatase 2A and is also inhibited by H7. Therefore, APRF serine phosphorylation is likely to represent a crucial event in IL-6 signal transduction leading to target gene induction.

Natural languages arise in an unpremeditated fashion resulting in words and syntax as individual units of information content that combine in a manner that is both complex and contextual, yet intuitive to a native reader. In an analogous manner, protein interaction domains such as the Src Homology 2 (SH2) domain recognize and “read” the information contained within their cognate peptide ligands to determine highly selective protein–protein interactions that underpin much of cellular signal transduction. Herein, we discuss how contextual sequence information, which combines the use of permissive and non-permissive residues within a parent motif, is a defining feature of selective interactions across SH2 domains. Within a system that reads phosphotyrosine modifications this provides crucial information to distinguish preferred interactions. This review provides a structural and biochemical overview of SH2 domain binding to phosphotyrosine-containing peptide motifs and discusses how the diverse set of SH2 domains is able to differentiate phosphotyrosine ligands.

In this study we examined the role of rho p21 in neuropeptide-stimulated tyrosine phosphorylation. Intact Swiss 3T3 cells were treated with the Clostridium botulinum C3 exoenzyme which specifically ADP ribosylates and inactivates rho p21. C3 exoenzyme treatment of cells caused a marked decrease in both bombesin- and endothelin-stimulated tyrosine phosphorylation of multiple proteins, including p125 focal adhesion kinase (FAK) and paxillin. Our results suggest that rho p21 is a component of the signal transduction pathway linking seven transmembrane domain receptors with tyrosine phosphorylation and cytoskeletal events.

Collagen is an important primary stimulus of platelets during the process of hemostasis. As with many other platelet stimuli, collagen signal transduction involves the hydrolysis of inositol phospholipids; however, the mechanisms which underlies this event is not well understood. Neither the collagen receptor nor the isoform of phospholipase C that is activated have been identified. We report that collagen-activation of platelets induces tyrosine phosphorylation of phospholipase C-gamma 2 but not phospholipase C-gamma 1. We also show that the platelet low affinity Fc receptor (Fc gamma RII), which mediates activation of platelets by immune complexes, and wheat germ agglutinin, which binds non-specifically to glycoprotein, stimulate phospholipase C-gamma 2 tyrosine phosphorylation. In contrast, we could not detect phospholipase C-gamma 2 tyrosine phosphorylation in platelets stimulated by either thrombin or a stable thromboxane A2 analogue, U46619.

Phosphorylation of the catalytic subunit of protein phosphatase 2A (PP2A) on threonines with a distinct autophosphorylation-activated protein kinase [Guo and Damuni (1993) Proc. Natl. Acad. Sci. USA 90, 2500–2504] inactivated the phosphatase with 32P-labelled myelin basic protein prepared by incubation with the kinase domain of the epidermal growth factor receptor, the src-family protein kinases p56lck and p60c-src, myelin basic protein kinase-1, or protamine kinase. Phosphoamino acid analysis demonstrated that the kinase domain of the epidermal growth factor receptor, p56lck and p60c-src phosphorylated myelin basic protein on tyrosines, that the protamine kinase phosphorylated myelin basic protein on serines, and that myelin basic protein kinase-1 phosphorylated myelin basic protein on threonines. The results demonstrate that the autophosphorylation-activated protein kinase not only inactivates the protein serine/threonine phosphatase, but also the protein tyrosine phosphatase activity of PP2A. This autophosphorylation-activated protein kinase-mediated inactivation of PP2A may, in response to extracellular stimuli, not only contribute to the enhanced phosphorylation of cellular proteins on serines and threonines but also on tyrosines.

Site-directed mutagenesis of the three binding sites for the mammary factor MPBF in the β-lactoglobulin (BLG) promoter demonstrates that MPBF is a transcriptional activator of the BLG gene in mammary cells. MPBF requires phosphorylation on tyrosine for maximum binding activity and binds to GAS (interferon γ-activation site) elements which are similar to the MPBF binding sites. Prolactin induces MPBF binding activity in CHO cells and is not antigenically related to Stat1 (p91) and Stat2 (p113), suggesting that this transcription factor is likely to be another member of the STAT family of cytokine/growth factor-induced transcription factors.

The role of low M r phosphotyrosine protein phosphatase (PTPase) in the control of cell proliferation was studied. A synthetic gene coding for PTPase was transfected and expressed in NIH/3T3 fibroblasts. The effects of the enzyme were particularly evident when cells were stimulated by platelet-derived growth factor (PDGF). The mitogenic response to PDGF was decreased and the inhibition reached 90%. This effect was more pronounced with respect to fetal calf serum stimulation. Hormone-dependent autophosphorylation of the PDGF receptor was significantly reduced. These results demonstrate that low M r PTPase, a cytosolic enzyme, not only affects cellular response to PDGF but also reduces the membrane receptor autophosphorylation.

We have investigated the regulation of tyrosine proteins phosphorylation by intracellular Ca 2+ level ([Ca 2+ ] i ) and protein kinase C (PKC) during platelet stimulation. We found that chelation of extracellular calcium completely prevented phosphorylation of tyrosine proteins induced by thapsigargin and phorbol 12-myristate 13-acetate (PMA), whereas, when induced by thrombin, it prevented a subset of tyrosine proteins. The selective inhibition of PKC by OF 109203X did not abolish tyrosine protein phosphorylation when induced by thrombin and thapsigargin. The results suggest that in human platelets tyrosine protein phosphorylation is dependent on [Ca 2+ ] i , although direct PKC activation can also induce phosphorylation of tyrosine proteins.

Beside 4- and 5-phosphatases playing a role in the interconversion between the D-3 phosphorylated polyphosphoinositides, the only enzyme described so far to be responsible for a phosphomonoesterasic activity on the D-3 position of inositol lipids is a specific 3-phosphatase that hydrolyzes PtdIns(3)P in NIH 3T3 cells. We report here the presence of a potent 3-phosphatase activity in different cell types. This activity is detected both in cytosol and membranes of A431 cells and is inhibited by VO43− and Zn2+. Interestingly, the cytosolic activity from A431 cells selectively hydrolyzes in vitro PtdIns(3)P and PtdIns(3,4)P2, whereas PtdIns(3,4,5)P3 remains a very poor substrate under the same conditions. Finally, assays of phosphatidylinositol 3-kinase and 3-phosphatase activities in the pool of phosphotyrosine-containing proteins isolated from EGF-stimulated A431 cells suggest a compartmentation of these two antagonistic activities during cell activation.

Incubation of HER 14 cells with phorbol myristate acetate (PMA) decreases epidermal growth factor (EGF)-dependent protein tyrosine phosphorylation, except for a 40-kDa MAP kinase II-like protein, whose tyrosine phosphorylation is further enhanced. The inhibitory effect of PMA on EGF-dependent protein tyrosine phosphorylation is reversed if cells are pre-incubated with a combination of Na3VO4 and NaF, two known inhibitors of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity of cell homogenate was measured on immunopurified EGF receptor, and was found to be enhanced in PMA-treated cells. These data suggest that the inhibitory effect of PMA on EGF-dependent protein tyrosine phosphorylation in HER14 cells may be mediated by protein tyrosine phosphatase activity.

Occupancy of CD72 on resting tonsillar B cells by monoclonal antibodies (mAb) promotes entry into the G1 phase of the cell cycle with an accompanying increase in MHC Class II expression and provides a co-stimulus to immobilized anti-μ for driving DNA synthesis. We now report that engagement of CD72 by mAb stimulates tyrosine phosphorylation in B cells with a peak of activity seen at 5–10 min. Two major substrates of 29 and 57 kDa showed a basal level of phosphorylation which increased with time, while a 40 kDa protein and several other minor components were phosphorylated de novo on the addition of mAb to CD72. Inositol lipid hydrolysis was found to be unperturbed, although a shallow rise in the basal level of intracellular free Ca2+ was provoked on engaging CD72. Receptor cross-linking was not a requirement for signaling human B cells through CD72: simple occupancy by univalent antibody was sufficient both to trigger the rise in basal [Ca2+]i and to promote DNA synthesis.

Previously, we have demonstrated that the tyrosine phosphorylated hepatocyte growth factor receptor (Met) binds to the c-Cbl phosphotyrosine-recognition, tyrosine kinase binding (TKB) domain in a reverse orientation compared to other c-Cbl binding partners. A Met peptide with the DpYR motif changed to RpYD (MetRD) retains a similar TKB binding affinity as the native Met peptide. However, the TKB: MetRD complex crystal structure reveals a complete reversal of the binding orientation. Collated data indicates that both binding and orientation is dictated by the phosphorylated tyrosine and an adjacent arginine forming intra-peptide hydrogen bonds and aligning unidirectionally with complementary charges in the phosphotyrosine binding pocket of c-Cbl.Structured summaryc-Cbl and MetRD bind: shown by x-ray crystallography (view interaction)c-Cbl and MetRD bind: shown by mass spectrometry studies of complexes (view interaction)c-Cbl bind to Met: shown by surface plasmon resonance (view interactions 1,2)

Macropinocytosis is regulated by Abl kinase via an unknown mechanism.We previously demonstrated that Abl kinase activity is, itself, regulated by Abi1 subsequent to Abl kinase phosphorylation of Abi1 tyrosine 213 (pY213) [1].Here we show that blocking phosphorylation of Y213 abrogated the ability of Abl to regulate macropinocytosis, implicating Abi1 pY213 as a key regulator of macropinocytosis.Results from screening the human SH2 domain library and mapping the interaction site between Abi1 and the p85 regulatory domain of PI-3 kinase, coupled with data from cells transfected with loss-of-function p85 mutants, support the hypothesis that macropinocytosis is regulated by interactions between Abi1 pY213 and the C-terminal SH2 domain of p85—thereby linking Abl kinase signaling to p85-dependent regulation of macropinocytosis.Structured summaryMINT-7908602:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to SHIP2 (uniprotkb:O15357) by array technology (MI:0008)MINT-7908362:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Emt (uniprotkb:Q08881) by array technology (MI:0008)MINT-7908235:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Lyn (uniprotkb:P07948) by array technology (MI:0008)MINT-7908075:Abi1 (uniprotkb:Q8IZP0)binds(MI:0407) to Fgr (uniprotkb:P09769) by array technology (MI:0008)MINT-7908330, MINT-7908522:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Vav1 (uniprotkb:P15498) by array technology (MI:0008)MINT-7907962:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fyn (uniprotkb:P06241) by array technology (MI:0008)MINT-7908203:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Src(uniprotkb:P12931) by array technology (MI:0008)MINT-7908570:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to SHP-2 (uniprotkb:P35235) by array technology (MI:0008)MINT-7908187, MINT-7908586:Abi1(uniprotkb:Q8IZP0) binds (MI:0407) to Gap (uniprotkb:P20936) by array technology (MI:0008)MINT-7907981, MINT-7907995:Abi1 (uniprotkb:Q8IZP0) physically interacts (MI:0915) with p85a(uniprotkb:P26450) by anti tag coimmunoprecipitation (MI:0007)MINT-7908251:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to PLCG1 (uniprotkb:P19174) by array technology (MI:0008)MINT-7908346:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Grb2 (uniprotkb:P62993) by array technology (MI:0008)MINT-7907945:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Abl (uniprotkb:P00519) by array technology (MI:0008)MINT-7908474:Abi1(uniprotkb:Q8IZP0)binds (MI:0407) to p85b (uniprotkb:O00459) by array technology (MI:0008)MINT-7908107:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Hck (uniprotkb:P08631) by array technology (MI:0008)MINT-7908011: p85a (uniprotkb:P26450) physically interacts (MI:0915) with Abi1 (uniprotkb:Q8IZP0) by pull down (MI:0096)MINT-7908155:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to FynT (uniprotkb:P06241-2) by array technology (MI:0008)MINT-7908283, MINT-7908490:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to p55g (uniprotkb:Q92569) by array technology (MI:0008)MINT-7907929, MINT-7907815, MINT-7907832, MINT-7907865, MINT-7907897, MINT-7907913, MINT-7907881, MINT-7907848:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to p85a (uniprotkb:P27986) by array technology (MI:0008)MINT-7908059:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Frk (uniprotkb:P42685) by array technology (MI:0008)MINT-7908378:Abi1(uniprotkb:Q8IZP0) binds (MI:0407) to CblC (uniprotkb:Q9ULV8) by array technology (MI:0008)MINT-7908618:Abi1(uniprotkb:Q8IZP0) binds (MI:0407) to CblA (uniprotkb:B5MC15) by array technology (MI:0008)MINT-7908139, MINT-7908538:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Nap4 (uniprotkb:O14512) by array technology (MI:0008)MINT-7908426:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblB (uniprotkb:Q13191) by array technology (MI:0008)MINT-7908506:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Crk (uniprotkb:P46108) by array technology (MI:0008)MINT-7908554:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to mAbl (uniprotkb:P00520) by array technology (MI:0008)MINT-7908043, MINT-7908394:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Vav2 (uniprotkb:P52735) by array technology (MI:0008)MINT-7908458:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to mSck/ShcB (uniprotkb:Q8BMC3) by array technology (MI:0008)MINT-7908091:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Yes (uniprotkb:P07947) by array technology (MI:0008)MINT-7908219:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Src (uniprotkb:P00523) by array technology (MI:0008)MINT-7908123:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fer (uniprotkb:P16591) by array technology (MI:0008)MINT-7908410:Abi1(uniprotkb:Q8IZP0) binds (MI:0407) to CrkL (uniprotkb:P46109) by array technology (MI:0008)MINT-7908314, MINT-7908442:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Arg (uniprotkb:P42684) by array technology (MI:0008)MINT-7908299:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to PLCG1 (uniprotkb:P10686) by array technology (MI:0008)MINT-7908171:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fes (uniprotkb:P07332) by array technology (MI:0008)MINT-7908027:Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Lck (uniprotkb:P06239) by array technology (MI:0008)

Src homology 2 (SH2) regions are short (approximately 100 amino acids), non-catalytic domains conserved among a wide variety of proteins involved in cytoplasmic signaling induced by growth factors. It is thought that SH2 domains play an important role in the intracellular response to growth factor stimulation by binding to phosphotyrosine containing proteins. In this paper we apply the techniques of multiple sequence alignment, secondary structure prediction and conservation analysis to 67 SH2 domain amino acid sequences. This combined approach predicts seven core secondary structure regions with the pattern beta-alpha-beta-beta-beta-beta-alpha, identifies those residues most likely to be buried in the hydrophobic core of the native SH2 domain, and highlights patterns of conservation indicative of secondary structural elements. Residues likely to be involved in phosphotyrosine binding are shown and orientations of the predicted secondary structures suggested which could enable such residues to cooperate in phosphate binding. We propose a consensus pattern that encapsulates the principal conserved features of the SH2 domains. Comparison of the proposed SH2 domain of akt to this pattern shows only 12/40 matches, suggesting that this domain may not exhibit SH2-like properties.

We have studied the role of tyrosine kinase in PMA-stimulated T cells. Protein kinase C (PKC)-mediated D10A cell proliferation is inhibited by the specific inhibitor of tyrosine kinase, tyrphostin. This inhibitor selectively blocks the mRNA expression of the proto-oncogene e-mye in response to the phorbol ester. On the other hand, the same doses of this inhibitor do not affect the mRNA expression of the proto-oncogene c-for in PMA-stimulated D10A cells. Phorbol esters induce in this T cell line the tyrosine phosphorylation of a unique protein of 42 kDa and the enzyme PKC is required for this activity.

Activation of the receptor tyrosine kinase DDR1 by collagen results in robust and sustained phosphorylation, however little is known about its downstream mediators.Using phosphopeptide mapping and site-directed mutagenesis, we here identified multiple tyrosine phosphorylation sites within DDR1. We found that Nck2 and Shp-2, two SH2 domain-containing proteins, bind to DDR1 in a collagen-dependent manner. The binding site of Shp-2 was mapped to tyrosine-740 of DDR1 within an ITIM-consensus sequence. Lastly, ablation of DDR1 in the mouse mammary gland resulted in delocalized expression of Nck2, suggesting that defects observed during alveologenesis are caused by the lack of the DDR1–Nck2 interaction.

To test the role of the leukocyte common antigen-related protein tyrosine phosphatase (LAR) as a regulator of insulin receptor (IR) signalling, an siRNA probe against LAR was developed. Knock-down of LAR induced post-receptor insulin resistance with the insulin-induced activation of PKB/Akt and MAP kinases markedly inhibited. The phosphorylation and dephosphorylation of the IR and insulin receptor substrate (IRS) proteins were unaffected by LAR knock-down. These results identify LAR as a crucial regulator of the sensitivity of two key insulin signalling pathways to insulin. Moreover, the siRNA probe provides a molecular tool of general applicability for further dissecting the precise targets and roles of LAR.

In rat adipocytes, phorbol ester-induced activation of PKC did not inhibit insulin signalling through IRS-1-dependent phosphatidylinositol (PI) 3-kinase activation. Moreover, phorbol esters alone provoked an increase in membrane PI 3-kinase activity. These findings may be relevant to the failure of phorbol esters to inhibit insulin effects on glucose transport and glycogen synthesis in rat adipocytes.