Biomedical subjects

R B Birge

Publications and source records attributed to R B Birge.

At least 19 recordsLinked to original sources

PubMed2004

A role for schwann cells in the neuroregenerative effects of a non-immunosuppressive fk506 derivative, jnj460.

UNLABELLED: FK506 and its non-immunosuppressive derivatives represent a class of pharmacological agents referred to as immunophilin ligands that have been reported to promote neuroregeneration and survival in several experimental models; however their cellular and molecular mechanisms of action have not been well established. Here we characterize a new immunophilin ligand that interacts with both FK506 binding protein 12 (FKBP12) and FKBP52, and demonstrate that JNJ460 induces neurite outgrowth from freshly explanted dorsal root ganglia (DRG) in a Schwann cell-dependent manner. Purified cultures of neurons fail to respond to these drugs, but cultures containing Schwann cells and neurons respond with neurite outgrowth, as do neurons grown in conditioned medium from JNJ460-treated Schwann cells. Using microarray analysis and a transcription reporter assay, we show that JNJ460 induces a series of transcriptional changes that occur in a temporal cascade. Among the Schwann cell-expressed genes upregulated following JNJ460 treatment is the POU transcription factor SCIP, which has been shown to regulate Schwann cell gene transcription and differentiation. JNJ460 potentiated transforming growth factor beta (TGF-beta)-induced transcriptional activation and SCIP induction in Schwann cells, by altering the interaction between FKBP12 and the TGF-beta type I receptor, TbetaR1. Finally, to test whether JNJ460 enhances neurite regeneration in vivo, we treated animals with JNJ460 for 30 days following mechanical transection of the sciatic nerve and demonstrated myelin and axonal hypertrophy at the ultrastructural level. Collectively, these data suggest that Schwann cells play an important role in the biological effects of immunophilin ligands by affecting neuron-glial signaling during regeneration. SUMMARY: The cellular and molecular mechanisms responsible for the regenerative effects of immunophilin ligands are not well understood. Here we show that the neuritogenic effects of JNJ460 in a DRG model depend on interactions between neurons and Schwann cells. Treatment of purified Schwann cells with JNJ460 alters Schwann cell gene expression, and promotes the generation of factors that act on neurons. These data indicate that Schwann cells play an important role in the actions of immunophilin ligands.

Animals↗

PubMed2001

Activation of the focal adhesion kinase signaling pathway by structural alterations in the carboxyl-terminal region of c-Crk II.

The Crk II adaptor protein encodes an SH2/SH3-domain containing adaptor protein with an SH2-SH3-SH3 domain structure that transmits signals from tyrosine kinases. The two SH3 domains are separated by a 54 amino acid linker region, whose length is highly conserved in xenopus, chicken, and mamalian Crk II proteins. To gain a better understanding into the role of the C-terminal region of Crk, we generated a series of C-terminal SH3 domain and SH3 linker mutants and examined their role in tyrosine kinase pathways. Expression of point mutations in the C-terminal SH3 domain (W276K Crk), at the tyrosine phosphorylation site (Y222F Crk II), or truncation of the entire C-terminus (Crk I or Crk Delta242), all increased c-Abl binding to the N-terminal SH3 domain of Crk and, where relevant, increased Tyr(222) phosphorylation. Deletion analysis of c-Crk II also revealed the presence of a C-terminal segment important for trans-activation of FAK. Such mutants, Crk Delta255 or Crk Delta242 Extended Linker (Crk Delta242([EL])), characterized by a disruption in the SH3 linker/C-terminal SH3 boundary, induced robust hyperphosphorylation of focal adhesion kinase (FAK) on Tyr(397), hyperphosphorylation of focal adhesion proteins p130(cas) and paxillin and increased focal adhesion formation in NIH3T3 cells. The effects of Crk Delta242([EL]) could be abrogated by co-expression of dominant negative c-Src or the protein tyrosine phosphatase PTP-PEST, but not by dominant negative Abl. Our results suggest that the C-terminal region of Crk contains negative regulatory elements important for both Abl and FAK dependent signal pathways, and offers a paradigm for an autoinhibitory region in the SH3 linker/C-terminal SH3 domain.

3T3 Cells↗

PubMed2000

The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction.

CMS/CD2AP is a cytoplasmic protein critical for the integrity of the kidney glomerular filtration and the T cell function. CMS contains domains and motifs characteristic for protein-protein interactions, and it is involved in the regulation of the actin cytoskeleton. We report here that the individual SH3 domains of CMS bind to phosphotyrosine proteins of approximately 80, 90, and 180 kDa in cell lysates stimulated with epidermal growth factor. The second SH3 domain of CMS bound specifically to a tyrosine-phosphorylated protein of 120 kDa, which we identified as the proto-oncoprotein c-Cbl. The c-Cbl-binding site for CMS mapped to the carboxyl terminus of c-Cbl and is different from the proline-rich region known to bind SH3-containing proteins. CMS binding to c-Cbl was markedly attenuated in a tyrosine phosphorylation-defective c-Cbl mutant indicating that this interaction is dependent on the tyrosine phosphorylation of CMS. It also implies that CMS interacts with c-Cbl in an inducible fashion upon stimulation of a variety of cell-surface receptors. Immunofluorescence analysis revealed that both proteins colocalize at lamellipodia and leading edges of cells, and we propose that the interaction of CMS with c-Cbl offers a mechanism by which c-Cbl associates and regulates the actin cytoskeleton.

Actins↗

PubMed2000

Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis.

The Crk family of adaptor proteins participate in diverse signaling pathways that regulate growth factor-induced proliferation, anchorage-dependent DNA synthesis, and cytoskeletal reorganization, important for cell adhesion and motility. Using kidney epithelial 293T cells for transient co-transfection studies and the nerve growth factor (NGF)-responsive PC12 cell line as a model system for neuronal morphogenesis, we demonstrate that the non-receptor tyrosine kinase c-Abl is an intermediary for NGF-inducible c-Crk II phosphorylation on the negative regulatory Tyr(222). Transient expression of a c-Crk II Tyr(222) point mutant (c-Crk Y222F) in 293T cells induces hyperphosphorylation of paxillin on Tyr(31) and enhances complex formation between c-Crk Y222F and paxillin as well as c-Crk Y222F and c-Abl, suggesting that c-Crk II Tyr(222) phosphorylation induces both the dissociation of the Crk SH2 domain from paxillin and the Crk SH3 domain from c-Abl. Interestingly, examination of the early kinetics of NGF stimulation in PC12 cells showed that c-Crk II Tyr(222) phosphorylation preceded paxillin Tyr(31) phosphorylation, followed by a transient initial dissociation of the c-Crk II paxillin complex. PC12 cells overexpressing c-Crk Y222F manifested a defect in cellular adhesion and neuritogenesis that led to detachment of cells from the extracellular matrix, thus demonstrating the biological significance of c-Crk II tyrosine phosphorylation in NGF-dependent morphogenesis. Whereas previous studies have shown that Crk SH2 binding to paxillin is critical for cell adhesion and migration, our data show that the phosphorylation cycle of c-Crk II determines its dynamic interaction with paxillin, thereby regulating turnover of multiprotein complexes, a critical aspect of cytoskeletal plasticity and actin dynamics.

Animals↗

PubMed2000

Association of the Abl tyrosine kinase with the Trk nerve growth factor receptor.

Nerve growth factor (NGF) initiates the majority of its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. In addition to rapid increases in the phosphorylation of phosphatidylinositol 3'-kinase (PI 3-kinase) and phospholipase C-gamma and increased ras activity, phosphorylation of c-Crk and paxillin proteins has been observed upon TrkA activation. The c-Abl tyrosine kinase is involved in the control of the axonal cytoskeleton and is known to interact with c-Crk proteins. Here we have tested the possibility that TrkA receptors might form an association with the c-Abl protein. After transfection in 293T cells, TrkA and c-Abl kinases could be coimmunoprecipitated. This interaction did not require TrkA receptors to be autophosphorylated. Mapping analysis indicated that the region of c-Abl association was confined to the juxtamembrane region of TrkA. The interaction of c-Abl with TrkA was also observed in differentiated pheochromocytoma PC12 cells. These results suggest that c-Abl may be recruited to the NGF receptor complex and be involved in regulating specific phosphorylation events that occur during neuronal differentiation.

Animals↗

PubMed2000

alphavbeta5 integrin recruits the CrkII-Dock180-rac1 complex for phagocytosis of apoptotic cells.

Integrin receptors are important for the phagocytosis of apoptotic cells. However, little is known about their function in mediating internalization, as previous studies used blocking antibodies for the inhibition of binding. Here we show that the alphavbeta5 receptor mediates both binding and internalization of apoptotic cells. Internalization is dependent upon signalling through the beta5 cytoplasmic tail, and engagement of the alphavbeta5 heterodimer results in recruitment of the p130cas-CrkII-Dock180 molecular complex, which in turn triggers Rac1 activation and phagosome formation. In addition to defining integrin-receptor signalling as critical for the internalization of apoptotic material, our results also constitute the first evidence in human cells that the CED-2-CED-5-CED-10 complex defined in Caenorhabditis elegans is functionally analagous to the CrkII-Dock180-Rac1 molecular complex in mammalian cells. By linking the alphavbeta 5 receptor to this molecular switch, we reveal an evolutionarily conserved signalling pathway that is responsible for the recognition and internalization of apoptotic cells by both professional and non-professional phagocytes.

Apoptosis↗

PubMed2000

A nuclear function for the tumor suppressor BRCA1.

The breast and ovarian cancer susceptibility gene BRCA1 has been recently cloned and revealed an open reading frame of 1863 amino acids, but a lack of significant homology to any known protein in the database has led to few clues about its functions. One of the first steps to investigate the function of BRCA1 was to define its subcellular localization. Several reports have led to contradictory findings that include: nuclear localization in normal cells and cytoplasmic in breast and ovarian cancer cells; nuclear in both normal and cancer cells; cytoplasmic and secreted to the extracellular space; present in tube-like invaginations of the nucleus; and colocalizing with the centrosome. As is apparent, the subcellular localization has been the most controversial aspect of BRCA1 biology and is a key point to uncover its functions. In this paper we review the published data on subcellular localization of BRCA1 with special emphasis on the antibodies and techniques used. We conclude that there is now overwhelming evidence to support a nuclear localization for BRCA1, both in normal and cancer cells. In addition, several BRCA1-interacting proteins have been isolated and they are preferentially located in the nucleus. Evidence supporting a physiological function for BRCA1 during DNA repair and transcriptional activation is also discussed.

BRCA1 Protein↗

PubMed1999

Targeted expression of an oncogenic adaptor protein v-Crk potentiates axonal growth in dorsal root ganglia and motor neurons in vivo.

The ability of neurons to survive and to target axonal growth requires a coordinated series of cell extrinsic and intrinsic events. Previously, in a cellular model for neuronal differentiation, we showed that pheochromocytoma (PC12) cells expressing v-Crk, an oncogenic form of the SH2/SH3-containing c-Crk adaptor protein, potentiates axonal growth and prolongs nerve growth factor (NGF)-independent survival. In the present study, we have generated transgenic mice that express v-Crk in sensory, motor, and enteric neurons by placing v-crk under the control of the neuron-specific peripherin promoter. In contrast to wild-type (wt) mice, dorsal root ganglia (DRG) neurons explanted from post-natal day 1 transgenic mice demonstrated a reduced dependence on trophic factors for both survival and axonogenesis. v-Crk also caused an increase in the number of surviving spinal motor neurons (SMN), and interestingly, upon staining of sternomastoid muscle fibers with rhodamine conjugated alpha-bungarotoxin, many muscle fibers displayed an apparent increase in volume of motor end plates, and an increase in complexity of neuromuscular junctions (NMJ). Our data suggest that v-Crk may be involved in transducing extracellular signals to regulate cytoskeletal organization, and may act on an intrinsic determinant for axonal growth in a variety of neural types including sensory and motor neurons during development.

Animals↗

PubMed1999

Integrin-mediated activation of focal adhesion kinase is required for signaling to Jun NH2-terminal kinase and progression through the G1 phase of the cell cycle.

The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Herein, we provide evidence that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK required the association of focal adhesion kinase (FAK) with a Src kinase and p130(CAS), the phosphorylation of p130(CAS), and subsequently, the recruitment of Crk. Ras and PI-3K were not required. FAK-JNK signaling was necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation.

Animals↗

PubMed1998

Drug resistance results in alterations in expression of immune recognition molecules and failure to express Fas (CD95).

It is demonstrated that methotrexate/cisplatin-sensitive L1210 cells express low levels of major histocompatibility complex (MHC) class II relative to the high levels expressed on methotrexate (MTX)/cisplatin-resistant L1210/DDP cells. L1210 cells express cell-surface Fas, while the L1210/DDP cells express no cell-surface Fas. Expression of costimulatory molecules B7-1/B7-2 and Fas is increased on L1210 cells, but not L1210/DDP, in the presence of methotrexate or trimetrexate (TMTX). Therefore, a component of the mechanism of action of some anti-cancer agents may be to facilitate immune recognition and T cell-directed, Fas-induced cell death. Loss of cell-surface Fas expression and failure of Fas (CD95)-dependent apoptotic death has been observed when cells develop drug resistance. The defect in apoptosis can be overcome by anti-cancer agents or experimental manipulation that induce Fas expression on the drug-resistant cells.

Animals↗

PubMed1998

Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein.

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.

Animals↗

PubMed1997

v-Crk, an effector of the nerve growth factor signaling pathway, delays apoptotic cell death in neurotrophin-deprived PC12 cells.

v-Crk is a member of a class of SH2 and SH3-containing adaptor proteins that have been implicated in regulating the TrkA receptor tyrosine kinase and potentiating Nerve Growth Factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells (Hempstead et al, Mol. Cell Biol. 14: 1964 - 1971). Given the fact that NGF induces both differentiation and survival by binding to TrkA, we examined the rate of apoptotic cell death elicited by NGF-withdrawal in native, v-Crk, and TrkA-expressing PC12 cells. While more than 50% of native PC12 cells underwent apoptosis within 48 h of NGF withdrawal, the v-Crk and TrkA-expressing cells were much more resistant to apoptosis under these conditions, whereby approximately 70 and 95%, respectively, of the cells were alive. The ability of v-Crk to delay apoptosis required prior NGF-dependent differentiation, since naive undifferentiated v-Crk expressing PC12 cells or cells that express v-Crk mutants that are defective in NGF signaling were not protected from apoptosis during growth factor withdrawal. Moreover, addition of 50 ng/ml EGF to serum and NGF deprived v-Crk expressing cells, which also causes neurite outgrowth, promoted complete and long-term survival, although such EGF replacement had no neurotrophic effect on wild-type PC12 cells or PC12 cells overexpressing Human Bcl-2. These experiments suggest that v-Crk potentiation of a receptor tyrosine kinase under conditions of growth factor deprivation is essential for preventing apoptosis. However, unlike native PC12 cells, neither v-Crk or TrkA-expressing PC12 cells exhibited a G1 arrest when incubated for 2 weeks in NGF. Thus, v-Crk and TrkA may protect NGF deprived PC12 by preventing cell cycle arrest and hence an aborted entry into a defective cell cycle. Moreover, during NGF-withdrawal, v-CrkPC12 cells exhibited down regulation in MAP kinase and JNK activities while in native cells, these activities increased within 6 - 8 h after NGF deprivation. Thus, unlike v-Crk-mediated augmentation of differentiation, sustained activation of MAP kinase may not be required for v-Crk-induced cell survival.

Journal Article↗

PubMed1997

v-Crk-induced cell transformation: changes in focal adhesion composition and signaling.

v-Crk is an oncogene product in which a viral Gag sequence is fused to a cellular Crk sequence. It contains one SH2 and one SH3 domain. To gain insight into the molecular mechanisms underlying v-Crk-induced cell transformation, we studied the subcellular localization and molecular interactions of v-Crk in v-Crk-transformed NIH-3T3 cells. Our results show that v-Crk specifically localizes to focal adhesions where it induces protein tyrosine phosphorylation. Subcellular fractionation studies indicated that a significant amount of v-Crk is present in the cytoskeletal cell fraction, a fraction that includes focal adhesions. Tyrosine phosphorylated proteins, including p130CAS, were also predominantly found in the cytoskeletal fraction. We show that v-Crk induces a translocation of p130CAS to the cytoskeleton, which is accompanied by hyperphosphorylation of this protein. Mutational analyses showed that functional v-Crk SH2 domain is required for the localization of v-Crk in focal adhesions. Functional v-Crk SH2 and SH3 domains were both found to be required for the observed increase in tyrosine phosphorylation of focal adhesion proteins and for the translocation and hyperphosphorylation of p130CAS. v-Crk immunoprecipitation studies revealed that cytoskeleton-associated v-Crk interacts with both p130CAS and an unidentified tyrosine kinase. These findings suggest that formation of a focal adhesion-located complex consisting of v-Crk, a tyrosine kinase and p130CAS, which may lead to the hyperphosphorylation of p130CAS. These specific and localized signaling events may represent initial steps in the process of v-Crk-induced cell transformation.

3T3 Cells↗

PubMed1996

SH2 and SH3-containing adaptor proteins: redundant or independent mediators of intracellular signal transduction.

Molecules which contain Src Homology 2 (SH2) and SH3 domains provide one of the principal ways by which signals are transduced in cells using protein-protein interactions between proline-rich motifs and SH3 domains and induced interactions between phosphotyrosine residues and SH2 domains. The simplest of SH2/SH3-containing proteins are the Crk, Grb2 and Nck adaptor proteins which contain SH2 and SH3 domains but no intrinsic catalytic activity. Whereas Grb2 connects activated receptor tyrosine kinases with Sos and activates p21ras, recent evidence suggests that this may not be the major mechanism by which Crk and Nck signal to downstream effectors. Identification of novel binding partners for Crk, Grb2 and Nck indicate that these adaptor proteins control distinct aspects of tyrosine kinase signalling.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Dissociation of NGF induced signal transduction from neurite elongation by expression of a mutant adaptor protein v-Crk in PC12 cells.

Expression of the adaptor protein v-Crk in PC12 cells results in sustained activation of NGF signaling pathways and augmented neuritogenesis. However, the inhibitory effect of the v-Crk SH2 domain mutant on neurite elongation does not correlate with impaired Trk A dependent signaling events or gene induction. In contrast, immunofluorescence studies and Triton X-100 extraction experiments indicate that v-Crk co-localizes with the cytoskeletal protein paxillin in the actin cytoskeleton whereas the v-Crk SH2 mutant causes aberrant aggregration of actin filaments at the growth cones. Interestingly, the neurotrophin receptor p75 in v-CrkPC12 cells also displays enhanced localization to the cytoskeleton and these cells exhibit an increased rate of NGF internalization. Together our data suggest that v-Crk might target the NGF-activated receptor signaling complex to the cytoskeleton, thereby potentiating neuritogenesis at the growth cone level. However, mutation in the v-Crk SH2 domain uncouples NGF signaling from the cytoskeletal interactions necessary for neurite elongation.

Adaptor Proteins, Signal Transducing↗

PubMed1995

v-Crk modulation of growth factor-induced PC12 cell differentiation involves the Src homology 2 domain of v-Crk and sustained activation of the Ras/mitogen-activated protein kinase pathway.

Nerve growth factor (NGF) and epidermal growth factor (EGF) elicit contrasting actions on PC12 pheochromocytoma cells; NGF causes neuronal differentiation, and EGF induces proliferation. However, ectopic expression of the Src homology 2 (SH2) and SH3-containing oncogenic adaptor protein v-Crk in PC12 cells results in EGF-inducible neuronal differentiation (Hempstead, B. L., Birge, R. B., Fajardo, J. E., Glassman, R., Mahadeo, D., Kraemer, R., and Hanafusa, H. (1994) Mol. Cell. Biol. 14, 1964-1971). Here we show that v-Crk complexes with both the tyrosine-phosphorylated EGF receptor and the Ras guanine nucleotide exchange factor SOS in PC12 cells and is involved in an pathway analogous to that of Grb2. Expression of v-Crk results in an enhanced and sustained activation of Ras and mitogen-activated protein (MAP) kinase following EGF or NGF stimulation, implying that v-Crk can couple divergent tyrosine kinase pathways to Ras. To investigate the causal relationship between EGF receptor binding, MAP kinase activation, and neurite outgrowth, we stably expressed two v-Crk SH2 point mutants, v-Crk(R273N) and v-Crk(H294R) in PC12 cells. Mutations within the SH2 domain of v-Crk block binding of v-Crk to the tyrosine phosphorylated EGF receptor, compromise v-Crk's ability to cause EGF-dependent neurite outgrowth, and act in a dominant negative manner for NGF-induced neurite outgrowth. However, the kinetics of MAP kinase activation in EGF- or NGF-treated v-Crk-(R273N)PC12 cells was comparable with that in v-CrkPC12 cells. These data are consistent with a model in which v-Crk regulates the strength of a tyrosine kinase signal leading to prolonged activation of Ras and MAP kinase. However, the experiments with the SH2 mutants suggest that sustained activation, by itself, may not be sufficient to switch the fate of v-CrkPC12 cells from proliferation toward differentiation.

Amino Acid Sequence↗

PubMed1995

Identification of the mouse liver 44-kDa acetaminophen-binding protein as a subunit of glutamine synthetase.

Identification of proteins that covalently bind acetaminophen (APAP) is essential for a clearer understanding of the hepatotoxicity that results after an APAP overdose. Birge et al. (1991) have reported that in our mouse model system a membrane-associated 44-kDa acetaminophen-binding protein is the earliest target detected immunochemically following administration of an hepatoxic dose of APAP. To identify this 44-kDa protein, liver microsomes from mice administered 600 mg APAP/kg were extracted with Triton X-114 and the resulting aqueous fraction was adsorbed to DEAE-cellulose. Further purification of the DEAE eluate by reverse-phase HPLC and by two-dimensional (2D) gel electrophoresis indicated that four proteins of approximately 44 kDa with pIs ranging from 6.7-7.0 were targeted by APAP. The most highly arylated of these 44-kDa isoforms (pI 7.0) was excised from 2D gels, digested with trypsin, and the resulting peptides were separated by reverse-phase HPLC. The two best resolved peptides were sequenced and 14 of the 15 amino acids detected were found to be identical to subunits of mouse liver glutamine synthetase (EC 6.3.1.2). Purification of glutamine synthetase from APAP-treated mice confirmed that the enzyme is indeed targeted by APAP in vivo. Since the intracellular activity of glutamine synthetase is significantly decreased after the administration of APAP to hepatocytes in culture, it is likely that the arylation of this enzyme may be involved in the ensuing hepatotoxicity.

Acetaminophen↗

PubMed1994

Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.

Animals↗