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J Biol Chem, Vol. 273, Issue 36, 23542-23548, September 4, 1998
andFrom the Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
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ABSTRACT |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Previous characterization of the nonreceptor tyrosine kinase FER identified a tight physical association with the catenin pp120 and led to the suggestion that FER may be involved in cell-cell signaling. To further understand the function of FER, we have continued our analyses of the interaction of FER with pp120 and other proteins. The majority of FER is localized to the cytoplasmic fraction where it forms a complex with the actin-binding protein cortactin. The Src homology 2 sequence of FER is required for directly binding cortactin, and phosphorylation of the FER-cortactin complex is up-regulated in cells treated with peptide growth factors. Using a dominant-negative mutant of FER, we provided evidence that FER kinase activity is required for the growth factor-dependent phosphorylation of cortactin. These data suggest that cortactin is likely to be a direct substrate of FER. Our observations provide additional support for a role of FER in mediating signaling from the cell surface, via growth factor receptors, to the cytoskeleton. The nature of the FER-cortactin interaction, and their putative enzyme-substrate relationship, support the previous proposal that one of the functions of the Src homology 2 sequences of nonreceptor tyrosine kinases is to provide a binding site for their preferred substrates.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The cytoplasmic tyrosine kinase FER belongs to a relatively small family of nonreceptor tyrosine kinases that consist of only one other member, FES (1, 2). The FES gene product in expressed only in cells of myeloid and endothelial lineages and has been implicated in cytokine signaling (3-9). In contrast, FER is expressed ubiquitously and its function remains poorly understood. FER is structurally distinct from members of the other cytoplasmic tyrosine kinase families; its amino acid sequence contains a catalytic domain adjacent to a single Src homology 2 (SH2)1 domain and an extensive coiled coil sequence in the amino terminus (2, 10). We previously showed that FER is tightly associated with the novel catenin pp120 and that the FER·pp120 complex is phosphorylated on tyrosines upon treatment of cells with growth factors (10). However, the significance of the interaction remains to be elucidated, because the function of pp120 is also not well understood.
Tyrosine phosphorylation has been demonstrated to be an important
mechanism for regulating cell-cell and cell-substrate adhesions. For
example, inhibition of tyrosine phosphatases in Madin-Darby canine
kidney cells results in the deterioration of adherens junctions and
dramatic changes in cell morphology (11). Treatment of epithelial cells
with epidermal growth factor leads to the tyrosine phosphorylation of
-catenin, plakoglobin and pp120, as well as changes in cell morphology (12, 13). More importantly, the major targets of v-src kinase activity have been shown to be proteins that
are directly implicated in cell adhesion or cytoskeletal function. These include focal adhesion kinase, cortactin, p130cas, and
pp120 (14). Results of subcellular localization and functional characterization have implicated these proteins in regulating cell
motility or membrane cytoskeleton dynamics. Focal adhesion kinase and
p130cas are localized to focal adhesion complexes and focal
adhesion kinase has been demonstrated to be required for the turnover
of the cellular structures (15-19). Cortactin has been shown to bind F-actin in vitro and is localized to the cortical
cytoskeleton (20, 21). pp120 is a component of the cadherin-catenin
complexes and, as such, may be involved in membrane-cytoskeleton
interactions (22, 23).
The association of FER and pp120, and their phosphorylation in cells treated with growth factors, led us to speculate that FER may be involved in a signaling pathway linking growth factor receptors with cell adhesion or the cytoskeleton. In this report, we describe studies aimed at further characterization of the interaction of FER with other cellular components. We also report on the results of experiments, using a dominant-negative FER mutant, that seek to analyze the function of the FER kinase.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cells and Antibodies-- NIH3T3 and human embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 10% fetal bovine serum, respectively. Glutathione S-Sepharose was from Amersham Pharmacia Biotech. Monoclonal anti-phosphotyrosine and anti-pp120 antibodies were from Transduction Laboratories. Monoclonal anti-cortactin antibody (4F11) was from Upstate Biotechnology, Inc. Anti-hemagglutinin (12CA5) antibodies were from Boehringer Mannheim. Texas Red-conjugated goat anti-mouse IgG and fluorescein-conjugated goat anti-rabbit IgG were from Jackson ImmunoResearch Laboratory. Polyclonal anti-dynamin antibodies were provided by Dr. Mark McNiven of the Mayo Clinic. Polyclonal FER1 and FER5 antisera were described previously (10). They were generated by immunizing rabbits with fusion proteins that contain codons 502-675 and codons 451-564 of FER, respectively. Antiserum FER2 was prepared also using a TrpE fusion protein containing codons 502-675.
Expression Plasmid Construction-- Full-length FER cDNA was isolated from a human foreskin fibroblast cDNA library (CLONTECH) using as probe a polymerase chain reaction fragment that encompasses codons 149-332. A single lysine to arginine mutation was introduced at codon 591, using the SculptorTM in vitro mutagenesis kit (Amersham Pharmacia Biotech). Both the wild-type and K591R mutant cDNA sequences were subcloned in a CMV-based eukaryotic expression vector to yield pCMV-HA-FER(WT) and pCMV-HA-FER(K591R). The subcloning was designed to introduce an influenza virus hemagglutinin (HA) epitope at the amino terminus of the FER sequences. The human CSF-1 receptor cDNA was kindly provided by Dr. C. Sherr of the Howard Hughes Medical Institute at St. Jude's Medical Center and was subcloned into the CMV expression vector to yield pCMV-CSF1R. Details of the subcloning procedures will be provided upon request.
Subcellular Fractionation-- Confluent cultures of NIH3T3 cells were serum-starved and stimulated with platelet-derived growth factor (PDGF) as described before (10). Cells were rinsed twice with cold PBS and scraped into ice-cold hypotonic buffer (20 mM Tris·HCl, pH 7.7, 15 mM NaCl, 2 mM MgCl2, 1 mM sodium orthovanadate, and 40 µM ammonium molybdate) (1 ml/10-cm plate). The cell suspension was allowed to swell on ice for 15 min, and cell lysis was achieved by homogenization in a tight-fitting glass homogenizer. The cell lysates were centrifuged for 10 min at 2000 rpm in a tabletop centrifuge to remove nuclei and unbroken cells. The postnuclear supernatants were centrifuged for 90 min at 100,000 × g to yield an S100 supernatant. The pelleted material was resuspended by homogenization in 3 ml of buffer that contained 5% glycerol, 20 mM Tris·HCl, pH 7.7, 1 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, and 40 mM ammonium molybdate. To that suspension was added 10% Triton X-100 to a final concentration of 1%. The mixture was clarified by centrifugation (10,000 × g, 10 min) to yield a solubilized membrane (P100) fraction. Protein concentrations were determined using the microBCA reagents (Pierce). Whole-cell lysates were prepared as described previously (10).
Immunoprecipitation and Protein Binding Assays-- Immunoprecipitations were performed as described before, using typically 300 µg of S100 and 30 µg of P100 fractions and 5 µg of purified antibodies or 5 µl of antisera (10). Immunoblotting was performed using standard techniques, and binding was detected with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech). For immune complex kinase assays, whole-cell lysates were immunoprecipitated with FER5 antiserum, and kinase assays were performed as described before (10).
Preparation of GST fusion proteins and in vitro binding assays were performed as described before (10). In binding assays that involved GST-CCD, the detection of cortactin required an intermediate immunoprecipitation, because the GST-CCD polypeptide comigrates with cortactin on SDS gels and prohibits the unambiguous detection of cortactin. In those experiments, proteins bound to immobilized GST fusion proteins were eluted by boiling in 1% SDS. The samples were diluted with 1% Triton X-100 to bring the final SDS concentration to 0.2%. The eluted materials were then immunoprecipitated with anti-cortactin antibody before analyses by Western blotting. GST pull-down experiments in denaturing conditions were performed by first immunoprecipitating S100 fractions with 5 µg of anti-phosphotyrosine antibody. The immunoprecipitates were denatured by boiling in SDS and diluted with 1% Triton X-100 as above. The immunoprecipitated proteins were then incubated with GST fusion proteins immobilized on Sepharose beads, and bound proteins were analyzed by Western blotting.Immunofluorescence Staining-- NIH3T3 cells were seeded on glass coverslips and were serum-starved before treatment with PDGF for 5 min. Cells were fixed with 4% paraformaldehyde in PBS (room temperature for 5 min) and were permeabilized with 0.5% Triton X-100 in PBS (10 min). Following blocking with 2% bovine serum albumin in PBS, cells were stained with FER5 antiserum (1:400) and anti-cortactin mAb 4F11 (1:2000). This was followed by staining with labeled second antibodies and mounting of the coverslips. Cells were examined using an Olympus BH2 microscope and photographed.
Transfection-- 293 cells (106 cells) were seeded in 10-cm dishes and were transfected 24 h later using the modified calcium phosphate procedure (24). Each culture received 2 µg of pCMV-CSF1R in the presence of 14 µg of either pCMV vector, pCMV-HA-FER, or pCMV-HA-FER(K591R). The amounts and ratio of cDNA used were optimized in prior experiments to obtain maximal expression of both CSF-1R and the FER cDNAs. At 14-18 h after transfection, cells were rinsed and fed with fresh culture medium. After another 4-12 h, the culture medium was replaced with starvation medium (Dulbecco's modified Eagle's medium plus 0.5% fetal bovine serum and 10 mM Hepes). The cultures were starved overnight and stimulated with M-CSF (100 ng/ml, R & D Systems) for 5 min. Cells were harvested and lysed as described above.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Identification of a Novel Interaction between FER and Cortactin-- We previously used an immunological approach to identify the association of FER with pp120 (10). To further identify other components that may interact with FER, we prepared additional FER-specific antisera and analyzed their ability to immunoprecipitate FER-containing complexes from lysates of NIH3T3 cells. As shown previously, antiserum FER1 immunoprecipitated a complex of FER and pp120 (Fig. 1, lanes 3 and 4). PDGF treatment resulted in an increase in the phosphorylation of both FER and pp120. Two other FER-specific antisera, FER2 and FER5, also immunoprecipitated FER, as well as a small amount of the 180-kDa activated PDGF receptor (Fig. 1, lanes 5-8). However, little or no pp120 was co-immunoprecipitated by either of these two antisera. FER5 immunoprecipitated additional polypeptides of 125 kDa and >205 kDa. The identity of these polypeptides has yet to be determined. These anti-FER antisera may therefore recognize distinct subpopulations or conformations of FER.
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Cortactin Binds the SH2 Sequence of FER-- The molecular basis of the FER-cortactin association was further examined in in vitro binding assays. A GST fusion protein containing the SH2 sequence of FER specifically precipitated cortactin from a cytosolic fraction (Fig. 4, A and B). The CCD of FER, which was previously shown to mediate interaction with pp120, did not interact with cortactin. Cortactin is one of the major cytosolic proteins that are subject to PDGF-regulated tyrosine phosphorylation and which bind the FER SH2 sequence in vitro (Fig. 4, C and D). We also tested the ability of the FER SH2 sequence to directly interact with cortactin. Cytosolic proteins were immunoprecipitated with anti-phosphotyrosine antibodies and denatured. Cortactin was the predominant component that, following denaturation and renaturation, retained the ability to bind the FER SH2 sequence (Fig. 4, E and F). This suggests that the FER SH2 sequence has the capacity to directly interact with cortactin.
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The Novel 102-kDa Polypeptide Is Closely Related to FER-- In addition to cortactin, a 102-kDa polypeptide was found to co-immunoprecipitate with FER from the cytosolic fraction. Although dynamin can be detected in the FER immunoprecipitates, its abundance in the immune complexes suggests that it is distinct from the 102-kDa component detected in the anti-phosphotyrosine Western blots. The identity of the 102-kDa polypeptide was further investigated by dissecting the interactions among the components of the immune complex. The immune complex was isolated using FER2 antiserum and was denatured. The immunoprecipitated polypeptides were then subjected to a second round of immunoprecipitation using antiserum FER5, which we found to be able to recognize denatured FER (Fig. 5C). Western blotting with anti-phosphotyrosine antibodies revealed that the 97-102-kDa doublet was retrieved by immunoprecipitation with FER5 antibodies (Fig. 5A, lanes 1-4). Cortactin, which was co-immunoprecipitated by FER2, was not found in the FER5 immunoprecipitates, but was recovered in a subsequent immunoprecipitation with anti-phosphotyrosine antibodies (lanes 5 and 6). Dynamin, which was present in the original FER2 immunoprecipitates, was not recovered in any of the subsequent re-immunoprecipitations. Furthermore, the dynamin that was co-immunoprecipitated by the FER2 antibodies was not phosphorylated. These results rule out the possibility that dynamin is the 102-kDa phosphoprotein co-immunoprecipitating with FER. They also suggest that the 102-kDa polypeptide is closely related to FER or that its association with FER is mediated by a novel interaction, which is resistant to denaturation.
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Cortactin Is a Direct Substrate of FER Kinase-- To help analyze the functional implication of the FER-cortactin interaction, we prepared an enzymatically inactive FER by introducing a point mutation in the conserved lysine (Lys591) of the predicted ATP binding site of the kinase. Transient expression of the K591R FER mutant cDNA showed that it encodes a polypeptide that is slightly larger than the endogenous FER kinase, as a result of the addition of an HA epitope tag to the amino terminus (Fig. 6, A and B). In an immune complex kinase assay, the lysate of cells transfected with the K591R mutant showed no autophosphorylation of the mutant FER protein and reduced autophosphorylation of the endogenous kinase (C, lane 6). In contrast, transfection with an analogous wild-type FER cDNA resulted in a significant autophosphorylation of the tagged kinase (C, lane 5). These results suggest that the K591R FER protein may function as a dominant interfering mutant.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We previously demonstrated the stable association of FER with the catenin pp120 and estimated that no more than 10-15% of the total FER protein was involved in that complex (10). As shown in the subcellular fractionation studies here, that previous estimate is consistent with the abundance of FER that is associated with the membrane fraction. The use of subcellular fractionation and the FER2 antiserum permitted the identification of a novel interaction between FER and the cytoskeletal protein cortactin. Immunoprecipitation of FER resulted in the coprecipitation of either cortactin or pp120, but not both. Thus, FER appears to interact with pp120 and cortactin in a mutually exclusive manner, although binding to the two polypeptides requires distinct FER sequences. These observations suggest that the FER·pp120 and FER-cortactin complexes may be involved in different signaling function. The FER-cortactin complex is detectable in a cytosolic fraction, but not in a lysate prepared using detergent-containing buffers. The association was verified by functional and structural analyses using independent assays and is therefore unlikely to be an artifact of the lysis procedure. The FER-cortactin association is unambiguously demonstrated by immunofluorescence colocalization in the membrane ruffles of PDGF-treated cells. Most significantly, the effect of the FER K591R mutant on cortactin phosphorylation strongly supports a direct physical interaction between the two proteins. We therefore believe that the interaction involves cytoskeletal structures that are detergent insoluble. Our analyses suggest that between 10 and 0% of the cellular cortactin is associated with FER.
In addition to cortactin, we identified also a novel 102-kDa polypeptide that co-immunoprecipitated with FER. We recently showed that cortactin interacts with dynamin 2, which has an apparent molecular mass of ~100 kDa.2 Our analyses showed that dynamin could be detected in immunoprecipitates prepared with anti-FER antibodies, presumably by virtue of the interaction of FER with cortactin. However, the results also clearly showed that the 102-kDa polypeptide is distinct from dynamin. The ability of the FER5 antiserum to recover both the 97-kDa FER and the 102-kDa polypeptide under denaturing conditions suggests that the latter is a highly modified form of FER or a closely related polypeptide. As is evident in the transient expression assays, there is a pronounced size heterogeneity in the cytosolic pool of wild-type FER protein. The fact that the size heterogeneity is absent in the case of the FER mutant suggests that it is dependent on FER kinase activity. It therefore seems plausible that the 102-kDa polypeptide may be a highly phosphorylated form of FER that is present in such low abundance as to be undetectable by Western blotting with anti-FER antibodies. The functional significance of this polypeptide, and its selective localization in the cytosol, remain to be investigated further.
In vitro binding assays demonstrated that the SH2 sequence of FER is capable of directly binding cortactin. This contrasts with our previous finding that the coiled coil sequence at the amino terminus of FER is required for binding pp120 (10). Expression of the FER K591R mutant was found to result in an inhibition of the autophosphorylation of the endogenous FER kinase, thus suggesting that the mutant was capable of functioning in a dominant interfering manner. The FER mutation has no apparent effect on its ability to interact with cortactin and pp120, and expression of the FER mutant has no effect on the expression of the two cytoskeletal proteins (data not shown). The mutation did result in an inhibition of the tyrosine phosphorylation of cortactin, but not of pp120, in response to growth factor receptor signaling. These data argue that cortactin, but not pp120, is a direct substrate of FER kinase. However, we cannot exclude the possibility that a residual amount of endogenous FER activity is sufficient to effect the phosphorylation of pp120. It should be noted that we have never been able to demonstrate a direct binding between FER and pp120.
Recently, peptide libraries were used to determine the substrate specificity of protein tyrosine kinases (27). Such analyses led to the conclusion that nonreceptor tyrosine kinases tend to phosphorylate peptide sequences that are recognized by their SH2 domains. Our observations on the FER-cortactin interaction and their putative enzyme-substrate relationship would be consistent with the specificity proposed on the basis of the peptide substrate analyses. Most significantly, our data suggest that the regulation of cortactin phosphorylation by growth factor receptors may be mediated in part by FER.
Although cortactin was originally identified as a target of tyrosine phosphorylation by v-Src, a role for c-Src in cortactin phosphorylation has not been established. The subcellular localization of c-Src to endosomal membranes suggests that c-Src and cortactin are localized to different cellular compartments (28). In addition to v-Src and FER, the cytoplasmic kinase FYN has also been implicated in the regulation of cortactin phosphorylation (29). It is therefore plausible that cortactin may be a substrate of multiple tyrosine kinases that serve to relay different extracellular signals. The significant basal phosphorylation of cortactin may be carried out by an unidentified tyrosine kinase. The partial phosphorylation allows cortactin to bind FER, and the FER-cortactin complex becomes a subject of regulation by growth factor signaling. The localization of cortactin to the cortical cytoskeleton, and its affinity for filamentous actin, led to the suggestion that cortactin may be involved in transmitting signals from the membrane to the actin cytoskeleton. That proposal is further substantiated by the observation that cortactin phosphorylation is induced upon integrin signaling in cell adhesion and in the cytoskeletal reorganization that accompanies bacterial entry of epithelial cells (30, 31). Recently, cortactin was shown to cross-link F-actin in vitro, and the cross-linking activity was down-regulated by the tyrosine phosphorylation of cortactin (32). These observations have consolidated a role for and FER and cortactin in the transmission of signals for reorganization of the actin cytoskeleton.
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ACKNOWLEDGEMENT |
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We thank Dr. M. McNiven for the anti-dynamin antibodies.
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FOOTNOTES |
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* This work was supported by Grant BE-202 from the American Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: National Institutes of Health, Bldg. 6, Rm.
B1-22, Bethesda, MD 20892.
§ To whom correspondence should be addressed. Present address: Dept. of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, P. O. Box 4000, Princeton, NJ 08543. Tel.: 609-252-4187; Fax: 609-252-6051.
The abbreviations used are: SH2, Src homology 2; PDGF, platelet-derived growth factor; M-CSF, macrophage-colony stimulating factor; S100, 100,000 × g supernatantP100, 100,000 × g pelletHA, hemagglutininGST, glutathione S-transferaseCCD, coiled coiled domainCMV, cytomegalovirusPBS, phosphate-buffered saline.
2 M. McNiven, L. Kim, and T. W. Wong, manuscript in preparation.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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-32P]ATP, and the products
of FER autophosphorylation are shown after SDS-polyacrylamide gel
electrophoresis, alkaline treatment, and autoradiography. Results shown
are representative of three independent experiments.
