共查询到20条相似文献,搜索用时 31 毫秒
1.
Protein tyrosine phosphorylation in response to fertilization 总被引:4,自引:0,他引:4
The sea urchin egg contains one or more protein tyrosine kinases which are active during the response of the egg to fertilization. In the present study, we have used an antibody specific for phosphotyrosine to determine which egg proteins are phosphorylated on tyrosine in response to fertilization. Analysis of immunoblots prepared from fertilized and unfertilized eggs revealed that fertilization results in a major increase in the phosphotyrosine content of a 350-kDa egg protein. Increased phosphorylation of this protein was detected as early as 1 min after fertilization, at which time it represented the most prominent phosphotyrosine containing protein in the egg. Tyrosine phosphorylation of this protein was transient however, and after 5 min post-insemination, the protein was dephosphorylated or otherwise degraded. Egg membrane proteins of approximately 40, 75, and 145 kDa were also found to act as substrates for protein tyrosine kinases in vitro, but did not exhibit significant changes in phosphotyrosine content during egg activation. 相似文献
2.
Anne-Marie Hansen Raghothama Chaerkady Jyoti Sharma J. Javier Díaz-Mejía Nidhi Tyagi Santosh Renuse Harrys K. C. Jacob Sneha M. Pinto Nandini A. Sahasrabuddhe Min-Sik Kim Bernard Delanghe Narayanaswamy Srinivasan Andrew Emili James B. Kaper Akhilesh Pandey 《PLoS pathogens》2013,9(6)
While phosphotyrosine modification is an established regulatory mechanism in eukaryotes, it is less well characterized in bacteria due to low prevalence. To gain insight into the extent and biological importance of tyrosine phosphorylation in Escherichia coli, we used immunoaffinity-based phosphotyrosine peptide enrichment combined with high resolution mass spectrometry analysis to comprehensively identify tyrosine phosphorylated proteins and accurately map phosphotyrosine sites. We identified a total of 512 unique phosphotyrosine sites on 342 proteins in E. coli K12 and the human pathogen enterohemorrhagic E. coli (EHEC) O157:H7, representing the largest phosphotyrosine proteome reported to date in bacteria. This large number of tyrosine phosphorylation sites allowed us to define five phosphotyrosine site motifs. Tyrosine phosphorylated proteins belong to various functional classes such as metabolism, gene expression and virulence. We demonstrate for the first time that proteins of a type III secretion system (T3SS), required for the attaching and effacing (A/E) lesion phenotype characteristic for intestinal colonization by certain EHEC strains, are tyrosine phosphorylated by bacterial kinases. Yet, A/E lesion and metabolic phenotypes were unaffected by the mutation of the two currently known tyrosine kinases, Etk and Wzc. Substantial residual tyrosine phosphorylation present in an etk wzc double mutant strongly indicated the presence of hitherto unknown tyrosine kinases in E. coli. We assess the functional importance of tyrosine phosphorylation and demonstrate that the phosphorylated tyrosine residue of the regulator SspA positively affects expression and secretion of T3SS proteins and formation of A/E lesions. Altogether, our study reveals that tyrosine phosphorylation in bacteria is more prevalent than previously recognized, and suggests the involvement of phosphotyrosine-mediated signaling in a broad range of cellular functions and virulence. 相似文献
3.
Jeffrey S. Cleaveland Peter A. Kiener David J. Hammond Bernice Z. Schacter 《Analytical biochemistry》1990,190(2)
A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation. 相似文献
4.
Jeanine F. Amacher Helen T. Hobbs Aaron C. Cantor Lochan Shah Marco‐Jose Rivero Sarah A. Mulchand John Kuriyan 《Protein science : a publication of the Protein Society》2018,27(5):923-932
Cbl proteins are E3 ubiquitin ligases specialized for the regulation of tyrosine kinases by ubiquitylation. Human Cbl proteins are activated by tyrosine phosphorylation, thus setting up a feedback loop whereby the activation of tyrosine kinases triggers their own degradation. Cbl proteins are targeted to their substrates by a phosphotyrosine‐binding SH2 domain. Choanoflagellates, unicellular eukaryotes that are closely related to metazoans, also contain Cbl. The tyrosine kinase complement of choanoflagellates is distinct from that of metazoans, and it is unclear if choanoflagellate Cbl is regulated similarly to metazoan Cbl. Here, we performed structure‐function studies on Cbl from the choanoflagellate species Salpingoeca rosetta and found that it undergoes phosphorylation‐dependent activation. We show that S. rosetta Cbl can be phosphorylated by S. rosetta Src kinase, and that it can ubiquitylate S. rosetta Src. We also compared the substrate selectivity of human and S. rosetta Cbl by measuring ubiquitylation of Src constructs in which Cbl‐recruitment sites are placed in different contexts with respect to the kinase domain. Our results indicate that for both human and S. rosetta Cbl, ubiquitylation depends on proximity and accessibility, rather than being targeted toward specific lysine residues. Our results point to an ancient interplay between phosphotyrosine and ubiquitin signaling in the metazoan lineage. 相似文献
5.
Kathrin H Kirsch Margaret Kensinger Hidesaburo Hanafusa Avery August 《BMC cell biology》2002,3(1):18-15
Background
The adaptor protein p130 Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals. 相似文献6.
We have sought to identify candidate substrates for src family protein-tyrosine kinases potentially important for transformation. Transfected NIH/3T3 cells, each overexpressing a normal or activated version of the fyn, fgr, or src translational product, were examined using antibody to phosphotyrosine as a probe. Expression of each cDNA induced similar but distinct patterns of tyrosine phosphorylated cellular proteins, with the extent of phosphorylation being greatest in cells expressing an activated kinase. A 70-kDa tyrosine-phosphorylated protein was found to associate with the activated fyn gene product. A protein designated p130, tyrosine phosphorylated in vitro, and in vivo, was found to physically associate with the activated product of each src family gene examined. Physical interaction of three different highly transforming tyrosine kinases with a common cellular protein suggests that p130 may play an important role in transformation induced by src family kinases. 相似文献
7.
C.Hugh Reynolds Malcolm G. Willson John Groffen Nora Heisterkamp Timothy C. Peakman Martin J. Page 《生物化学与生物物理学报:疾病的分子基础》1993,1181(2):122-130
Mouse c-Abl type IV and human BCR/ABL proteins have been expressed in insect cells using the baculovirus system. The proteins were expressed as full-length polypeptides as judged by electrophoresis in denaturing gels. They were identified by immunoprecipitation and immunoblotting with antibodies against ABL peptides and, for BCR/ABL, against a BCR peptide. In these immunoprecipitates both proteins gave autophosphorylation principally on tyrosine. Both proteins were active tyrosine kinases, phosphorylating a variety of tyrosine-containing substrates. In fresh extracts both proteins contained phosphotyrosine as shown by Western blots with antiphosphotyrosine antibodies. Partial purification could be achieved readily using ion exchange columns, and the BCR/ABL protein, p210BCR/ABL, could be further purified to near-homogeneity using an antiphosphotyrosine column. Both enzymes required a divalent metal ion for activity. At low concentrations of ATP (2 μM) and with angiotensin II as substrate both enzymes were activated by Mn2+ or by Mg2+. No major differences in catalytic properties were found between the two isolated enzymes in solution. The oncogenic properties of p210BCR/ABL may be due to its different subcellular location, or to the presence of an intracellular inhibitor of c-Abl that does not inhibit BCR/ABL, or to altered substrate-specificity such that it can phosphorylate a unique substrate which is not recognised by c-Abl. 相似文献
8.
《Plant Physiology and Biochemistry》2000,38(11):825-836
The presence of tyrosine kinase and tyrosine-phosphorylated proteins was investigated in coconut tissues cultured in vitro. In order to study this phenomenon, plumular explants were taken from mature zygotic embryos and cultured in a medium that induces somatic embryogenesis. Immunoblot analyses of soluble proteins of coconut cultured tissues with a recombinant monoclonal antibody against phosphotyrosine detected protein bands with molecular masses ranging from 170 to 27 kDa. The highest response was exhibited by plumule-forming callus, which decreased both in number and intensity of bands with a longer time of in vitro culture. The specific immunodetection was corroborated by incubating the membranes with anti-phosphotyrosine antibody in the presence of 1 mM phosphotyrosine. Tyrosine phosphorylated proteins was also suggested by the presence of phosphoproteins resistant to alkaline treatment. In plumule, plumular callus and callus with globular embryos and shoots, a 41-kDa protein remained phosphorylated after alkaline treatment. In plumule, most [32P]-proteins remained phosphorylated after alkaline treatment. Phosphoaminoacid analysis in protein hydrolysates from [32P]-labelled 41-kDa protein showed the presence of [32P]-tyrosine and [32P]-threonine. Evaluation of tyrosine kinase activity in these tissues by the use of RR-SRC, a synthetic peptide substrate (derived from the amino acid sequence surrounding the phosphorylation site), showed that the activity was highest in plumule forming callus and initial explant, whereas in other tissues, tyrosine kinase activity decreased to values close to zero. Genistein, a specific tyrosine kinase inhibitor, diminished the ability of soluble extracts from coconut tissues cultured in vitro to incorporate 32P into RR-SRC. These results suggest the presence of tyrosine phosphorylated proteins and tyrosine kinase activity in coconut tissues that have been cultured in vitro. 相似文献
9.
Dalit Ben-Yosef Anat Talmor Lior Shwartz Yosef Granot Ruth Shalgi 《Molecular reproduction and development》1998,49(2):176-185
Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P-Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P-Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P-Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P-Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl-phosphorylated, mitogen-activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P-Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin-induced Ca2+ rise is sufficient to imitate fertilization-induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg. Mol. Reprod. Dev. 49:176–185, 1998. © 1998 Wiley-Liss, Inc. 相似文献
10.
Gajanan Sathe Chan Hyun Na Santosh Renuse Anil Madugundu Marilyn Albert Abhay Moghekar Akhilesh Pandey 《Clinical proteomics》2018,15(1):29
Background
Cerebrospinal fluid (CSF) is an important source of potential biomarkers that affect the brain. Biomarkers for neurodegenerative disorders are needed to assist in diagnosis, monitoring disease progression and evaluating efficacy of therapies. Recent studies have demonstrated the involvement of tyrosine kinases in neuronal cell death. Thus, neurodegeneration in the brain is related to altered tyrosine phosphorylation of proteins in the brain and identification of abnormally phosphorylated tyrosine peptides in CSF has the potential to ascertain candidate biomarkers for neurodegenerative disorders.Methods
In this study, we used an antibody-based tyrosine phosphopeptide enrichment method coupled with high resolution Orbitrap Fusion Tribrid Lumos Fourier transform mass spectrometer to catalog tyrosine phosphorylated peptides from cerebrospinal fluid. The subset of identified tyrosine phosphorylated peptides was also validated using parallel reaction monitoring (PRM)-based targeted approach.Results
To date, there are no published studies on global profiling of phosphotyrosine modifications of CSF proteins. We carried out phosphotyrosine profiling of CSF using an anti-phosphotyrosine antibody-based enrichment and analysis using high resolution Orbitrap Fusion Lumos mass spectrometer. We identified 111 phosphotyrosine peptides mapping to 66 proteins, which included 24 proteins which have not been identified in CSF previously. We then validated a set of 5 tyrosine phosphorylated peptides in an independent set of CSF samples from cognitively normal subjects, using a PRM-based targeted approach.Conclusions
The findings from this deep phosphotyrosine profiling of CSF samples have the potential to identify novel disease-related phosphotyrosine-containing peptides in CSF.11.
Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells. 总被引:14,自引:7,他引:7
L Naldini A Stacchini D M Cirillo M Aglietta F Gavosto P M Comoglio 《Molecular and cellular biology》1986,6(5):1803-1811
Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene. 相似文献
12.
《Cell communication & adhesion》2013,20(2):91-100
As cells adhere to extracellular matrix proteins, several focal adhesion proteins become tyrosine phosphorylated. One of the most prominent of these has been identified as the tyrosine kinase p125FAK (focal adhesion kinase, FAK). An interaction between FAK and members of the Src family tyrosine kinases p59fyn, pp60v-src, and activated pp60c-src (527F) has been demonstrated, raising the possibility that these kinases may regulate FAK activity. To explore the role of Src family kinases in focal adhesions and in the regulation of FAK activity, we isolated fibroblasts from transgenic mice that lack either pp60c-src p59fyn, or pp62c-yes. These primary fibroblasts, and those of a control mouse, were passaged numerous times and resulted in spontaneously immortalized cell lines without the addition of transforming agents. After confirming the absence of the appropriate nonreceptor tyrosine kinases in the fyc¯, srn¯ and yes¯ fibroblasts, the ability of these fibroblasts to form focal adhesions and stress fibers was assessed by immunofluorescence microscopy and found to be comparable to that of normal fibroblasts. We investigated phosphotyrosine levels in response to adhesion to fibronectin and identified the pp60src substrate p130 as the one major protein with reduced levels of tyrosine phosphorylation in the cells lacking p59fyn and pp62c-yes, and particularly in those lacking pp60c-scr. We examined FAK phosphorylation and kinase activity and found that there were no significant differences between these cells. 相似文献
13.
A I Kharitonenkov N G Kudriavtseva T V Bulargina 《Biokhimii?a (Moscow, Russia)》1989,54(10):1732-1739
Protein kinases phosphorylating proteins at tyrosine residues play an essential role in the cell growth regulation and neoplastic transformation. However, the functions of the majority of tyrosine protein kinases are still obscure, thus creating hindrances in the identification and isolation of phosphotyrosine-containing proteins. The use of the phosphotyrosine structural analog, aminobenzyl phosphonate, as a hapten group enabled the preparation of monoclonal antibodies capable of reacting to phosphotyrosine. The phosphotyrosine specificity of six clones of monoclonal antibodies was tested by a competitive solid phase immunoenzymatic assay. Using fluorescence quenching, the values of constants of binding for antibodies of four clones to phosphotyrosine (2.5-4.0 x 10(6) M-1) were determined. Using two independent methods, it was shown that clone B4 antibodies reveal the highest specificity towards phosphotyrosine. An immunoadsorbent based on clone B4 antibodies was obtained; this immunoadsorbent possessed an ability to selectively interact with an EFR receptor phosphorylated at tyrosine residue. Using eluate acid hydrolysis from the immunoadsorbent, it was demonstrated that clone B4 antibodies interact only with the phosphotyrosine-containing proteins. The experimental results are suggestive of clone B4 monoclonal antibody specificity to phosphotyrosine and of the feasibility of their application for the isolation and identification of tyrosine protein kinases and their substrates. 相似文献
14.
Stimulation of factor-dependent myeloid cell lines with interleukin 3 induces tyrosine phosphorylation of several cellular substrates 总被引:18,自引:0,他引:18
R Isfort R D Huhn A R Frackelton J N Ihle 《The Journal of biological chemistry》1988,263(35):19203-19209
Interleukin 3 (IL-3) is required for the proliferation of growth factor-dependent myeloid cell lines. To determine the possible signal transduction mechanisms involved in IL-3 growth regulation, we have examined the effects of IL-3 on tyrosine phosphorylation. Using a monoclonal antibody against phosphotyrosine, IL-3 was found to specifically and rapidly induce tyrosine phosphorylation of cytoplasmic proteins of 70, 56, and 38 kDa and a membrane-associated glycoprotein of 140 kDa. Minor and/or variable detected phosphoproteins of 120, 85, 51, and 28 kDa were also seen. Oncogenes encoding tyrosine protein kinases abrogate the requirement of factor-dependent myeloid cells for IL-3. We therefore compared the phosphoprotein profiles of a transformed, IL-3-independent cell line with the IL-3-induced profile. In cells transformed with trk, the 56-, 51-, and 38-kDa cytoplasmic phosphoproteins were constitutively phosphorylated, whereas the 140-kDa phosphoprotein was only phosphorylated in the presence of IL-3. Taken together, these results support a role for tyrosine phosphorylation in the IL-3 signal transduction pathway and suggest that growth factor abrogation by oncogenes encoding tyrosine protein kinases may be due to the phosphorylation of substrates which are normally phosphorylated in response to IL-3. 相似文献
15.
Cheng Heung-Chin Matsuura Isao Wang Jerry H. 《Molecular and cellular biochemistry》1993,127(1):103-112
Synthetic peptides such as P60stc autophosphorylation site peptides and angiotensin are indiscriminately phosphorylated by protein tyrosine kinases. The observation has led to the general belief that protein tyrosine kinases are highly promiscuous, displaying littlein vitro site specificity. In recent years, evidence has been accumulating to indicate that such a belief requires close examination. Synthetic peptides showing high substrate activity for specific groups of protein tyrosine kinases have been obtained. Systematic modification of certain substrate peptides suggests that kinase substrate determinants reside with specific amino acid residues proximal to the target tyrosine. A number of protein kinases have been shown to be regulated by tyrosine phosphorylation at specific sites by highly specific protein tyrosine kinases. These and other selected biochemical studies that contribute to the evolving view ofin vitro substrate specificity of protein tyrosine kinases are reviewed. 相似文献
16.
Nicholas A. Flint Kurt E. Amrein Thomas Jascur Paul Burn 《Journal of cellular biochemistry》1994,55(3):389-397
The lymphocyte-specific, nonreceptor protein tyrosine kinase Lck has been purified from an Escherichia coli expression system using a monoclonal antibody column followed by dye-affinity chromatography. Polyacrylamide gel electrophoretic analysis of purified protein revealed a single 56 kDa band, indicating that recombinant Lck was purified to near-homogeneity. The purified enzyme displayed tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties including protein phosphorylation and kinetic characteristics of the enzyme have been assessed. Peptide map analysis revealed that bacterially expressed Lck is phosphorylated predominantly on the autophosphorylation site (tyrosine-394), which is characteristic for activated protein tyrosine kinases. Indeed, we found that the recombinant enzyme is approximately fivefold more active than Lck from resting T cells, which is extensively phosphorylated at the regulatory carboxy-terminal tyrosine residue (tyrosine-505). Thus, we have overproduced recombinant human Lck in E. coli and developed a simple two-step purification procedure which yields highly active enzyme. This will enable the identification and characterization of potential regulators and targets of Lck and thereby greatly facilitate studies which will clarify its role in T cell signal transduction. © 1994 Wiley-Liss, Inc. 相似文献
17.
Phase- and Age-Related Differences in Protein Tyrosine Phosphorylation in Sequoia sempervirens 总被引:1,自引:0,他引:1
L.-C. Huang S.-Y. Pu T. Murashige S.-F. Fu T.-T. Kuo D.-D. Huang H.-J. Huang 《Biologia Plantarum》2003,47(4):601-603
Apical and basal halves of 3 cm long apical segments of in vitro cultured juvenile, adult and rejuvenated Sequoia sempervirens shoots were analyzed for total and tyrosine phosphorylated proteins. The latter was detected by a phosphotyrosine specific antibody. Younger tissues, or the apical halves of shoot terminals, showed larger amounts of 36, 44, 46 kDa proteins and lesser amounts of 29 kDa proteins. These are proposed as age-related changes. Phase-related proteins were also evident. Adult tissues contained more of the 34 and 36 kDa proteins than juvenile and rejuvenated shoots. Western blotting with a phosphotyrosine specific antibody revealed more of 25, 39, and 54 kDa protein in the younger tissues. In addition, tyrosine phosphorylated proteins of 25 and 34 kDa were higher in the adult, than in juvenile or rejuvenated tissues. Our findings showed that protein tyrosine phosphorylation, or the signal transduction pathway, is involved in phase- and age-related processes. 相似文献
18.
Inhibition of phosphotyrosine phosphatases reveals candidate substrates of the PDGF receptor kinase 总被引:3,自引:0,他引:3
R Zippel L Morello R Brambilla P M Comoglio L Alberghina E Sturani 《European journal of cell biology》1989,50(2):428-434
In normal fibroblasts stimulated by platelet derived growth factor (PDGF), PDGF receptors are transiently phosphorylated on tyrosine and represent the major phosphotyrosine containing protein. The phosphate of the phosphotyrosine groups turns over rapidly, and extensive evidence indicates a dynamic balance between phosphorylation and dephosphorylation reactions. Thus, the effect of an inhibitor of phosphatases, orthovanadate, on the pattern of the tyrosine phosphorylations induced by PDGF in Swiss 3T3 fibroblasts was investigated. Western blot analysis with antibodies against phosphotyrosine indicated that whereas in unstimulated cells no phosphotyrosine containing proteins were detected, treatment of cells with orthovanadate alone elicited the slow phosphorylation of several proteins including a 170 kDa component that was recognized to be the phosphorylated PDGF receptor. Addition of PDGF to cells shortly pretreated with vanadate highly increased the intensity of the 170 kDa band corresponding to the phosphorylated receptor and caused its stabilization during time. In addition, the phosphorylation on tyrosine of other proteins (molecular mass 116, 80, 73, 60, 50 and 39 kDa) was also induced. Both the receptor and the other tyrosine phosphorylated proteins appeared to be associated with the detergent insoluble matrix. 相似文献
19.
Oncogenes,protein tyrosine kinases,and signal transduction 总被引:1,自引:0,他引:1
Many oncogenes encode protein tyrosine kinases (PTKs). Oncogenic mutations of these genes invariably result in constitutive activation of these PTKs. Autophosphorylation of the PTKs and tyrosine phosphorylation of their cellular substrates are essential events for transmission of the mitogenic signal into cells. The recent discovery of the characteristic amino acid sequences, of thesrc homology domains 2 and 3 (SH2 and SH3), and extensive studies on proteins containing the SH2 and SH3 domains have revealed that protein tyrosine-phosphorylation of PTKs provides phosphotyrosine sites for SH2 binding and allows extracellular signals to be relayed into the nucleus through a chain of protein-protein interactions mediated by the SH2 and SH3 domains. Studies on oncogenes, PTKs and SH2/SH3-containing proteins have made a tremendous contribution to our understanding of the mechanisms for the control of cell growth, oncogenesis, and signal transduction. This review is intended to provide an outline of the most recent progress in the study of signal transduction by PTKs. 相似文献
20.
《Cell biology international reports》1984,8(11):959-969
The RSV oncogene v-src is known to transform host cells through the action of a single gene protein product (pp60src) endowed with tyrosine specific kinase activity. The nature and cellular localization of substrates of pp60src kinase are largely unknown. Detergent insoluble cell matrix was prepared by treating RSV-transformed mouse fibroblasts with the non-ionic detergent NP-40. These preparations, highly enriched in cytoskeletal proteins, when observed in the E M consisted mainly of a meshwork of filaments; fragments of plasma membrane and nuclear “ghosts” were also present. Antibodies against phosphotyrosine -previously shown to be reactive with protein phosphorylated at tyrosine residues- were prepared and affinity purified using a synthetic hapten (azobenzyl phosphonic acid, ABP). By means of the immunogold techniques applied to electron microscopy, phosphotyrosine containing molecules were found to be present in RSV transformed, but not in control fibroblasts. Gold particles were mostly bound to electron dense granular material associated with the filaments. These results are consistent with the idea that pp60src itself and some tyrosine phosphorylated proteins may be found among detergent-insoluble cell structures. 相似文献