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1.
Protein kinase activity, including activity specific for the phosphorylation of tyrosine residues, can be detected among particulate fraction proteins of T cell lymphomas after separation by SDS-polyacrylamide gel electrophoresis. Putative protein kinases are detected by renaturation of enzyme activity directly within the gel following removal of detergent. LSTRA, a cell line that exhibits elevated levels of protein-tyrosine kinase activity, was found to express a predominant protein-tyrosine kinase of molecular weight 30,000. This same enzyme was present in T lymphocytes and other T lymphoid cell lines. Studies involving rapid preparation of protein fractions, limited proteolysis and one-dimensional peptide mapping did not demonstrate a direct relationship between the phosphorylated 30,000 dalton protein and the predominant 56,000 dalton phosphotyrosine containing protein that is observed following phosphorylation of LSTRA cell particulate fractions in vitro. 相似文献
2.
Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells. 总被引:30,自引:11,他引:19
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A F Voronova J E Buss T Patschinsky T Hunter B M Sefton 《Molecular and cellular biology》1984,4(12):2705-2713
The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid. 相似文献
3.
The LSTRA cell line has been shown to have an exceptionally high level of a tyrosine protein kinase (pp56lck). We now report that LSTRA cells also have a much higher level of proteins phosphorylated on tyrosine residues in comparison to several other cell lines with normal levels of pp56lck. The level of phosphotyrosine-containing proteins in LSTRA cells was comparable to that seen in K562 cells, a cell line known to have a constitutively active tyrosine protein kinase. These results provide evidence that LSTRA cells have an elevated level of in vivo tyrosine protein kinase activity, probably due to the overexpression and activation of pp56lck. 相似文献
4.
Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck). 总被引:57,自引:23,他引:34
J D Marth J A Cooper C S King S F Ziegler D A Tinker R W Overell E G Krebs R M Perlmutter 《Molecular and cellular biology》1988,8(2):540-550
The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56lck is phosphorylated in vivo at a carboxy-terminal tyrosine residue (Tyr-505) analogous to Tyr-527 of pp60c-src. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56lck in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60c-src, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56lck in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquisition of a posttranslationally activated version of pp56lck. 相似文献
5.
Remi Fagard Grard Gacon Jean-Paul Boissel Louise Reibel Jean-Pierre Piau Siegmund Fischer Georges Schapira Jean-Pierre Accary 《Journal of biochemical and biophysical methods》1985,10(5-6):307-314
Gastrin was recently shown to be phosphorylated on its single tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine protein kinase (TPK). The TPK previously detected in the murine lymphoma (LSTRA) induced by the Moloney murine leukemia virus phosphorylates gastrin, the apparent Km is 65 μM and the maximum rate 1900 pmol/min per mg; the kinase is more efficeint with MnCl2 than with MgCl2, is stimulated by NaVO3 and inhibited by ZnCl2. Gastrin phosphorylation is observed only when a TPK is expressed by the cell: extracts of fibroblasts infected with a temperature-sensitive mutant of the Rous sarcoma virus had no gastrin kinase activity when grown at the non-permissive temperature whereas cells grown at the permissive temperature were transformed and disclosed a clear gastrin kinase activity. Gastrin kinases were detected in various transformed cells; human lymphomas, K562 cells, cells from a patient with acute proliferative leukemia, and normal cels; human T and B lymphocytes. 相似文献
6.
J M Trevillyan Y Lin S J Chen C A Phillips C Canna T J Linna 《Biochimica et biophysica acta》1986,888(3):286-295
7.
Phosphorylations of the subcellular matrix in cells transformed by Rous' sarcoma virus 总被引:3,自引:0,他引:3
G Gacon S Gisselbrecht J P Piau M Y Fiszman S Fischer 《European journal of biochemistry》1982,125(2):453-456
The transforming protein of Rous' sarcoma virus (RSV) is a phosphoprotein of Mr 60 000 (pp60src) which displays protein kinase activity specific for tyrosine residues; pp60src is associated with the plasma membrane and is recovered in the detergent-insoluble material which represents the subcellular matrix of the cell. After phosphorylation of this material of RSV-transformed cells with [gamma-32P]ATP, five phosphoproteins have been detected which are not seen in normal cells. These proteins (Mr = 135 000, 125 000, 75 000, 70 000, 60 000) contain phosphotyrosine. Their phosphorylation is strongly inhibited by anti-pp60src antibodies. In cells transformed by a temperature-sensitive mutant of RSV, these phosphoproteins, present at the permissive temperature, are no longer detected at the non-permissive temperature. It is concluded that these phosphorylations are mediated by pp60src protein kinase activity. This supports a possible role of the phosphorylation of cytoskeletal proteins in the transformation process. 相似文献
8.
9.
Protein kinase activity of FSV (Fujinami sarcoma virus) P130gag-fps shows a strict specificity for tyrosine residues 总被引:10,自引:0,他引:10
A number of oncogenic viruses encode transforming proteins with protein kinase activities apparently specific for tyrosine residues. Recent evidence has raised questions as to the substrate specificity of these kinases in general and the physiological relevance of tyrosine phosphorylation in particular. The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) is strongly phosphorylated at 2 tyrosine residues in FSV-transformed cells of which 1 (Tyr-1073) is also the major site of P130gag-fps intermolecular autophosphorylation in vitro. We have investigated the specificity of the protein kinase activity intrinsic to FSV P130gag-fps by using site-directed mutagenesis to change the codon for Tyr-1073 to those for the other commonly phosphorylated hydroxyamino acids, serine and threonine. This approach has some advantages over the use of synthetic peptides to define protein kinase recognition sites in that the protein containing the altered target site can be expressed in intact cells. In addition it allows higher order as well as primary structure of the enzyme recognition site to be considered. Neither serine nor threonine were phosphorylated when substituted for tyrosine at position 1073 of P130gag-fps indicating a stringent specificity for tyrosine as a substrate of the P130gag-fps protein kinase autophosphorylating activity. Consistent with the suggestion that tyrosine phosphorylation is of functional significance we find that these and other FSV Tyr-1073 mutants have depressed enzymatic and oncogenic capacities. 相似文献
10.
Conservation of function of Drosophila melanogaster abl and murine v-abl proteins in transformation of mammalian cells.
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G D Holland M J Henkemeyer D A Kaehler F M Hoffmann R Risser 《Journal of virology》1990,64(5):2226-2235
The Drosophila melanogaster abl and the murine v-abl genes encode tyrosine protein kinases (TPKs) whose amino acid sequences are highly conserved. To assess functional conservation between the two gene products, we constructed Drosophila abl/v-abl-chimeric Abelson murine leukemia viruses. In these chimeric Abelson murine leukemia viruses, the TPK and carboxy-terminal regions of v-abl were replaced with the corresponding regions of D. melanogaster abl. The chimeric Abelson murine leukemia viruses were able to mediate morphological and oncogenic transformation of NIH 3T3 cells and were able to abrogate the interleukin-3 dependence of a lymphoid cell line. We also found that a virus that contained both TPK and carboxy-terminal Drosophila abl regions had no in vitro transforming activity for primary bone marrow cells and lacked the ability to induce tumors in susceptible mice. A virus that replaced only a portion of the v-abl TPK region with that of Drosophila abl had low activity in in vitro bone marrow transformation and tumorigenesis assays. These results indicate that the transforming functions of abl TPKs are only partially conserved through evolution. These results also imply that the TPK region of v-abl is a major determinant of its efficient lymphoid cell-transforming activity. 相似文献
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.
D A Tinker E A Krebs I C Feltham S K Attah-Poku V S Ananthanarayanan 《The Journal of biological chemistry》1988,263(11):5024-5026
An attempt has been made at defining the secondary structural requirement for phosphorylation of substrates of a protein tyrosine kinase from the leukemia virus-transformed LSTRA cell line. An examination of the sites of phosphorylation of substrates of protein tyrosine kinases indicated a relatively high probability of the beta-turn as the secondary structural feature at these sites. We have, therefore, synthesized three tyrosine peptides: Ala-Pro-Tyr-Gly-NHCH3, Leu-Pro-Tyr-Ala-NHCH3, and Pro-Gly-Ala-Tyr-NH2, of which the first two peptides, but not the third, would be expected to contain the tyrosine residue in a beta-turn. Circular dichroism and infrared spectral data on the peptides confirmed this expectation. Phosphorylation data on the peptides by the tyrosine kinase showed that the two beta-turn peptides were phosphorylated with Vmax and Km values comparable to those of the 13-residue-long arginine-containing synthetic peptide substrate having a sequence homologous to the autophosphorylation site of the LSTRA kinase. The peptides used here contain the shortest sequence length among the reported synthetic peptide substrates for protein tyrosine kinases. Their preference for the beta-turn indicated that this conformation may serve as the recognition site for tyrosine phosphorylation. 相似文献
13.
Localization of the Abelson murine leukemia virus protein in a detergent-insoluble subcellular matrix: architecture of the protein. 总被引:17,自引:6,他引:11
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We examined the interaction of Abelson murine leukemia virus protein P120 with other cellular components after extraction with the nonionic detergent Triton X-100. Most of the Abelson murine leukemia virus P120-associated kinase activity was found in the detergent-insoluble matrix in both lymphoid and fibroblast cell lines. The P120 labeled during a short exposure of cells to [35S]-methionine was mainly in the detergent-insoluble matrix (lymphoid cells) or equally distributed in the detergent-insoluble matrix and the soluble fraction (fibroblasts). Steady-state-labeled P120 was distributed equally in the two fractions (lymphoid cells) or mostly in the soluble portion (fibroblasts). Thus, there was an apparent movement of P120 from the detergent-insoluble matrix to the detergent-soluble fraction and a concomitant loss of enzymatic activity. When the detergent-insoluble matrix was incubated with [32P]ATP in situ, phosphorylation of tyrosine residues of P120 was observed. We found an 80,000-molecular-weight fragment of P120 (designated F80) after extraction of fibroblast cells with detergent. F80 was not found in extracted lymphoid cells, but mixing labeled lymphoid cells and unlabeled fibroblasts before extraction produced the fragment. F80 contained the gag determinants of P120 but did not react with Abelson-specific serum. These data allowed us to assign various features of the protein to regions of the P120 molecule and to localize the Abelson-specific antigenic determinants to the C-terminal region of the molecule. 相似文献
14.
Cloning and characterization of an envelope-specific probe from xenotropic murine leukemia proviral DNA. 总被引:26,自引:21,他引:5
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C E Buckler M D Hoggan H W Chan J F Sears A S Khan J L Moore J W Hartley W P Rowe M A Martin 《Journal of virology》1982,41(1):228-236
UR2 is a newly characterized avian sarcoma virus whose genome contains a unique sequence that is not related to the sequences of other avian sarcoma virus transforming genes thus far identified. This unique sequence, termed ros, is fused to part of the viral gag gene. The product of the fused gag-ros gene of UR2 is a protein of 68,000 daltons (P68) immunoprecipitable by antiserum against viral gag proteins. In vitro translation of viral RNA and in vivo pulse-chase experiments showed that P68 is not synthesized as a large precursor and that it is the only protein product encoded in the UR2 genome, suggesting that it is involved in cell transformation by UR2. In vivo, P68 was phosphorylated at both serine and tyrosine residues. Immunoprecipitates of P68 with anti-gag antisera had a cyclic nucleotide-independent protein kinase activity that phosphorylated P68, rabbit immunoglobulin G in the immune complex, and alpha-casein. The phosphorylation by P68 was specific to tyrosine of the substrate proteins. P68 was phosphorylated in vitro at only one tyrosine site, and the tryptic phosphopeptide of in vitro-labeled P68 was different from those of Fujinami sarcoma virus P140 and avian sarcoma virus Y73-P90. A comparison of the protein kinases encoded by UR2, Rous sarcoma virus, Fujinami sarcoma virus, and avian sarcoma virus Y73 revealed that UR2-P68 protein kinase is distinct from the protein kinases encoded by those viruses by several criteria. Our results suggest that several different protein kinases encoded by viral transforming genes have the same functional specificity and cause essentially the same cellular alterations. 相似文献
15.
S Fischer R Fagard P Comoglio G Gacon 《Biochemical and biophysical research communications》1985,132(2):481-489
We studied the ability to phosphorylate phosphoinositides by 3 different subcellular preparations, and immunopurified tyrosine protein kinase (TPK) from two murine lymphoma cell lines induced by the Moloney murine leukemia virus: LSTRA with a very active TPK and MBL2 without significant TPK activity. We could not find any difference in the phosphorylation of phosphoinositides by these preparations. The TPK purified with two antibodies which phosphorylate actively tyrosine on exogenous substrate were unable to phosphorylate phosphoinositides. 相似文献
16.
Activation of the c-abl oncogene by viral transduction or chromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties. 总被引:19,自引:15,他引:19
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The v-abl protein of Abelson murine leukemia virus is a tyrosine-specific kinase. Its normal cellular homolog, murine c-abl, does not possess detectable tyrosine kinase activity in vitro. Previously, we have detected tyrosine kinase activity in vitro for an altered c-abl gene product (c-abl P210) in the K562 human chronic myelogenous leukemia cell line. The expression of this variant c-abl gene product correlates with chromosomal translocation and amplification of the c-abl gene in K562 cells. Like v-abl, c-abl P210 is a fusion protein containing non-abl sequences near the amino terminus of c-abl. We compared the in vitro tyrosine kinase activity of c-abl P210 with that of wild-type murine v-abl. The remarkable similarities of these two proteins with respect to cis-acting autophosphorylation, trans-acting phosphorylation of exogenous substrates, and kinase inhibition, using site-directed abl-specific antisera, suggested that c-abl P210 could function similarly to v-abl in vivo. In addition, c-abl P210 possessed an associated serine kinase activity in immunoprecipitates. The serine kinase activity was not inhibited by site-directed, abl-specific antisera that inhibit the tyrosine kinase activity, suggesting that the serine kinase activity is not an intrinsic property of c-abl P210. Thus, the activation of the c-abl gene in a human leukemia cell line may have functional consequences analogous to activation of the c-abl gene in Abelson murine leukemia virus. 相似文献
17.
T cell activation induces rapid tyrosine phosphorylation of a limited number of cellular substrates 总被引:43,自引:0,他引:43
E D Hsi J N Siegel Y Minami E T Luong R D Klausner L E Samelson 《The Journal of biological chemistry》1989,264(18):10836-10842
Activation of murine T cells by antigen, antibodies binding the T cell antigen receptor, or stimulatory anti-Thy-1 antibodies results in rapid phosphorylation of the T cell receptor zeta chain on tyrosine residues. The T cell receptor is itself unlikely to be a tyrosine kinase; rather, it is probable that this receptor is coupled to a nonreceptor tyrosine kinase. To understand further this protein kinase pathway, additional targets of the tyrosine kinase have been sought by comparing anti-phosphotyrosine antibody immunoblots of cellular proteins from unactivated and activated T cell hybridomas. In addition to the T cell receptor zeta chain, two proteins of 53 and 62 kDa are phosphorylated on tyrosine residues after T cell activation. These phosphorylations require stimulatory anti-Thy-1 antibodies, antigen, or antireceptor antibody stimulation. The 53-kDa protein is preferentially phosphorylated by antigen or antireceptor antibody. Of interest is that variants of the murine T cell hybridoma lacking the T cell receptor zeta chain or lacking surface antigen receptor can nonetheless be stimulated by anti-Thy-1 antibodies to phosphorylate the 62-kDa substrate. In contrast to the tyrosine kinases of oncogenic viruses, the kinase coupled to the T cell antigen receptor appears to have a limited number of targets. These proteins are candidates for critical substrates in this protein tyrosine kinase pathway. 相似文献
18.
Lck-dependent tyrosyl phosphorylation of the phosphotyrosine phosphatase SH-PTP1 in murine T cells. 总被引:15,自引:2,他引:13
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U Lorenz K S Ravichandran D Pei C T Walsh S J Burakoff B G Neel 《Molecular and cellular biology》1994,14(3):1824-1834
The phosphorylation and dephosphorylation of proteins on tyrosyl residues are key regulatory mechanisms in T-cell signal transduction and are controlled by the opposing activities of protein tyrosine kinases and phosphotyrosyl phosphatases (PTPs). In T cells, several nontransmembrane protein tyrosine kinases are associated with receptors; for example, Lck is bound to the coreceptors CD4 and CD8 and becomes activated upon their stimulation. In comparison, little is known about the role of nontransmembrane PTPs in early T-cell signaling. SH-PTP1 (PTP1C, HCP, SHP) is a nontransmembrane PTP expressed primarily in hematopoietic cells, including T cells. We have found that SH-PTP1 is basally phosphorylated on serine in resting T cells. Upon stimulation of CD4 or CD8 either in a T-cell hybridoma cell line or in primary thymocytes, SH-PTP1 becomes tyrosyl phosphorylated. Moreover, SH-PTP1 is constitutively phosphorylated on tyrosine in the Lck-overexpressing lymphoma cell line LSTRA. SH-PTP1 is also a good substrate for recombinant Lck in vitro. Comparisons of the tryptic phosphopeptide maps of wild-type SH-PTP1 and deletion and point mutations establish that the two sites (Y-536 and Y-564) which are directly phosphorylated by Lck in vitro are also phosphorylated in vivo in LSTRA cells. One of these sites (Y-564) is phosphorylated in T cells in response to Lck activation. We conclude that SH-PTP1 undergoes Lck-dependent tyrosyl phosphorylation in T cells and likely plays a role in early T-cell signaling. 相似文献
19.
Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src. 总被引:74,自引:42,他引:32
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We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells. 相似文献