Abelson murine leukaemia virus protein is phosphorylated in vitro to form phosphotyrosine

Witte, Owen N.; Dasgupta, Asim; Baltimore, David

doi:10.1038/283826a0

Cited by 531 publications

(256 citation statements)

References 22 publications

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“…The enzymatic activity of the p210c-abl protein was assayed in immunocomplexes under experimental conditions known to allow v-abl tyrosine kinase activity (52 (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

“…While the catalytic domain is fully contained in the c-abl protein and can be activated in properly designed in vitro assays (31), no in vivo occurrence of tyrosine kinase activity has yet been demonstrated for normal c-abl gene product (30,31,34). On the other hand, the acquisition of constitutive tyrosine kinase activity has been shown to be a requisite for the transforming activity of v-abl oncogene products (35,39,40,52,53). Ph' translocation of the c-abl gene in CML cells then offers a valuable opportunity for comparing, at the gene product level, a spontaneously activated human proto-oncogene with its retrovirus-transduced acutely transforming version.…”

Section: Discussionmentioning

confidence: 99%

“…Detergent-insoluble proteins were labeled in situ as described by Burr et (52). The reaction was stopped by the addition of excess ice-cold RIPA buffer containing 10 mM EDTA, and the immunocomplexes were washed four more times in the same buffer.…”

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confidence: 99%

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Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Naldini¹,

Stacchini²,

Cirillo³

et al. 1986

Mol. Cell. Biol.

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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.

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“…The enzymatic activity of the p210c-abl protein was assayed in immunocomplexes under experimental conditions known to allow v-abl tyrosine kinase activity (52 (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Naldini¹,

Stacchini²,

Cirillo³

et al. 1986

Mol. Cell. Biol.

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“…Aliquots of the anti-JAK2 immunoprecipitated material were then subjected to in vitro radiolabelling autokinase assays, which provide a basic measure of JAK2's enzymatic activity [33][34][35]. No detectable activity was associated with the kinaseinactive EGFP/rJAK2(K882E), as anticipated.…”

Section: Characteristics Of the Egfp/rjak2 Chimeras Produced In Cos-7mentioning

confidence: 99%

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

Lee¹,

Duhé²

2006

J Biomed Sci

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SummaryJanus kinase 2 (JAK2) is an essential intracellular signal transducer for numerous cytokines and hormones. To examine how JAK2 structural modifications can affect cellular physiology, we created expression vectors for chimeric proteins containing an enhanced green fluorescent protein (EGFP) fused to rat JAK2 (EGFP/rJAK2), and a kinase-inactive variant, EGFP/rJAK2(K882E). The properties of EGFP/rJAK2 were examined following transient transfection of COS-7 cells. EGFP/rJAK2 was expressed throughout the cell, and was found in subcellular membrane, cytosolic and nuclear fractions. Interestingly, EGFP/rJAK2 phosphorylated other proteins in situ without additional cytokine stimulation. Furthermore, despite a much higher level of tyrosine phosphorylation arising from in situ autophosphorylation, the in vitro radiolabelling autokinase activity of EGFP/rJAK2 was significantly less than that of the endogenous JAK2. These results reveal a technical limitation of the application of the ''conventional'' in vitro radiolabelling autokinase assay to hyperphosphorylated forms of the enzyme and illustrate the potential weaknesses in individual assays commonly used to determine JAK2's enzymatic activity and subcellular distribution. We also suggest that the EGFP/rJAK2 model can be very useful in studying JAK2-related cancers, because its ubiquitous distribution and abnormal constitutive hyperphosphorylation may distinguish it from the cytokine-regulated, membrane-proximal form of JAK2 associated with normal physiology.

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“…Although P145"4/ P210b"/"b' and P1906cr'"b' have tyrosine kinase activity, which is increased in the abnormal proteins. Tyrosine kinase activity is a feature of some cellular oncogenes, and it is responsible for the transforming activity of AbMLV [99] and Rous sarcoma virus (RSV) [loo].…”

Section: Proteinmentioning

confidence: 99%

Oncogenes and human leukemias

Butturini¹,

Gale

1988

The International Journal of Cell Cloning

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Abstract. Eukaryotic cells contain a family of genes termed "cellular oncogenes" or "proto-oncogenes," thought to regulate normal cell growth and development. In some circumstances, such as following transduction by retroviruses, activation of these genes causes tumors and leukemias in animals. Possible mechanisms of cellular oncogene activation include: 1) DNA point mutation, deletion or insertion, 2) gene amplification, 3) gene activation by internal rearrangement, chromosomal translocation or promoter insertion, 4) recombinative events resulting in the formation of novel chimeric genes, and others.

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Abelson murine leukaemia virus protein is phosphorylated in vitro to form phosphotyrosine

Cited by 531 publications

References 22 publications

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

Oncogenes and human leukemias

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