Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

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Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1553 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1553-1565 ◽

Cited By ~ 190

Author(s):

J R McWhirter ◽

J Y Wang

Keyword(s):

Amino Acids ◽

Tyrosine Kinase ◽

Fusion Proteins ◽

Kinase Activity ◽

Lymphoblastic Leukemia ◽

Myelogenous Leukemia ◽

Tyrosine Kinase Activity ◽

Abl Tyrosine Kinase ◽

Binding Function

Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis.

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Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1553-1565.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1553-1565

Author(s):

J R McWhirter ◽

J Y Wang

Keyword(s):

Amino Acids ◽

Tyrosine Kinase ◽

Fusion Proteins ◽

Kinase Activity ◽

Lymphoblastic Leukemia ◽

Myelogenous Leukemia ◽

Tyrosine Kinase Activity ◽

Abl Tyrosine Kinase ◽

Binding Function

Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis.

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Effect of herbimycin A, an antagonist of tyrosine kinase, on bcr/abl oncoprotein-associated cell proliferations: abrogative effect on the transformation of murine hematopoietic cells by transfection of a retroviral vector expressing oncoprotein P210bcr/abl and preferential inhibition on Ph1-positive leukemia cell growth

Blood ◽

10.1182/blood.v80.5.1330.1330 ◽

1992 ◽

Vol 80 (5) ◽

pp. 1330-1338 ◽

Cited By ~ 53

Author(s):

M Okabe ◽

Y Uehara ◽

T Miyagishima ◽

T Itaya ◽

M Tanaka ◽

...

Keyword(s):

Protein Kinase ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Myelogenous Leukemia ◽

Human Leukemia ◽

In Vitro Growth ◽

Tyrosine Kinase Activity ◽

Abl Tyrosine Kinase ◽

Herbimycin A

Abstract Herbimycin A, a benzoquinoid ansamycin antibiotic, was demonstrated to decrease intracellular phosphorylation by protein tyrosine kinase (PTK). In Philadelphia chromosome (Ph1)-positive leukemias such as chronic myelogenous leukemia (CML) and Ph1-positive acute lymphoblastic leukemia (ALL), both of which express bcr-abl fused gene products (P210bcr-abl or P190bcr-abl protein kinase) with augmented tyrosine kinase activities, herbimycin A markedly inhibited the in vitro growth of the Ph1-positive ALL cells and the leukemic cells derived from CML blast crisis. However, the same dose of herbimycin A did not inhibit in vitro growth of a broad spectrum of Ph1-negative human leukemia cells, and several other protein kinase antagonists also displayed no preferential inhibition. Furthermore, we demonstrated that herbimycin A has an antagonizing effect on the growth of transformed cells by a transfection of retroviral amphotrophic vector expressing P210bcr/abl into a murine interleukin (IL)-3-dependent myeloid FDC-P2 cell line. This inhibition was abrogated by the addition of sulfhydryl compounds, similar to the reaction previously described for Rous sarcoma virus transformation. The inhibitory effect of herbimycin A on the growth of Ph1-positive cells was associated with decreased bcr/abl tyrosine kinase activity, but no decrease of bcr-abl mRNA and protein, suggesting that the inactivation of bcr-abl tyrosine kinase activity by herbimycin A may be induced by its binding to the bcr-abl protein portion that is rich with sulfhydryl groups. The present study indicates that herbimycin A is a beneficial agent for the investigation of the role of the bcr-abl gene in Ph1-positive leukemias and further suggests that the development of agents inhibiting the bcr-abl gene product may offer a new therapeutic potential for Ph1-positive leukemias.

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Constitutive Association of BRCA1 and c-Abl and Its ATM-Dependent Disruption after Irradiation

Molecular and Cellular Biology ◽

10.1128/mcb.22.12.4020-4032.2002 ◽

2002 ◽

Vol 22 (12) ◽

pp. 4020-4032 ◽

Cited By ~ 49

Author(s):

Nicolas Foray ◽

Didier Marot ◽

Voahangy Randrianarison ◽

Nicole Dalla Venezia ◽

Didier Picard ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Activity ◽

Direct Interaction ◽

Cell Cycle Checkpoint ◽

Dependent Manner ◽

Tyrosine Kinase Activity ◽

Abl Tyrosine Kinase ◽

C Terminus

ABSTRACT BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.

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Effect of herbimycin A, an antagonist of tyrosine kinase, on bcr/abl oncoprotein-associated cell proliferations: abrogative effect on the transformation of murine hematopoietic cells by transfection of a retroviral vector expressing oncoprotein P210bcr/abl and preferential inhibition on Ph1-positive leukemia cell growth

Blood ◽

10.1182/blood.v80.5.1330.bloodjournal8051330 ◽

1992 ◽

Vol 80 (5) ◽

pp. 1330-1338 ◽

Cited By ~ 1

Author(s):

M Okabe ◽

Y Uehara ◽

T Miyagishima ◽

T Itaya ◽

M Tanaka ◽

...

Keyword(s):

Protein Kinase ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Myelogenous Leukemia ◽

Human Leukemia ◽

In Vitro Growth ◽

Tyrosine Kinase Activity ◽

Abl Tyrosine Kinase ◽

Herbimycin A

Herbimycin A, a benzoquinoid ansamycin antibiotic, was demonstrated to decrease intracellular phosphorylation by protein tyrosine kinase (PTK). In Philadelphia chromosome (Ph1)-positive leukemias such as chronic myelogenous leukemia (CML) and Ph1-positive acute lymphoblastic leukemia (ALL), both of which express bcr-abl fused gene products (P210bcr-abl or P190bcr-abl protein kinase) with augmented tyrosine kinase activities, herbimycin A markedly inhibited the in vitro growth of the Ph1-positive ALL cells and the leukemic cells derived from CML blast crisis. However, the same dose of herbimycin A did not inhibit in vitro growth of a broad spectrum of Ph1-negative human leukemia cells, and several other protein kinase antagonists also displayed no preferential inhibition. Furthermore, we demonstrated that herbimycin A has an antagonizing effect on the growth of transformed cells by a transfection of retroviral amphotrophic vector expressing P210bcr/abl into a murine interleukin (IL)-3-dependent myeloid FDC-P2 cell line. This inhibition was abrogated by the addition of sulfhydryl compounds, similar to the reaction previously described for Rous sarcoma virus transformation. The inhibitory effect of herbimycin A on the growth of Ph1-positive cells was associated with decreased bcr/abl tyrosine kinase activity, but no decrease of bcr-abl mRNA and protein, suggesting that the inactivation of bcr-abl tyrosine kinase activity by herbimycin A may be induced by its binding to the bcr-abl protein portion that is rich with sulfhydryl groups. The present study indicates that herbimycin A is a beneficial agent for the investigation of the role of the bcr-abl gene in Ph1-positive leukemias and further suggests that the development of agents inhibiting the bcr-abl gene product may offer a new therapeutic potential for Ph1-positive leukemias.

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Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽

10.1182/blood.v106.11.438.438 ◽

2005 ◽

Vol 106 (11) ◽

pp. 438-438 ◽

Cited By ~ 1

Author(s):

Xiaoyan Jiang ◽

Kyi Min Saw ◽

Allen Eaves ◽

Connie Eaves

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Point Mutations ◽

Chronic Phase ◽

Kinase Domain ◽

Tyrosine Kinase Activity ◽

Abl Kinase ◽

Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

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The Discovery of Novel BCR-ABL Tyrosine Kinase Inhibitors Using a Pharmacophore Modeling and Virtual Screening Approach

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.649434 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ting-Ting Huang ◽

Xin Wang ◽

Shao-Jia Qiang ◽

Zhen-Nan Zhao ◽

Zhuo-Xun Wu ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Pharmacophore Modeling ◽

Myelogenous Leukemia ◽

Lipinski’S Rule ◽

Abl Tyrosine Kinase ◽

Pharmacophore Models

Chronic myelogenous leukemia (CML) typically results from a reciprocal translocation between chromosomes 9 and 22 to produce the bcr-abl oncogene that when translated, yields the p210 BCR-ABL protein in more than 90% of all CML patients. This protein has constitutive tyrosine kinase activity that activates numerous downstream pathways that ultimately produces uncontrolled myeloid proliferation. Although the use of the BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, dasatinib, bosutinib, and ponatinib have increased the overall survival of CML patients, their use is limited by drug resistance and severe adverse effects. Therefore, there is the need to develop novel compounds that can overcome these problems that limit the use of these drugs. Therefore, in this study, we sought to find novel compounds using Hypogen and Hiphip pharmacophore models based on the structures of clinically approved BCR-ABL TKIs. We also used optimal pharmacophore models such as three-dimensional queries to screen the ZINC database to search for potential BCR-ABL inhibitors. The hit compounds were further screened using Lipinski’s rule of five, ADMET and molecular docking, and the efficacy of the hit compounds was evaluated. Our in vitro results indicated that compound ZINC21710815 significantly inhibited the proliferation of K562, BaF3/WT, and BaF3/T315I leukemia cells by inducing cell cycle arrest. The compound ZINC21710815 decreased the expression of p-BCR-ABL, STAT5, and Crkl and produced apoptosis and autophagy. Our results suggest that ZINC21710815 may be a potential BCR-ABL inhibitor that should undergo in vivo evaluation.

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Inhibition of Src tyrosine kinase activity by squamosamide derivative FLZ attenuates neuroinflammation in both in vivo and in vitro Parkinson's disease models

Neuropharmacology ◽

10.1016/j.neuropharm.2013.07.020 ◽

2013 ◽

Vol 75 ◽

pp. 201-212 ◽

Cited By ~ 33

Author(s):

Wenjiao Tai ◽

Xuan Ye ◽

Xiuqi Bao ◽

Baozhong Zhao ◽

Xiaoliang Wang ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Disease Models ◽

Tyrosine Kinase Activity ◽

Src Tyrosine Kinase

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p145, a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line

Molecular and Cellular Biology ◽

10.1128/mcb.8.8.3510-3517.1988 ◽

1988 ◽

Vol 8 (8) ◽

pp. 3510-3517

Author(s):

S Giordano ◽

M F Di Renzo ◽

R Ferracini ◽

L Chiadò-Piat ◽

P M Comoglio

Keyword(s):

Gastric Carcinoma ◽

Tyrosine Kinase ◽

Cell Line ◽

Kinase Activity ◽

Carcinoma Cell ◽

Tyrosine Kinase Activity ◽

Gastric Carcinoma Cell ◽

Gastric Carcinoma Cell Line

A protein with an Mr of 145,000 (p145) was detected by antibodies to phosphotyrosine by Western blot (immunoblot) analysis. This protein was phosphorylated on tyrosine in a gastric carcinoma cell line. In cells that were metabolically labeled with 32Pi, this protein was phosphorylated on tyrosine and serine. p145 is a cysteine-rich transmembrane glycoprotein. The extracellular domain could be labeled by 125I under nonpermeating conditions and was cleaved by mild trypsin treatment of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed a shift of p145 mobility to an apparent Mr of 190,000. After immunoprecipitation with phosphotyrosine antibodies, p145 displayed a strong associated protein kinase activity in vitro, becoming phosphorylated on tyrosine. There was no immunological cross-reaction between p145 and known tyrosine kinases. Both in vivo and in vitro tyrosine phosphorylations were unaffected by the addition of known growth factors. However, p145 was rapidly dephosphorylated in vivo when cells were exposed to low pH, a condition that is known to dissociate ligands from their receptors. These data suggest that p145 is associated with a protein tyrosine kinase activity which, in the tumor cell line studied, is activated by an as yet unidentified factor.

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