c-Abl Tyrosine Kinase Is Regulated Downstream of the Cytoskeletal Protein Synemin in Head and Neck Squamous Cell Carcinoma Radioresistance and DNA Repair

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

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The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function.

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4131 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4131-4140 ◽

Cited By ~ 49

Author(s):

C A Koch ◽

M Moran ◽

I Sadowski ◽

T Pawson

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Specific Activity ◽

Intramolecular Interaction ◽

Kinase Domain ◽

Sh2 Domain ◽

Homology Region ◽

Src Homology ◽

Type V

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

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The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4131-4140.1989 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4131-4140

Author(s):

C A Koch ◽

M Moran ◽

I Sadowski ◽

T Pawson

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Specific Activity ◽

Intramolecular Interaction ◽

Kinase Domain ◽

Sh2 Domain ◽

Homology Region ◽

Src Homology ◽

Type V

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

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Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.2883-2894.1994 ◽

1994 ◽

Vol 14 (5) ◽

pp. 2883-2894

Author(s):

B J Mayer ◽

D Baltimore

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Sh3 Domain ◽

Sh2 Domain ◽

Sh3 Domains ◽

Sh2 Domains ◽

Abl Tyrosine Kinase ◽

Transforming Activity

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

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The SH2 Domain of BCR-ABL1 Regulates Kinase Autophosphorylation By Controlling Activation Loop Accessibility

Blood ◽

10.1182/blood.v124.21.2209.2209 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2209-2209

Author(s):

Oliver D. Hantschel ◽

Allan Joaquim Lamontanara ◽

Sandrine Georgeon ◽

Giancarlo Tria ◽

Dmitri Svergun

Keyword(s):

Drug Resistance ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Binding Pocket ◽

Kinase Domain ◽

Sh2 Domain ◽

Activation Loop ◽

Phosphorylation Sites

Abstract Chronic myelogenous leukemia (CML) is caused by BCR-ABL1, which is a constitutively active form of the Abelson tyrosine kinase (ABL1). While treatment with the tyrosine kinase inhibitors imatinib, nilotinib, dasatinib, bosutinib or ponatinib that target the ATP binding pocket of BCR-ABL1 leads to durable cytogenetic and molecular remissions in the majority CML patients, primary and secondary drug resistance remains a clinical problem. Targeting additional sites in the BCR-ABL1 kinase outside the highly conserved ATP binding pocket may be an alternative strategy to restrict drug resistance and limit side effects of ATP-competitive drugs with low selectivity. Our recent work has shown that an allosteric intramolecular interaction of the BCR-ABL1 SH2 domain with its kinase domain is critical for leukemogenesis and can be targeted with an engineered high-affinity binding protein. We have now elucidated the molecular mechanisms responsible for the regulation of BCR-ABL1 kinase activity by its SH2 domain: To this end, we set-up an efficient expression system for the BCR-ABL1 SH2-kinase domain unit in E.coli with excellent yield, purity and activity. Detailed biophysical and biochemical analysis of the purified recombinant proteins in vitro recapitulated SH2-dependent regulation of BCR-ABL1 in CML cells and enabled a quantitative enzymatic analysis of BCR-ABL1 activation. Unexpectedly, we found that the interaction of the SH2 domain with the kinase domain is the critical switch that shifts the BCR-ABL1 activation loop from an otherwise closed to a fully open conformation and enables its autophosphorylation. The activation loop is a central and almost universally used control element that regulates the activity of protein kinases, as the conformation and phosphorylation status of the activation loop determines substrate binding to the active site. In BCR-ABL1, activation loop phosphorylation is required for transformation of fibroblasts and haematopoietic progenitors. We show that the SH2-kinase interaction enables autophosphorylation of the activation loop in trans by rendering a key phosphorylation site (Tyr-412) highly accessible. This requires prior phosphorylation of Tyr-245 in the SH2-kinase linker of BCR-ABL1. Mutational disruption of the SH2-kinase interaction abolished activation loop phosphorylation. Importantly, this effect was independent of the phosphotyrosine binding ability of the SH2 domain, which indicated that the SH2 domain is a true allosteric activator of BCR-ABL1 kinase activity. We also show that the spectrum of tyrosine phosphorylation sites that we mapped by mass spectrometry in vitro were vastly overlapping with the observed BCR-ABL1 phosphorylation sites in CML cells indicating that BCR-ABL1 autophosphorylation might be the major mechanism that determines its cellular phosphorylation status. In summary, our study demonstrates a novel mechanism by which a protein-protein interaction domain may allosterically mediate the transition of an inactive to an active kinase conformation in a key oncoprotein. This work may serve as an archetype to identify further allosteric regulatory mechanisms in other tyrosine kinases that are activated in haematological malignancies and facilitate the development of new allosteric inhibitors targeting oncogenic tyrosine kinases. Disclosures No relevant conflicts of interest to declare.

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Novel Receptor Tyrosine Kinase Pathway Inhibitors for Targeted Radionuclide Therapy of Glioblastoma

Pharmaceuticals ◽

10.3390/ph14070626 ◽

2021 ◽

Vol 14 (7) ◽

pp. 626

Author(s):

Julie Bolcaen ◽

Shankari Nair ◽

Cathryn H. S. Driver ◽

Tebatso M. G. Boshomane ◽

Thomas Ebenhan ◽

...

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Tyrosine Kinases ◽

Radionuclide Therapy ◽

Kinase Inhibitors ◽

Nuclear Imaging ◽

Therapy Resistance ◽

Targeted Radionuclide Therapy ◽

Therapy Strategy ◽

Dismal Prognosis

Glioblastoma (GB) remains the most fatal brain tumor characterized by a high infiltration rate and treatment resistance. Overexpression and/or mutation of receptor tyrosine kinases is common in GB, which subsequently leads to the activation of many downstream pathways that have a critical impact on tumor progression and therapy resistance. Therefore, receptor tyrosine kinase inhibitors (RTKIs) have been investigated to improve the dismal prognosis of GB in an effort to evolve into a personalized targeted therapy strategy with a better treatment outcome. Numerous RTKIs have been approved in the clinic and several radiopharmaceuticals are part of (pre)clinical trials as a non-invasive method to identify patients who could benefit from RTKI. The latter opens up the scope for theranostic applications. In this review, the present status of RTKIs for the treatment, nuclear imaging and targeted radionuclide therapy of GB is presented. The focus will be on seven tyrosine kinase receptors, based on their central role in GB: EGFR, VEGFR, MET, PDGFR, FGFR, Eph receptor and IGF1R. Finally, by way of analyzing structural and physiological characteristics of the TKIs with promising clinical trial results, four small molecule RTKIs were selected based on their potential to become new therapeutic GB radiopharmaceuticals.

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Development of the Sensing Platform for Protein Tyrosine Kinase Activity

Biosensors ◽

10.3390/bios11070240 ◽

2021 ◽

Vol 11 (7) ◽

pp. 240

Author(s):

Lan-Yi Wei ◽

Wei Lin ◽

Bey-Fen Leo ◽

Lik-Voon Kiew ◽

Chia-Ching Chang ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Significant Inhibition ◽

Tyrosine Kinase Activity ◽

Protein Tyrosine ◽

Sensing Platform ◽

Relative Standard ◽

Protein Tyrosine Kinase Activity

A miniature tyrosinase-based electrochemical sensing platform for label-free detection of protein tyrosine kinase activity was developed in this study. The developed miniature sensing platform can detect the substrate peptides for tyrosine kinases, such as c-Src, Hck and Her2, in a low sample volume (1–2 μL). The developed sensing platform exhibited a high reproducibility for repetitive measurement with an RSD (relative standard deviation) of 6.6%. The developed sensing platform can detect the Hck and Her2 in a linear range of 1–200 U/mL with the detection limit of 1 U/mL. The sensing platform was also effective in assessing the specificity and efficacies of the inhibitors for protein tyrosine kinases. This is demonstrated by the detection of significant inhibition of Hck (~88.1%, but not Her2) by the Src inhibitor 1, an inhibitor for Src family kinases, as well as the significant inhibition of Her2 (~91%, but not Hck) by CP-724714 through the platform. These results suggest the potential of the developed miniature sensing platform as an effective tool for detecting different protein tyrosine kinase activity and for accessing the inhibitory effect of various inhibitors to these kinases.

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Topology of Streptococcus pneumoniae CpsC, a Polysaccharide Copolymerase and Bacterial Protein Tyrosine Kinase Adaptor Protein

Journal of Bacteriology ◽

10.1128/jb.02106-14 ◽

2014 ◽

Vol 197 (1) ◽

pp. 120-127 ◽

Cited By ~ 4

Author(s):

Jonathan J. Whittall ◽

Renato Morona ◽

Alistair J. Standish

Keyword(s):

Streptococcus Pneumoniae ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Tyrosine Phosphatase ◽

Adaptor Protein ◽

Tyrosine Kinase Activity ◽

Content Type ◽

Protein Tyrosine ◽

C Terminus

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. InStreptococcus pneumoniae, this transmembrane adaptor is CpsC, with the C terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest that CpsC has two transmembrane domains, with the N and C termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal hemagglutinin (HA)-tagged Cps2C protein inS. pneumoniaestrain D39. Incubation of both protoplasts and membranes with carboxypeptidase B (CP-B) resulted in complete degradation of HA-Cps2C in all cases, indicating that the C terminus of Cps2C was likely extracytoplasmic and hence that the protein's topology was not as predicted. Similar results were seen with membranes fromS. pneumoniaestrain TIGR4, indicating that Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C terminus by flow cytometry indicated that it was extracytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this effect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C terminus flipping across the cytoplasmic membrane, where it interacts with CpsD in order to regulate tyrosine kinase activity.

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Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2493-2499.1989 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2493-2499

Author(s):

M Fujii ◽

D Shalloway ◽

I M Verma

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Cellular Transformation ◽

Membrane Association ◽

Protein Tyrosine Phosphorylation ◽

Promoter Activation ◽

Tyrosine Kinase Activity ◽

Atp Binding Site ◽

Src Gene

A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.

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