Mechanism of activation of the human trk oncogene.

The human trk oncogene was generated by a genetic rearrangement that replaced the extracellular domain of the normal trk tyrosine kinase receptor by sequences coding for the 221 amino-terminal residues of a nonmuscle tropomyosin. Molecular dissection of a cDNA clone of the trk oncogene indicated that both the tropomyosin and tyrosine kinase domains were required for proper transforming activity. Replacement of nonmuscle tropomyosin sequences with those of other tropomyosin isoforms had no deleterious effect. However, when tropomyosin sequences were replaced with those of another cytoskeletal gene, such as beta-actin or beta-globin, transforming activity was completely abolished. These results illustrate the important role of tropomyosin sequences in endowing the trk kinase with transforming properties. Functionally unrelated subdomains of the tropomyosin molecule were equally efficient in activating the trk gene. Moreover, the transforming activity of the trk oncogene was not affected when its subcellular localization was drastically altered. Therefore, tropomyosin sequences are likely to contribute to the malignant activation of the trk oncogene not by facilitating its interaction with defined cytoskeletal structures as initially suspected, but by allowing its kinase domain to fold into a constitutively active configuration.

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The amino-terminal 14 amino acids of v-src can functionally replace the extracellular and transmembrane domains of v-erbB

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4760-4770.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4760-4770

Author(s):

M McMahon ◽

R C Schatzman ◽

J M Bishop

Keyword(s):

Membrane Protein ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Integral Membrane Protein ◽

Kinase Domain ◽

Protein Tyrosine ◽

Amino Terminal ◽

Transforming Activity

The retroviral oncogene v-erbB encodes a truncated form of the receptor for epidermal growth factor, an integral membrane protein-tyrosine kinase. By contrast, the oncogene v-src encodes a protein-tyrosine kinase that is a peripheral membrane protein. The morphologies and spectra of cells transformed by these two oncogenes differ. In an effort to identify the functional determinant(s) of these differences, we constructed and tested first deletion mutants of v-erbB and then chimeras between v-src and v-erbB. As reported previously, the absence of any membrane anchorage eliminated transformation by v-erbB. Anchorage of the cytoplasmic kinase domain of v-erbB to membranes with amino-terminal portions of the v-src protein permitted transformation. The phenotype and spectrum of transformation were those expected for v-erbB rather than for v-src. The transforming chimeras lost their biological activity if the signal for myristylation at the amino terminus of v-src was compromised by mutation. Biochemical fractionations revealed a correlation between transforming activity and the association of chimeric gene products with the membrane fraction of the cell. For reasons not yet apparent, the combined presence of membrane anchorage domains of v-src, and the transmembrane domain of v-erbB in the same chimera typically (but not inevitably) impeded transformation. Our results suggest that the specificity of transformation by v-erbB resides in the selection of substrates by the cytoplasmic domain of the gene product. The protein retains access to those substrates even when anchored to the membrane in the manner of a peripheral rather than a transmembrane protein.

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Proapoptotic Function of the MET Tyrosine Kinase Receptor through Caspase Cleavage

Molecular and Cellular Biology ◽

10.1128/mcb.24.23.10328-10339.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 10328-10339 ◽

Cited By ~ 67

Author(s):

David Tulasne ◽

Julien Deheuninck ◽

Filipe Calheiros Lourenco ◽

Fabienne Lamballe ◽

Zongling Ji ◽

...

Keyword(s):

Epithelial Cells ◽

Tyrosine Kinase ◽

Cortical Neurons ◽

Kinase Domain ◽

Tyrosine Kinase Receptor ◽

Caspase Cleavage ◽

Cellular Targets ◽

Juxtamembrane Region ◽

Hepatocyte Growth

ABSTRACT The MET tyrosine kinase, the receptor of hepatocyte growth factor-scatter factor (HGF/SF), is known to be essential for normal development and cell survival. We report that stress stimuli induce the caspase-mediated cleavage of MET in physiological cellular targets, such as epithelial cells, embryonic hepatocytes, and cortical neurons. Cleavage occurs at aspartic residue 1000 within the SVD site of the juxtamembrane region, independently of the crucial docking tyrosine residues Y1001 or Y1347 and Y1354. This cleavage generates an intracellular 40-kDa MET fragment containing the kinase domain. The p40 MET fragment itself causes apoptosis of MDCK epithelial cells and embryonic cortical neurons, whereas its kinase-dead version is impaired in proapoptotic activity. Finally, HGF/SF treatment does not favor MET cleavage and apoptosis, confirming the known survival role of ligand-activated MET. Our results show that stress stimuli convert the MET survival receptor into a proapoptotic factor.

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The amino-terminal 14 amino acids of v-src can functionally replace the extracellular and transmembrane domains of v-erbB.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4760 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4760-4770 ◽

Cited By ~ 7

Author(s):

M McMahon ◽

R C Schatzman ◽

J M Bishop

Keyword(s):

Membrane Protein ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Integral Membrane Protein ◽

Kinase Domain ◽

Protein Tyrosine ◽

Amino Terminal ◽

Transforming Activity

The retroviral oncogene v-erbB encodes a truncated form of the receptor for epidermal growth factor, an integral membrane protein-tyrosine kinase. By contrast, the oncogene v-src encodes a protein-tyrosine kinase that is a peripheral membrane protein. The morphologies and spectra of cells transformed by these two oncogenes differ. In an effort to identify the functional determinant(s) of these differences, we constructed and tested first deletion mutants of v-erbB and then chimeras between v-src and v-erbB. As reported previously, the absence of any membrane anchorage eliminated transformation by v-erbB. Anchorage of the cytoplasmic kinase domain of v-erbB to membranes with amino-terminal portions of the v-src protein permitted transformation. The phenotype and spectrum of transformation were those expected for v-erbB rather than for v-src. The transforming chimeras lost their biological activity if the signal for myristylation at the amino terminus of v-src was compromised by mutation. Biochemical fractionations revealed a correlation between transforming activity and the association of chimeric gene products with the membrane fraction of the cell. For reasons not yet apparent, the combined presence of membrane anchorage domains of v-src, and the transmembrane domain of v-erbB in the same chimera typically (but not inevitably) impeded transformation. Our results suggest that the specificity of transformation by v-erbB resides in the selection of substrates by the cytoplasmic domain of the gene product. The protein retains access to those substrates even when anchored to the membrane in the manner of a peripheral rather than a transmembrane protein.

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Regulation of Jak2 Function by Phosphorylation of Tyr317 and Tyr637 during Cytokine Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.00278-09 ◽

2009 ◽

Vol 29 (12) ◽

pp. 3367-3378 ◽

Cited By ~ 23

Author(s):

Scott A. Robertson ◽

Rositsa I. Koleva ◽

Lawrence S. Argetsinger ◽

Christin Carter-Su ◽

Jarrod A. Marto ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Activity ◽

Feedback Inhibition ◽

Dominant Role ◽

Kinase Domain ◽

Cytokine Signaling ◽

Tyrosine Kinase Domain ◽

Phosphorylation Sites ◽

Cytokine Stimulation

ABSTRACT Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr317 in the FERM domain and Tyr637 in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr317 promotes increased Jak2 activity, and the phosphorylation of Tyr317 during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr317 in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr317 in the attenuation of Jak2 signaling. In contrast, mutation of Tyr637 decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr637 phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr317 and Tyr637 act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.

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Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2503 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2503-2512 ◽

Cited By ~ 144

Author(s):

G Heidecker ◽

M Huleihel ◽

J L Cleveland ◽

W Kolch ◽

T W Beck ◽

...

Keyword(s):

Structural Features ◽

Kinase Domain ◽

Full Length ◽

Wild Type ◽

Amino Terminal ◽

Raf Kinase ◽

Rich Domain ◽

Binding Residue ◽

Transforming Activity ◽

Mutational Activation

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.

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Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6711 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6711-6722 ◽

Cited By ~ 113

Author(s):

G A Rodrigues ◽

M Park

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Tyrosine Kinases ◽

Leucine Zipper ◽

Kinase Domain ◽

Site Directed Mutagenesis ◽

Leucine Zipper Motif ◽

Transforming Activity ◽

Met Oncogene ◽

Hepatocyte Growth

Oncogenic activation of the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase involves a genomic rearrangement that generates a hybrid protein containing tpr-encoded sequences at its amino terminus fused directly to the met-encoded receptor kinase domain. Deletion of Tpr sequences abolishes the transforming ability of this protein, implicating this region in oncogenic activation. We demonstrate, by site-directed mutagenesis and coimmunoprecipitation experiments, that a leucine zipper motif within Tpr mediates dimerization of the tpr-met product and is essential for the transforming activity of the met oncogene. By analogy with ligand-stimulated activation of receptor tyrosine kinases, we propose that constitutive dimerization mediated by a leucine zipper motif within Tpr is responsible for oncogenic activation of the Met kinase. The possibility that this mechanism of activation represents a paradigm for a class of receptor tyrosine kinase oncogenes activated by DNA rearrangement is discussed.

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Conformational dynamics of FERM-mediated autoinhibition in Pyk2 tyrosine kinase

10.1101/681932 ◽

2019 ◽

Author(s):

Hanna S. Loving ◽

Eric S. Underbakke

Keyword(s):

Tyrosine Kinase ◽

Conformational Dynamics ◽

Kinase Domain ◽

Ferm Domain ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Protein Scaffolding ◽

Hdx Ms

AbstractPyk2 is a non-receptor tyrosine kinase that evolved from gene duplication of focal adhesion kinase (FAK) and subsequent functional specialization in the brain and hemopoietic cells. Pyk2 shares a domain organization with FAK, with an N-terminal regulatory FERM domain adjoining the kinase domain. FAK regulation involves integrin-mediated membrane clustering to relieve autoinhibitory interactions between FERM and kinase domains. Pyk2 regulation remains cryptic, involving Ca2+ influx and protein scaffolding. While the mechanism of the FAK FERM domain in autoinhibition is well-established, the regulatory role of the Pyk2 FERM is ambiguous. We probed the mechanisms of FERM-mediated autoinhibition of Pyk2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and kinase activity profiling. The results reveal FERM-kinase interfaces responsible for autoinhibition. Pyk2 autoinhibition impacts activation loop conformation. In addition, the autoinhibitory FERM-kinase interface exhibits allosteric linkage with the FERM basic patch conserved in both FAK and Pyk2.Table of Contents graphic

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The Role of ERBB2/HER2 Tyrosine Kinase Receptor in the Regulation of Cell Death

Biochemistry (Moscow) ◽

10.1134/s0006297920100156 ◽

2020 ◽

Vol 85 (10) ◽

pp. 1277-1287

Author(s):

A. A. Daks ◽

O. A. Fedorova ◽

O. Y. Shuvalov ◽

S. E. Parfenev ◽

N. A. Barlev

Keyword(s):

Cell Death ◽

Tyrosine Kinase ◽

Tyrosine Kinase Receptor

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Favourable Prognosis Associated with FLT3 Tyrosine Kinase Domain Mutations in AML in Contrast to the Adverse Outcome Associated with Internal Tandem Duplications.

Blood ◽

10.1182/blood.v106.11.334.334 ◽

2005 ◽

Vol 106 (11) ◽

pp. 334-334 ◽

Cited By ~ 5

Author(s):

Adam Mead ◽

David Linch ◽

Robert Hills ◽

Keith Wheatley ◽

Alan Burnett ◽

...

Keyword(s):

Overall Survival ◽

Tyrosine Kinase ◽

Relapse Rate ◽

Poor Prognosis ◽

Disease Free Survival ◽

Kinase Domain ◽

Small Sample ◽

Tyrosine Kinase Receptor ◽

Tyrosine Kinase Domain ◽

Tandem Duplications

Abstract It is widely accepted that internal tandem duplications (ITDs) of the juxtamembrane domain of FLT3 occur in about one quarter of cases of acute myeloid leukaemia (AML) in young adults and predict for early relapse from complete remission (CR). Constitutive activation of FLT3 can also arise from mutations in the tyrosine kinase domain (TKD) but there is controversy as to the clinical significance of this class of mutation. This is partly due to the small sample size of some studies and the inclusion of elderly patients who have a poor prognosis regardless of their FLT3 status. To definitively resolve this issue we have screened for TKD mutations in AML blast cells from 1339 young adult patients included in the UK MRC AML 10 and 12 trials using a sensitive denaturing HPLC technique (Transgenomic WAVE®). Mutant samples were confirmed by sequencing or specific restriction digest. 161 of 1339 (12%) patients had a TKD mutation which is a higher frequency than previously reported both because of the sensitivity of the technique and the detection of mutations outside the EcoRV digest site. 91 patients (6.8%) were deemed to have high level mutant arbitrarily defined as ≥ 20% of all FLT3 alleles and 70 (5.2%) had a low level mutant. 79 of the 161 (49%) mutants were Asp835Tyr. 8 mutants occurred outside the EcoRV digest site and 3 novel mutations were characterised. 372 (28%) patients in this cohort had an ITD. There was a negative correlation between the presence of an ITD and a TKD mutation with only 2.5% having evidence of both mutations. Furthermore, high levels of both class of mutation in the same patient were not seen so that it is possible that both mutations never arise in the same cell. The demographics of patients with a TKD mutation differed from those with a FLT3 ITD in that the presence of TKD mutations were not correlated with FAB type and were infrequent in patients with secondary AML. Both types of mutation were more frequent in patients with a high white count but were infrequent in patients with adverse cytogenetics. The presence of TKD mutations did not impact on CR rate, the incidence of resistant disease or the induction death rate. In contrast to FLT3 ITDs, TKD mutations were associated with a reduced relapse rate (odds ratio [OR] 0.77, 95% confidence intervals [CI] 0.59–0.99, P.04), improved disease free survival (OR 0.75, CI 0.60–0.93, P.008) and increased overall survival (OR 0.79, CI 0.64–0.97, P.02). In patients with wild type FLT3 the actuarial relapse rate at 5 years was 46%, compared to 57% with an ITD (excluding rare double mutants) and 34% in those with a TKD mutation. The overall survival at 5 years was 44%, 35% and 55% respectively. In multivariate analysis, the presence of a TKD mutation still had an effect on the relapse rate (OR 0.82, CI 0.67–1.01, P.05) and overall survival (OR 0.83, CI 0.70–0.98, P.03). These data suggest that different classes of activating mutation of the same tyrosine kinase receptor can be associated with markedly different clinical outcomes. FLT3 ITDs are associated with a poor prognosis and FLT3 TKD mutations with a relatively good prognosis. This unexpected genotype-phenotype relationship has not previously been described with oncogenic mutations and is significant to the understanding of the pathophysiology of chemoresistance as well as prognostic stratification.

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