Stability and solubility engineering of the EphB4 tyrosine kinase catalytic domain using a rationally designed synthetic library

A human epithelial (HeLa) cDNA library was screened with degenerate oligonucleotides designed to hybridize to highly conserved regions of protein-tyrosine kinases. One cDNA from this screen was shown to contain a putative protein-tyrosine kinase catalytic domain and subsequently used to isolate another cDNA from a human keratinocyte library that encompasses the entire coding region of a 976-amino-acid polypeptide. The predicted protein has an external domain of 534 amino acids with a presumptive N-terminal signal peptide, a transmembrane domain, and a cytoplasmic domain of 418 amino acids that includes a canonical protein-tyrosine kinase catalytic domain. Molecular phylogeny indicates that this protein kinase is closely related to eph and elk and that this receptor family is more closely related to the non-receptor protein-tyrosine kinase families than to other receptor protein-tyrosine kinases. Antibodies raised against a TrpE fusion protein immunoprecipitated a 130-kDa protein that became phosphorylated on tyrosine in immune complex kinase assays, indicating that this protein is a bona fide protein-tyrosine kinase. Analysis of RNA from 13 adult rat organs showed that the eck gene is expressed most highly in tissues that contain a high proportion of epithelial cells, e.g., skin, intestine, lung, and ovary. Several cell lines of epithelial origin were found to express the eck protein kinase at the protein and RNA levels. Immunohistochemical analysis of several rat organs also showed staining in epithelial cells. These observations prompted us to name this protein kinase eck, for epithelial cell kinase.

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Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.4.1579 ◽

1994 ◽

Vol 91 (4) ◽

pp. 1579-1583 ◽

Cited By ~ 360

Author(s):

K. M. Carlson ◽

S. Dou ◽

D. Chi ◽

N. Scavarda ◽

K. Toshima ◽

...

Keyword(s):

Tyrosine Kinase ◽

Missense Mutation ◽

Multiple Endocrine Neoplasia ◽

Catalytic Domain ◽

Multiple Endocrine Neoplasia Type ◽

Ret Protooncogene ◽

Endocrine Neoplasia ◽

Endocrine Neoplasia Type ◽

Kinase Catalytic Domain

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cDNA cloning and characterization of eck, an epithelial cell receptor protein-tyrosine kinase in the eph/elk family of protein kinases.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6316 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6316-6324 ◽

Cited By ~ 155

Author(s):

R A Lindberg ◽

T Hunter

Keyword(s):

Protein Kinase ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Catalytic Domain ◽

Protein Tyrosine Kinases ◽

Receptor Protein ◽

Protein Tyrosine ◽

Kinase Catalytic Domain ◽

Receptor Protein Tyrosine Kinase

A human epithelial (HeLa) cDNA library was screened with degenerate oligonucleotides designed to hybridize to highly conserved regions of protein-tyrosine kinases. One cDNA from this screen was shown to contain a putative protein-tyrosine kinase catalytic domain and subsequently used to isolate another cDNA from a human keratinocyte library that encompasses the entire coding region of a 976-amino-acid polypeptide. The predicted protein has an external domain of 534 amino acids with a presumptive N-terminal signal peptide, a transmembrane domain, and a cytoplasmic domain of 418 amino acids that includes a canonical protein-tyrosine kinase catalytic domain. Molecular phylogeny indicates that this protein kinase is closely related to eph and elk and that this receptor family is more closely related to the non-receptor protein-tyrosine kinase families than to other receptor protein-tyrosine kinases. Antibodies raised against a TrpE fusion protein immunoprecipitated a 130-kDa protein that became phosphorylated on tyrosine in immune complex kinase assays, indicating that this protein is a bona fide protein-tyrosine kinase. Analysis of RNA from 13 adult rat organs showed that the eck gene is expressed most highly in tissues that contain a high proportion of epithelial cells, e.g., skin, intestine, lung, and ovary. Several cell lines of epithelial origin were found to express the eck protein kinase at the protein and RNA levels. Immunohistochemical analysis of several rat organs also showed staining in epithelial cells. These observations prompted us to name this protein kinase eck, for epithelial cell kinase.

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Identification of protein-tyrosine kinase catalytic domain conserved sequences in the ORF expressed sequence tags database

Nature Genetics ◽

10.1038/87090 ◽

2001 ◽

Vol 27 (S4) ◽

pp. 55-55

Author(s):

Daniel Giannella-Neto ◽

Maria Lúcia C. Correa ◽

Ana Mercedes S. Cavaleiro-Luna ◽

Maria Angela H.Z. Fortes ◽

Cassio N. Coimbra ◽

...

Keyword(s):

Tyrosine Kinase ◽

Expressed Sequence Tags ◽

Protein Tyrosine Kinase ◽

Catalytic Domain ◽

Conserved Sequences ◽

Protein Tyrosine ◽

Kinase Catalytic Domain ◽

Expressed Sequence

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A Unique GTP-Dependent Sporulation Sensor Histidine Kinase in Bacillus anthracis

Journal of Bacteriology ◽

10.1128/jb.01184-08 ◽

2008 ◽

Vol 191 (3) ◽

pp. 687-692 ◽

Cited By ~ 11

Author(s):

Francesca Scaramozzino ◽

Andrea White ◽

Marta Perego ◽

James A. Hoch

Keyword(s):

Bacillus Anthracis ◽

Histidine Kinase ◽

Catalytic Domain ◽

Coiled Coil ◽

Reverse Reaction ◽

Forward Reaction ◽

Critical Signal ◽

Sensor Histidine Kinase ◽

Virulence Plasmids ◽

Kinase Catalytic Domain

ABSTRACT The Bacillus anthracis BA2291 gene codes for a sensor histidine kinase involved in the induction of sporulation. Genes for orthologs of the sensor domain of the BA2291 kinase exist in virulence plasmids in this organism, and these proteins, when expressed, inhibit sporulation by converting BA2291 to an apparent phosphatase of the sporulation phosphorelay. Evidence suggests that the sensor domains inhibit BA2291 by titrating its activating signal ligand. Studies with purified BA2291 revealed that this kinase is uniquely specific for GTP in the forward reaction and GDP in the reverse reaction. The G1 motif of BA2291 is highly modified from ATP-specific histidine kinases, and modeling this motif in the structure of the kinase catalytic domain suggested how guanine binds to the region. A mutation in the putative coiled-coil linker between the sensor domain and the catalytic domains was found to decrease the rate of the forward autophosphorylation reaction and not affect the reverse reaction from phosphorylated Spo0F. The results suggest that the activating ligand for BA2291 is a critical signal for sporulation and in a limited concentration in the cell. Decreasing the response to it either by slowing the forward reaction through mutation or by titration of the ligand by expressing the plasmid-encoded sensor domains switches BA2291 from an inducer to an inhibitor of the phosphorelay and sporulation.

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Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

Biochemical Journal ◽

10.1042/bj20111536 ◽

2012 ◽

Vol 442 (1) ◽

pp. 105-118 ◽

Cited By ~ 30

Author(s):

Craig R. Pigott ◽

Halina Mikolajek ◽

Claire E. Moore ◽

Stephen J. Finn ◽

Curtis W. Phippen ◽

...

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

Functional Organization ◽

Elongation Factor ◽

Kinase Domain ◽

Conserved Residues ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Kinase Catalytic Domain ◽

Eukaryotic Elongation Factor

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

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Identification of tetracycline combinations as EphB1 tyrosine kinase inhibitors for treatment of neuropathic pain

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016265118 ◽

2021 ◽

Vol 118 (10) ◽

pp. e2016265118

Author(s):

Mahmoud S. Ahmed ◽

Ping Wang ◽

Ngoc Uyen Nhi Nguyen ◽

Yuji Nakada ◽

Ivan Menendez-Montes ◽

...

Keyword(s):

Neuropathic Pain ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Catalytic Domain ◽

Therapeutic Option ◽

Atp Binding ◽

Approved Drugs ◽

Fda Approved Drugs

Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.

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Characterization of elk, a brain-specific receptor tyrosine kinase

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2496-2502.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2496-2502

Author(s):

V Lhoták ◽

P Greer ◽

K Letwin ◽

T Pawson

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Catalytic Domain ◽

Signal Sequence ◽

Tyrosine Kinase Domain ◽

Rat Tissues ◽

Protein Tyrosine

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.

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