Structures of an Eph receptor tyrosine kinase and its potential activation mechanism

Wei, Qiang; Li, Jun; Wang, Nan; Zhang, Xiaoying; Jin, Jin; Chin-Sang, Ian D.; Zheng, Jimin; Jia, Zongchao

doi:10.1107/s1399004714021944

Cited by 11 publications

(19 citation statements)

References 33 publications

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“…No structural investigations were pursued. Recently, an EPHA2 partial‐JM‐KD construct (590–876) produced in E. coli with an N‐terminal His 6 ‐tag was crystallized in both phosphorylated form and after treatment with a dual‐specificity phosphatase PTEN . The homogeneity of the produced protein was not determined, neither was its suitability for co‐crystallization with ligands assessed.…”

Section: Introductionsupporting

confidence: 83%

Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies

et al. 2016

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The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non-small-cell lung cancer). Small-molecule-based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells). H, N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well-folded but unstable, and it did not crystallize. However, a construct (D596-G900) produced in Sf9 cells yielded homogenous, well-folded protein that crystallized readily, thereby resulting in eleven new EPHA2-ligand crystal structures. We have also established a strategy for selective and uniform N-amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2-drug interactions by NMR.

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Section: Introductionsupporting

confidence: 83%

Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies

et al. 2016

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“…Similar to c-KIT and CSF1R, we found that EPHA2 forms a potential autophosphorylation complex in a crystal structure of the kinase domain [PDB: 4PDO (23)], with a hydrogen bond between juxtamembrane region Tyr 594 and Asp 739 of the HRD motif (Fig. 1C).…”

Section: Resultsmentioning

confidence: 85%

“…Although Wei et al . (23) note that the juxtamembrane segments of each chain are ordered, point away from the kinase domain, and contact neighboring chains in the crystal, they do not mention the presence of the autophosphorylation site of these segments in the active site of the neighboring kinase protein.…”

Section: Resultsmentioning

confidence: 99%

“…The newly identified autophosphorylation events include: (i) the activation loop Tyr of human nonreceptor tyrosine kinase LCK [PDB: 2PL0 (20)], which is similar to the IGF1R structure (10); (ii) a second tyrosine autophosphorylation site (Tyr 1166 ) in the activation loop of human IGF1R [PDB: 3LVP (21)]; (iii) a Tyr in the N-terminal juxtamembrane region of human colony stimulating factor 1 receptor (CSF1R) [PDB: 3LCD (22)] that is homologous to the tyrosine observed in the c-KIT complex (7); (iv) a Tyr in the N-terminal juxtamembrane region of Ephrin receptor A2 (EPHA2) [PDB: 4PDO (23)] that represents a phosphorylation site near but distinct from that in the c-KIT and CSF1R complexes; and (v) a Ser in the C-terminal tail of human CDC2/CDC28-like kinase 2 (CLK2) [PDB: 3NR9 (24)]. We also identified several additional structures of autophosphorylation complexes of PAK1: PDB: 4O0R, 4O0T (25); 4P90 (26); 4ZY4, 4ZY5, 4ZY6 (27); and 4ZLO, 4ZJI, and 4ZJJ (28).…”

Section: Introductionmentioning

confidence: 99%

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Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

Malecka

Fink

et al. 2015

Sci. Signal.

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Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets.

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“…Since our previous research established the enzyme-substrate reciprocal relationship between EphA2 and PTEN14, we treated EphA2 using PTEN phosphatase to obtain an unphosphorylated autoinhibited EphA2 structure. The data collection and refinement statistics are summarized in Table S1.…”

Section: Resultsmentioning

confidence: 99%

A new autoinhibited kinase conformation reveals a salt-bridge switch in kinase activation

Wei

Yang

et al. 2016

Sci Rep

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In the structure of autoinhibited EphA2 tyrosine kinase reported herein, we have captured the entire activation segment, revealing a previously unknown role of the conserved Arg762 in kinase autoinhibition by interacting with the essential Mg2+-chelating Asp757. While it is well known that this Arg residue is involved in an electrostatic interaction with the phospho-residue of the activation loop to stabilize the active conformation, our structure determination revealed a new role for the Arg, acting as a switch between the autoinhibited and activated conformations. Mutation of Arg762 to Ala in EphA2 sensitized Mg2+ response, resulting in enhanced kinase catalytic activity and Mg2+ cooperativity. Furthermore, mutation of the corresponding Arg/Lys to Ala in PKA and p38MAPK also exhibited similar behavior. This new salt bridge-mediated switch may thus be an important mechanism of activation on a broader scope for kinases which utilize autophosphorylation.

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Structures of an Eph receptor tyrosine kinase and its potential activation mechanism

Cited by 11 publications

References 33 publications

Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies

Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

A new autoinhibited kinase conformation reveals a salt-bridge switch in kinase activation

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