Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • It is not precisely known which of the

    2019-12-04

    It is not precisely known which of the intramolecular interactions created by the SH2 domain, the SH3–SH2 linker and the SH2–kinase linker are essential for the catalytic activity of Csk or how these interactions regulate Csk activity. Thus far it has been shown that site-directed mutagenesis of SH3–SH2 linker residues decreases catalytic activity of Csk, indicating the importance of the SH3–SH2 linker for Csk activity [9]. The SH2–kinase linker of Src plays a critical role in the inactivation of Src. The side chain of Leu255, within the SH2–kinase linker, must be inserted into a hydrophobic pocket on the surface of the N-terminal lobe of the kinase domain to inactivate Src [10]. Interestingly, both Csk and CHK have in their SH2–kinase linker a residue with a long hydrophobic side chain at a position corresponding to Src Leu255 (Fig. 2A). These amino FLAG Peptide mg residues are Phe183 (Csk) and Leu223 (CHK), respectively. In the crystal structure of active and inactive Csk molecules, Phe183 lies within a short α-helix, designated αBC, and makes contact with the αC-helix in the N-terminal lobe of the kinase domain (Fig. 1A,B). Therefore, it is possible that conserved hydrophobicity in the SH2–kinase linker of Csk plays a role in the stabilization of the active conformation of the αC-helix.
    Materials and methods
    Results
    Discussion In the crystal structure of inactive Src, Leu255 within the SH2–kinase linker binds in a hydrophobic pocket on the surface of the N-terminal lobe of the kinase domain [16]. The interaction between Leu255 and the kinase domain leads to the rotation of the αC-helix and disruption of a salt bridge between Glu310 and Lys295. As a result, catalysis is inhibited because the displaced Glu310 can no longer align the side chain of Lys295 to coordinate the phosphate groups of ATP [10]. In contrast to Src, the SH2–kinase linker of Csk directly interacts with the αC-helix (Fig. 1B). This suggests that the SH2–kinase linker could control the movement of the αC-helix and therefore the formation of an ion pair between Glu236 (Src Glu310) and Lys222 (Src Lys295) (Fig. 1B). The decreased activity of Csk ΔV177-Y184 supports the idea that the contact of the SH2–kinase linker with the kinase domain is required to sustain the catalytic activity of Csk. However, the observed activity of the Csk ΔV177-Y184 mutant does not give conclusive evidence for the SH2–kinase linker involvement in Csk activity. Deletion of the SH2–kinase linker residues not only eliminates contacts made by the deleted residues but also results in the misplacement of the potential intramolecular interactions N-terminal to the deletion. Therefore the loss of these connections could also contribute to the observed activity of the Csk ΔV177-Y194 mutant. Sequence alignment of Src and Csk SH2–kinase linkers reveals that Csk has a residue with a long hydrophobic side chain (Phe183) in a position corresponding to Src Leu255 (Fig. 2A). Phe183 lies within the αBC-helix and the crystal structure of Csk reveals a hydrophobic interaction of the Phe183 indole ring with the αC-helix (Fig. 1B). This contact suggests that the SH2–kinase linker interacts with the αC-helix via Phe183. Our studies confirm that the hydrophobic interaction between Phe183 and the αC-helix is required to maintain the catalytic activity of Csk. Deletion of the Phe183 indole ring resulted in decreased activity, but the activity could be restored with a residue possessing a long hydrophobic side chain (Leu or Trp). The observed activity of the F183A mutant suggests that the loss of contact between the SH2–kinase linker and the αC-helix leads to a greater degree of freedom in the movement of the αC-helix and therefore to loss of functional orientation of Glu236. Consequently, the restoration of the activity of the F183L and F183W mutants indicates that the long hydrophobic side chains of leucine and tryptophan can make a contact with the αC-helix and control the orientation of the αC-helix. These results demonstrate that the orientation of the αC-helix regulates the catalytic activity of Csk and a critical parameter controlling the orientation of αC-helix is a hydrophobic contact between Phe183 and the αC-helix.