Research ArticleBiochemistry

A redox-active switch in fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily

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Science Signaling  07 Jul 2020:
Vol. 13, Issue 639, eaax6313
DOI: 10.1126/scisignal.aax6313
  • Fig. 1 FN3K adopts a protein kinase fold.

    Comparison of the overall fold of FN3K from T. fusca (TfFN3K; PDB code: 3F7W), aminoglycoside phosphotransferase (APH; PDB code: 1L8T) (85), and protein kinase A (PKA; PDB code: 1ATP) (86). The structures are shown as a cartoon where the N-lobe is colored in light blue and the C-lobe in olive green. The substrates are shown as either sticks (ribuloselysine and kanamycin) or cartoons (serine peptide) and colored green. The oxygen atom on the hydroxyl group where the phosphate group is transferred is colored in red. The P-loop and activation loop are colored in red and blue, respectively.

  • Fig. 2 WT AtFN3K is a β strand exchange disulfide-mediated dimer.

    (A) Cartoon representation of the crystal structure of A. thaliana FN3K (AtFN3K) homodimer. The two disulfide bridges between two Cys32 and two Cys236 as well as the ADP molecules are shown as sticks. (B) Simulated annealing omit difference maps (Fo-Fc) calculated at 2.4-Å resolution and contoured at 4.5 RMSD. Maps were calculated after substituting both cysteines with alanine (top and middle panels) and removing the ADP molecule (bottom). (C) Top view of AtFN3K showing the β strand exchange.

  • Fig. 3 Comparison of the ATP and substrate-binding regions in AtFN3K with APH and PKA.

    (A) Comparison of the P-loop of AtFN3K with that of APH and PKA. PDB codes 1L8T and 1ATP were used for APH and PKA, respectively. Carbon atoms of ADP and ATP molecules are colored in black, and the oxygen atoms are colored in red. Chain A and chain B of AtFN3K are colored in slate and salmon, respectively. (B) Comparison of the substrate-binding lobe of AtFN3K with APH and PKA. Catalytic aspartate is shown as sticks with carbon atoms colored in magenta. The activation loop is colored in lemon. The APH substrate kanamycin is shown as lines with carbon atoms colored in magenta. The PKA peptide substrate is shown as ribbon and colored in black. The serine residue in the peptide is modeled and shown as lines. PDB codes were used as in (A). (C) Surface representation of AtFN3K. Chain A and chain B and ADP-associated carbons are colored as described in (A). The two disulfide bridges, C32-C32 and C236-C236, are indicated with sticks with sulfur atoms colored in yellow.

  • Fig. 4 Geometric analysis of the disulfides in AtFN3K.

    (A) Distances and dihedral values labeled for the disulfide bridges between Cys32-Cys32 and Cys236-Cys236 between chain A and chain B. The angle (Cβ-Sγ-Sγ) is shown with a green dashed line. The dihedrals are shown with dashed line colored in black. PyMOL version 2.3 (87) was used to calculate the distances and the dihedral angles. (B) Distribution of the Cα-Cα distance and χ3 angle (Cβ-Sγ-Sγ-Cβ) for PDB structures with a resolution of less than 1.5 Å. The data were retrieved from (64). Values for the Cys32-Cys32 and Cys236-Cys236 disulfides in AtFN3K are represented by the purple vertical lines.

  • Fig. 5 P-loop cysteine of AtFN3K (Cys32) is critical for the formation of disulfide-linked dimer species.

    (A) Nonreducing SDS-PAGE of WT and Cys-to-Ala mutant AtFN3K. Fifteen micrograms of protein was incubated with 1 mM DTT or 1 mM H2O2 for 20 min and then subjected to SDS-PAGE under nonreducing conditions. DS-S, disulfide-linked dimer; MRed, monomer reduced; MS-S, monomer with intramolecular disulfide. Blots are representative of three experiments. (B) Multiple sequence alignment of FN3K orthologs. Two additional cysteines (Cys196 and Cys222) specific to plant FN3Ks are shown. The alignment was generated using MUSCLE (67).

  • Fig. 6 Both WT and triple cysteine mutant (C32A/C236A/C196A) AtFN3K exist as two distinct species in solution, and the WT dimer is redox sensitive.

    (A) Size exclusion chromatography (SEC) of AtFN3K WT protein. Each fraction was 1 ml in volume. A, aggregates; D, dimer; M, monomer; A280, absorbance at 280 nm. (B) PK/LDH assay using 1 μg of protein to assess the activity of WT protein in the presence or absence of 2 mM DTT. Ribulose-N-α-Ac-lysine was used as the substrate. Data are means ± SE of six independent experiments. (C and D) As in (A) and (B), respectively, for the triple cysteine mutant protein.

  • Fig. 7 P-loop cysteine contributes to redox sensitivity in HsFN3K.

    (A and B) PK/LDH assays performed with 10.0 μg of HsFN3K (A) or 1.0 μg each of TfFN3K and L. plantarum FN3K (LpFN3K) (B). Proteins were incubated with buffer (0 mM DTT) or 2 mM DTT, and ribulose-N-α-Ac-lysine was used as the substrate. Data are means ± SE of three independent experiments. (C) Effect of different diamide concentrations on transfected Flag-tagged WT and C24A HsFN3K in HEK 293 cells. Total cell lysates were immunoblotted for Flag. Blot is representative of three experiments.

  • Fig. 8 Redox-sensitive metabolites are altered in HsFN3K knockout cells.

    (A) 1H NMR spectra of WT and FN3K knockout (FN3K-KO) HepG2 cells. Traces are the average for each group (WT, n = 10; FN3K-KO, n = 9). Insets highlight examples of regions containing annotated metabolites observed to be significantly different between cell lines. (B) Box and whisker plots of significant (false discovery rate, P < 0.05) metabolites annotated with highest confidence. Black points indicate outliers. Two-tailed t test was performed, and the P values were corrected for false discovery rate using the Benjamini-Hochberg method.

  • Fig. 9 Proposed redox feedback regulation of plant and mammalian FN3Ks.

    Cartoon showing the possible relationship between redox regulation of FN3K activity and its physiological function. The disulfide is colored in yellow.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/639/eaax6313/DC1

    Fig. S1. Hydrogen bonds and disulfide bridges between chain A and chain B in WT AtFN3K.

    Fig. S2. Oxidized monomeric species are an artifact of the SDS-PAGE gel.

    Fig. S3. WT and triple cysteine mutant (C32A/C236A/C196A) AtFN3K exist as two distinct species.

    Fig. S4. Reducing and nonreducing SDS-PAGE of dimer and monomer fractions of WT and triple cysteine mutant AtFN3K.

    Fig. S5. Effects of thiol reagents on the activity of WT and cysteine mutant AtFN3K.

    Fig. S6. P-loop cysteine (Cys24) is critical for the formation of disulfide-linked dimer in HsFN3K.

    Fig. S7. Western blot of HsFN3K KO in HepG2 cells.

    Fig. S8. WT and C32A/C236A AtFN3K localize to the nucleus.

    Fig. S9. Multiple sequence alignment showing the conservation of P-loop cysteine in selected human ePKs and FN3Ks.

    Fig. S10. FN3K and FN3KRP expression levels in human tumors.

    Fig. S11. Spectral data of ribulose-N-α-Ac-lysine.

    Table S1. Data collection and refinement statistics of WT AtFN3K.

    Table S2. Metabolites identified in extracts of WT HepG2 and FN3K-KO cells.

    References (88, 89)

  • This PDF file includes:

    • Fig. S1. Hydrogen bonds and disulfide bridges between chain A and chain B in WT AtFN3K.
    • Fig. S2. Oxidized monomeric species are an artifact of the SDS-PAGE gel.
    • Fig. S3. WT and triple cysteine mutant (C32A/C236A/C196A) AtFN3K exist as two distinct species.
    • Fig. S4. Reducing and nonreducing SDS-PAGE of dimer and monomer fractions of WT and triple cysteine mutant AtFN3K.
    • Fig. S5. Effects of thiol reagents on the activity of WT and cysteine mutant AtFN3K.
    • Fig. S6. P-loop cysteine (Cys24) is critical for the formation of disulfide-linked dimer in HsFN3K.
    • Fig. S7. Western blot of HsFN3K KO in HepG2 cells.
    • Fig. S8. WT and C32A/C236A AtFN3K localize to the nucleus.
    • Fig. S9. Multiple sequence alignment showing the conservation of P-loop cysteine in selected human ePKs and FN3Ks.
    • Fig. S10. FN3K and FN3KRP expression levels in human tumors.
    • Fig. S11. Spectral data of ribulose-N-α-Ac-lysine.
    • Table S1. Data collection and refinement statistics of WT AtFN3K.
    • Table S2. Metabolites identified in extracts of WT HepG2 and FN3K-KO cells.
    • References (88, 89)

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