Figure 3.
Figure 3. P4HA2 hydroxylates Carabin on Pro306. (A-B) MS/MS spectra of 2 tryptic peptides (A, Pro116; B, Pro306) derived from MS/MS spectra of 2 tryptic peptides derived from Carabin. The b ion and y ion are fragment ions of tryptic peptide in tandem mass spectrometry. The x and y axes represent m/z and relative ion intensity, respectively. (C) P306A mutant of Carabin binds weakly to P4HA2. 293T cells were transfected with WT Carabin or mutant Flag Carabin as shown. Endogenous P4HA2 was detected in Flag-immunoprecipitated complexes by anti-P4HA2 antibody. (D) Diminished hydroxylation of P306A Carabin mutant. 293T cells were cotransfected with V5-P4HA2 and the illustrated plasmids. As shown, cell sample was treated with EDHB (200 μM for 12 hours). The Flag-immunoprecipitates were resolved by SDS-PAGE. Specific hydroxylation antibody (Hy-OH) for the substrates of hydroxylases was used to reveal the hydroxylation of Flag-Carabin. (E) P306A mutant of Carabin is stable. 293T cells were cotransfected with plasmids as shown. Cell lysates were determined by WB. (F) Carabin can be polyubiquitinated. 293T cells were transfected as shown. Before harvest, cells were treated with MG132 (5 µM for 8 hours). Cell lysates were incubated with Flag beads. Then, the immunoprecipitates were resolved by SDS-PAGE. The polyubiquitinated forms of GST-Carabin were detected by WB with anti-hemagglutinin (HA) antibody. (G) Ubiquitination of Carabin is dependent on the hydroxylation in vitro. Purified GST-Carabin proteins were combined with hydroxylate substrates and His-Ub as shown. After His pulldown, complexes were analyzed by WB. (H) Declined ubiquitination of the P306A Carabin mutant. 293T cells were cotransfected as shown. After MG132 treatment (5 μM for 8 hours), cell lysates were prepared and subjected to immunoprecipitation with Flag beads or GST beads, respectively. The immunoprecipitates were analyzed by WB with anti-Flag and anti-GST antibodies. (I) Growth of SU-DHL-6 cell expressed P306A mutant of Carabin is slow. Overexpression of WT or mutant Carabin in SU-DHL-6 cells was carried out by lentivirus and detected by WB. Cell growth assay was performed as depicted in Figure 1B. Points, mean (n = 6); bar, SD; *P < .05, ***P < .001. (J) SU-DHL-6 cell-expressed P306A mutant of Carabin has the lowest kinase activity of Erk. Cells depicted as in panel I were stimulated with PMA (40 ng/mL) for 10 min (+) or not (−). Cell lysates were analyzed using an anti-phospho Erk antibody. Erk were used as a loading marker.

P4HA2 hydroxylates Carabin on Pro306. (A-B) MS/MS spectra of 2 tryptic peptides (A, Pro116; B, Pro306) derived from MS/MS spectra of 2 tryptic peptides derived from Carabin. The b ion and y ion are fragment ions of tryptic peptide in tandem mass spectrometry. The x and y axes represent m/z and relative ion intensity, respectively. (C) P306A mutant of Carabin binds weakly to P4HA2. 293T cells were transfected with WT Carabin or mutant Flag Carabin as shown. Endogenous P4HA2 was detected in Flag-immunoprecipitated complexes by anti-P4HA2 antibody. (D) Diminished hydroxylation of P306A Carabin mutant. 293T cells were cotransfected with V5-P4HA2 and the illustrated plasmids. As shown, cell sample was treated with EDHB (200 μM for 12 hours). The Flag-immunoprecipitates were resolved by SDS-PAGE. Specific hydroxylation antibody (Hy-OH) for the substrates of hydroxylases was used to reveal the hydroxylation of Flag-Carabin. (E) P306A mutant of Carabin is stable. 293T cells were cotransfected with plasmids as shown. Cell lysates were determined by WB. (F) Carabin can be polyubiquitinated. 293T cells were transfected as shown. Before harvest, cells were treated with MG132 (5 µM for 8 hours). Cell lysates were incubated with Flag beads. Then, the immunoprecipitates were resolved by SDS-PAGE. The polyubiquitinated forms of GST-Carabin were detected by WB with anti-hemagglutinin (HA) antibody. (G) Ubiquitination of Carabin is dependent on the hydroxylation in vitro. Purified GST-Carabin proteins were combined with hydroxylate substrates and His-Ub as shown. After His pulldown, complexes were analyzed by WB. (H) Declined ubiquitination of the P306A Carabin mutant. 293T cells were cotransfected as shown. After MG132 treatment (5 μM for 8 hours), cell lysates were prepared and subjected to immunoprecipitation with Flag beads or GST beads, respectively. The immunoprecipitates were analyzed by WB with anti-Flag and anti-GST antibodies. (I) Growth of SU-DHL-6 cell expressed P306A mutant of Carabin is slow. Overexpression of WT or mutant Carabin in SU-DHL-6 cells was carried out by lentivirus and detected by WB. Cell growth assay was performed as depicted in Figure 1B. Points, mean (n = 6); bar, SD; *P < .05, ***P < .001. (J) SU-DHL-6 cell-expressed P306A mutant of Carabin has the lowest kinase activity of Erk. Cells depicted as in panel I were stimulated with PMA (40 ng/mL) for 10 min (+) or not (−). Cell lysates were analyzed using an anti-phospho Erk antibody. Erk were used as a loading marker.

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