Fig. 4: P4HA2 hydroxylates SUFU to regulate the Hedgehog signaling.

A, B The regulation of Hh signaling by P4HA2 is dependent on P4HA2 hydroxylase activity. HEK293T P4HA2 KO cells were expressed with wild-type P4HA2 (P4HA2) or hydroxylase dominant negative mutant (P4HA2-HDH). Cells were treated with (+) or without (−) 200 nM SAG. qRT-PCR analysis of GLI1 (A) and GLI2 (B) transcripts was performed using RNA isolated from the indicated cell lines. Data are shown as the mean ± SEM (n = 3). *P < 0.05, ***P < 0.001. All experiments were repeated three times independently. C–E The hydroxylation levels of GLI1 and SUFU decrease in P4HA2 knockout cells. P4HA2 KO and the vehicle cells were transfected with Flag-tagged KIF7, GLI1, and SUFU. Cell lysates were subjected to Flag IP and then analyzed by WB. Hydroxylated proteins were recognized by the Pan-hydroxylation antibody (OH). The relative quantification of Flag-KIF7 (C), GLI1 (D), and SUFU (E) hydroxylation level was analyzed and normalized. The values shown are the means of three independent experiments. F–H LC-MS/MS spectra of tryptic peptides. 3 tryptic peptides Pro 46 (F), Pro 134 (G), and Pro 249 (H) from SUFU. 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. I Validation of the proline 46, proline 134, and proline 249 hydroxylation sites of SUFU. P46A, P134A, P249A, and P46/134/249A proline to alanine mutants of SUFU were constructed and transfected into HEK293T cells. Cell lysates were subjected to Flag IP. The relative quantification of SUFU WT and mutant hydroxylation levels was analyzed and normalized. The values shown are the means of three independent experiments. J P4HA2 regulates the protein stability of SUFU in response to the Hh pathway activation. HEK293T P4HA2 KO cells were expressed with wild-type P4HA2 or a P4HA2 dominant negative mutant in hydroxylase activity (HDH). Cell lysates were analyzed by WB with anti-P4HA2 and GAPDH antibodies. K Hydroxylated SUFU regulate the Hh signaling. SUFU knockout (KO) NIH/3T3 and control cell lines were constructed and then infected with SUFU WT and mutant (SUFU P46/134/627 A) lentivirus. qRT-PCR analysis of GLI1 transcripts was performed using RNA isolated from the above cell lines. L SUFU KO NIH/3T3 and control cell lines were constructed and then infected with SUFU WT and mutant (SUFU P46/134/627 A) lentivirus. Utilized a dual luciferase reporter gene system, GLI1-Firefly and Renilla plasmids were overexpressed in above cell lines. Relative fold change of GLI1 luciferase activity was measured and normalized. Data are shown as the mean ± SEM (n = 3). ***P < 0.001, ****P < 0.0001. All experiments were repeated three times independently.