TRIM27 cooperates with STK38L to inhibit ULK1-mediated autophagy and promote tumorigenesis (2022)
Figure 5

Fig 5. TRIM27-mediated ubiquitination activates STK38L to phosphorylate ULK1.

A. Western blot analysis of ULK1 levels in HeLa cells stably expressing Flag-TRIM27, the cells were transfected with either Myc-vector, Myc-STK38L, or Myc-STK38L-K119A. Then the cells were treated with starvation before lysis.

B. Phosphorylation of HA-ULK1 in HeLa STK38L knockout cells with or without expression of Flag-STK38L. HA-ULK1 was immunoprecipitated and then analyzed by western blot using anti-phosphoserine or anti-phosphothreonine, and the cell lysates were analyzed using anti-HA and anti-Myc.

C. Co-immunoprecipitation of Flag-TRIM27 with HA-ULK1 or HA-ULK1-S495A in HEK293T cells. HA-ULK1 or HA-ULK1 S495A was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with anti-HA or anti-Flag.

D. Ubiquitination analysis of HA-ULK1 WT or HA-ULK1 S495A in HEK293T cells co-expressing Flag-TRIM27 and His-Ub.

E. Phosphorylation of HA-ULK1 in HEK293T cells co-expressing Myc-STK38L with or without CIP treatment. HA-ULK1 was immunoprecipitated with anti-HA and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).

F. Phosphorylation of HA-ULK1 or HA-ULK1 S495A in HEK293T cells co-expressing Myc-STK38L. HA-ULK1 or HA-ULK1 S495A was immunoprecipitated with anti-HA and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).

G. In vitro kinase assay using GST-STK38L, or GST-STK38L K119A (a kinase-inactive mutant) and HA-ULK1 as substrate. GST-STK38L or GST-STK38L K119A was pulled down using glutathione Sepharose beads. HA-ULK1 was immunoprecipitated from HEK293T using anti-HA and treated with lambda phosphatase, the reaction product was analyzed by western blot using p-ULK1-S495 (Ser495).

H. Ubiquitination analysis of Myc-STK38L in HEK293T cells co-expressing Flag-TRIM27 and His-Ub.

I. Ubiquitination analysis of Flag-STK38L in HEK293T cells co-expressing His-Ub with or without TRIM27 knockdown, cells were cultured in fed or starved conditions as indicated.

J. Ubiquitination analysis of Flag-STK38L in HEK293T cells co-expressing Myc-TRIM27 with either His-Ub (wild-type) or His-Ub K6R.

K. Ubiquitination analysis of Myc-STK38L WT, Myc-STK38L-K432R+K434R or Myc-STK38L-K181R+K215R+K224R+K432R+K434R (Myc-STK38L-5KR) mutant in HEK293T cells co-expressing HA-TRIM27 and His-Ub.

L. Ubiquitination analysis of Myc-STK38L K1-38R mutant (All lysines were mutated to arginines), or Myc-STK38L-R181/215/224/432/434K (All lysines were mutated to arginines except the indicated five lysine residues) mutant in HEK293T cells co-expressing HA-TRIM27 and His-Ub.

M. Phosphorylation of HA-ULK1 S495(Ser495) in HEK293T cells co-expressing Myc-STK38L WT, or Myc-STK38L-5KR mutant in K. The cells were cultured in fed or starved conditions as indicated and HA-ULK1 was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-Myc, anti-HA, or anti-actin.

N. Phosphorylation of HA-ULK1 in HeLa cells with or without TRIM27-knockout, cells were cultured in fed or starved conditions as indicated, HA-ULK1 was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-HA, or anti-actin.