Fig. 5
PDHK1-241aa interacts with PPP1CA to inhibit AKT dephosphorylation and activate the AKT-mTOR signaling pathway. A Volcano plot of differentially expressed protein-coding genes following circPDHK1 interference and overexpression. B The RNA-seq analysis showed that relevant KEGG enrichment pathways following circPDHK1 interference and overexpression in Caki-1 cells. C GSEA results plotted to visualize correlations between circPDHK1 and AKT-mTOR pathway expression in Caki-1 cells. Western blot detection of proteins related to the AKT-mTOR pathway in (D) 8 pairs of ccRCC tissues and (E) Caki-1 and 786-O cells following circPDHK1 interference and overexpression as indicated. F Coimmunoprecipitation (co-IP) assays were used to verify the interaction between PDHK1-241aa and AKT or p-AKT (Thr308). G Total proteins of PDHK1-241aa-flag-transfected HEK293T cells separated by SDS‒PAGE. 9 interacting proteins of circPDHK1 were identified by LC‒MS and listed with their corresponding scores. The LC‒MS analysis showed that identification of PPP1CA in the PDHK1-241aa protein complex. H Co-IP used to verify binding between PPP1CA and PDHK1-241aa. I Immunofluorescence localization of PDHK1-241aa-flag and PPP1CA from overexpression vectors in Caki-1 and 786-O cells. The green (anti-flag) indicated the circPDHK1-3 × flag; The red(anti-PPP1CA) indicated the PPP1CA; The blue (hochest) indicated the nucleus. Bars = 20 μm. J Variations in PPP1CA and PDHK1-241aa localization verified by nuclear and cytoplasmic separation assays. K NCBI database (https://www.ncbi.nlm.nih.gov/cdd/) predicted that the PDHK1 domain is composed of BCDHK domain and HATPase domain, respectively. L Schematic illustrations of PDHK1 expression plasmids. M We also established truncation mutants BCDHK domain 3 × flag and HATPase domain 3 × flag plasmid. Immunoblot analysis following cotransfection with PDHK1-241aa and the indicated truncation mutants. N The specific binding between PDHK1-241aa and PPP1CA was predicted by PyMOL (https://pymol.org/2/) and HDOCK (http://hdock.phys.hust.edu.cn/). *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance