Fig 2: Graphical abstract depicting the regulation of AKT activation by TLK1-AKTIP signaling, and thereby, CRPC progression of PCa cells by apoptotic prevention. TLK1 phosphorylation of AKTIP increases AKT phosphorylation at its regulatory sites. AKT phosphorylates BAD, Caspase9, and FoxO3 and inhibits their pro-apoptotic function. AKT can also activate mTORC1 which will phosphorylate 4EBP1 and release eIF4E. Excess eIF4E will increase the translation of TLK1B isoform, and hence, initiate a positive feed-forward loop.

Fig 3: TLK1 phosphorylates AKTIP at T22 and S237. (a) AKTIP phosphopeptide determination by MS in control AKTIP (top panel) and TLK1B+ AKTIP samples (bottom panel). The unique phosphorylated residues in TLK1B+ AKTIP samples are marked by red circles. (b) Peptides containing unique phosphorylated sites detected in the TLK1B+AKTIP samples with their corresponding MOWSE scores. (c) Spectral analysis of AKTIP phosphoresidues T22 and S237 determination by MS. (d,e) In vitro phosphorylation of wild-type and mutant GST-AKTIP proteins by TLK1B and densitometric quantitation of the bands relative to amounts, as determined by WB. (f) Graphical outline of AKTIP domain structure and location of the phosphorylated residues.

Fig 4: TLK1>AKTIP axis regulates activating AKT phosphorylations. (a) Incubation of LNCaP cell with charcoal-stripped serum (CSS), which elevates the expression of TLK1B, elevates the phosphorylation of AKT at T308 and S473, and this is suppressed with the TLK1 inhibitor Thioridazine (THD). Depletion of AKTIP largely suppresses AKT phosphorylation in both FCS and CSS conditions. (c) Depletion of AKTIP and addition of THD synergistically suppresses pAKT (S473) in both HEK 293 and LNCaP cells. (e) Treatment of LNCaP cells with two different inhibitors of TLK1 (THD or J54) results in dose-dependent loss of pAKT (S473). (g) Depletion of TLK1 with shRNA results in reduction of pAKT (S473). (b,d,f,h) Quantitation of the respective WBs.