Interaction of TLK1 and AKTIP as a Potential Regulator of AKT Activation in Castration-Resistant Prostate Cancer Progression

Abstract

Prostate cancer (PCa) progression is characterized by the emergence of resistance to androgen deprivation therapy (ADT). AKT/PKB has been directly implicated in PCa progression, often due to the loss of PTEN and activation of PI3K>PDK1>AKT signaling. However, the regulatory network of AKT remains incompletely defined. Here, we describe the functional significance of AKTIP in PCa cell growth. AKTIP, identified in an interactome analysis as a substrate of TLK1B (that itself is elevated following ADT), enhances the association of AKT with PDK1 and its phosphorylation at T308 and S473. The interaction between TLK1 and AKTIP led to AKTIP phosphorylation at T22 and S237. The inactivation of TLK1 led to reduced AKT phosphorylation, which was potentiated with AKTIP knockdown. The TLK1 inhibitor J54 inhibited the growth of the LNCaP cells attributed to reduced AKT activation. However, LNCaP cells that expressed constitutively active, membrane-enriched Myr-AKT (which is expected to be active, even in the absence of AKTIP) were also growth-inhibited with J54. This suggested that other pathways (like TLK1>NEK1>YAP) regulating proliferation are also suppressed and can mediate growth inhibition, despite compensation by Myr-AKT. Nonetheless, further investigation of the potential role of TLK1>AKTIP>AKT in suppressing apoptosis, and conversely its reversal with J54, is warranted.

Keywords: AKTIP/FT1/FTS; PKB/AKT; TLK1; apoptosis; castration-resistant prostate cancer; prostate cancer.

Figure 1

Interaction of TLK1 and AKTIP. (a) In vitro pull-down of recombinant His-TLK1B and GST-AKTIP, isolated on Ni-NTA Sepharose; 1 µg of each protein were allowed to bind to each other and to the beads, followed by extensive washing and WB for AKTIP. (b) Ni-NTA Sepharose pull-down of endogenous AKTIP from HEK 293 cell extract, incubated with or without recombinant His-TLK1B. AKTIP pull-down was determined by WB. (c) Co-IP of TLK1 and AKTIP form extracts of LNCaP and HEK 293 cells using TLK1 antiserum, followed by WB for AKTIP. (d) Co-IP of AKTIP using AKTIP specific antibody from LNCaP cell extracts with or without THD treatment and sequential immunoblotting of AKT and AKTIP.

Figure 2

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.

Figure 2

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.

Figure 2

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.

Figure 3

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.

Figure 3

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.

Figure 4

TLK1 regulation of cell proliferation is mediated through AKT activation as well as other pathways. (a) Proliferation rates of LNCaP and Myr-AKT expressing LNCaP cells with or without two different concentrations of the TLK1 inhibitor J54 treatment. The growth curves with standard error of mean (SEM) were obtained by plotting confluence percentage over time using IncuCyte. (b) Cell lysates were prepared from LNCaP and Myr-AKT expressing LNCaP cells treated with two different concentrations of J54 for two different time points. WB was conducted to determine the level of pAKT S473, total AKT, pNEK1 T141, total NEK1, PCNA, PARP, and cleaved PARP. GAPDH was used as a loading control.

Figure 4

TLK1 regulation of cell proliferation is mediated through AKT activation as well as other pathways. (a) Proliferation rates of LNCaP and Myr-AKT expressing LNCaP cells with or without two different concentrations of the TLK1 inhibitor J54 treatment. The growth curves with standard error of mean (SEM) were obtained by plotting confluence percentage over time using IncuCyte. (b) Cell lysates were prepared from LNCaP and Myr-AKT expressing LNCaP cells treated with two different concentrations of J54 for two different time points. WB was conducted to determine the level of pAKT S473, total AKT, pNEK1 T141, total NEK1, PCNA, PARP, and cleaved PARP. GAPDH was used as a loading control.

Figure 5

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.