Activation of protein kinase C decreases equilibrative nucleobase transporter 1-mediated substrate uptake via phosphorylation of threonine 231

• Published in: Biochimica et biophysica acta. General subjects Vol. 1869; no. 3; p. 130765
• Main Authors: Ruel, Nicholas M., Hammond, James R.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2025
• Subjects: 6-mercaptopurine; Acute lymphoblastic leukemia; alanine; Animals; Biological Transport; drug therapy; drugs; Enzyme Activation; HEK293 Cells; Humans; kidneys; lymphocytic leukemia; Mercaptopurine - metabolism; Mercaptopurine - pharmacology; mutation; neoplasm cells; nucleobases; phosphorylation; Phosphorylation - drug effects; Protein kinase C; Protein Kinase C - metabolism; Tetradecanoylphorbol Acetate - pharmacology; threonine; Threonine - metabolism; toxicity; Transporter regulation; Transporter regulation; Protein kinase C; 6-mercaptopurine; Acute lymphoblastic leukemia
• DOI: 10.1016/j.bbagen.2025.130765
• ISSN: 0304-4165; 1872-8006

Protein kinase C (PKC) signalling has been shown to be dysregulated in various cancers including acute lymphoblastic leukemia (ALL). We have previously determined that changes in the expression levels of SLC43A3-encoded equilibrative nucleobase transporter 1 (ENBT1) can significantly alter 6-mercaptopurine (6-MP) toxicity in ALL cells. 6-MP is a common drug used in ALL chemotherapy. Furthermore, it has been reported that activation of PKC by phorbol 12-myristate 13-acetate (PMA) impacts nucleobase uptake via an ENBT1-like transporter in Lilly Laboratories Culture-Porcine Kidney 1 (LLC-PK1) cells. We hypothesized that activation of PKC would also alter ENBT1-mediated uptake of nucleobases in leukemia cell models. Using MOLT-4, SUP-B15, and K562 cells, we incubated the cells with PMA or its inactive isoform 4α-PMA for 30 min and determined changes to ENBT1-mediated substrate uptake. All of the cell lines tested showed decreased ENBT1-mediated substrate uptake when exposed PMA, relative to that observed using 4α-PMA. Pre-incubation with the broad-spectrum PKC inhibitor, Gö6983, reversed the decrease caused by PMA. Finally, to determine the residue responsible for this PKC-mediated effect, we transiently transfected HEK293 cells (which do not express endogenous ENBT1) with wild-type SLC43A3 transcript or constructs mutated to modify the predicted PKC sites in ENBT1. We found that the mutation of threonine 231 to alanine prevents the decrease in ENBT1-mediated uptake following incubation with PMA, suggesting its involvement. This study shows that activation of PKC decreases ENBT1-mediated uptake, suggesting that aberrant activation of PKC in ALL could decrease ENBT1-mediated 6-MP uptake potentially leading to decreased therapeutic efficacy. [Display omitted] •PKC activation decreases ENBT1-mediated nucleobase transport in leukemia cells.•Mutation of threonine 231 in ENBT1 abolishes this effect of PKC.•Enhanced PKC signalling in leukemia may impact therapy using nucleobase analogues.