Background: Metabolic reprogramming and redox homeostasis contribute to esophageal squamous cell carcinoma (ESCC). CDC-like kinase 4 (CLK4) is a dual-specificity kinase that can phosphorylate substrates' tyrosine or serine/threonine residue. However, the role and mechanism of CLK4 in ESCC remain unknown.
Methods: CLK4 expression was analysed using publicly available datasets and confirmed in ESCC tissues and cell lines. The biological roles of CLK4 were studied with gain and loss-of-function experiments. Mass spectrometry was employed to examine the effects of CLK4 on metabolic profiling. In vitro kinase assay, co-immunoprecipitation, glutathione S-transferase pulldown, chromatin immunoprecipitation and luciferase reporter were used to elucidate the relationship among CLK4, microphthalmia-associated transcription factor (MITF), COP1 and ZRANB1.
Results: CLK4 down-regulation was observed in ESCC cell lines and clinical samples and associated with the methylation of its promoter. Low levels of CLK4 promoted ESCC development by affecting the purine synthesis pathway and nicotinamide adenine dinucleotide phosphate (NADPH)/nicotinamide adenine dinucleotide phosphate (NADP+ ) ratio. Interestingly, CLK4 inhibited ESCC development by blocking MITF-enhanced de novo purine synthesis and redox balance. Mechanistically, wild type CLK4 (WT-CLK4) but not kinase-dead CLK4-K189R mutant phosphorylated MITF at Y360. This modification promoted its interaction with E3 ligase COP1 and its K63-linked ubiquitination at K308/K372, leading to sequestosome 1 recognition and autophagic degradation. However, the deubiquitinase ZRANB1 rescued MITF ubiquitination and degradation. In turn, MITF bound to E- rather than M-boxes in CLK4 promoter and transcriptionally down-regulated its expression in ESCC. Clinically, the negative correlations were observed between CLK4, MITF, and purine metabolic markers, which predicts a poor clinical outcome of ESCC patients. Notably, CLK4 itself was a redox-sensitive kinase, and its methionine oxidation at M307 impaired kinase activity, enhanced mitochondria length and inhibited lipid peroxidation, contributing to ESCC.
Conclusions: Our data highlight the potential role of CLK4 in modulating redox status and nucleotide metabolism, suggesting potential therapeutic targets in ESCC treatment.
Keywords: CDC-like kinase 4; esophageal squamous cell carcinoma; methionine oxidation; microphthalmia-associated transcription factor; purine synthesis.
© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.