Quantitative Phosphoproteomics Reveals the Requirement of DYRK1-Mediated Phosphorylation of Ion Transport- and Cell Junction-Related Proteins for Notochord Lumenogenesis in Ascidian

Abstract

The dual-specificity tyrosine phosphorylation-regulated kinase (DYRK1) phosphorylates diverse substrates involved in various cellular processes. Here, we found that blocking the kinase activity of DYRK1 inhibited notochord development and lumenogenesis in ascidian Ciona savignyi. By performing phosphoproteomics in conjunction with notochord-specific proteomics, we identified 1065 notochord-specific phosphoproteins that were present during lumen inflation, of which 428 differentially phosphorylated proteins (DPPs) were identified after inhibition of DYRK1 kinase activity. These DPPs were significantly enriched in metal ion transmembrane transporter activity, protein transport and localization, and tight junction. We next analyzed the downregulated phosphoproteins and focused on those belonging to the solute carrier (SLC), Ras-related protein (RAB), and tight junction protein (TJP) families. In vivo phospho-deficient study showed that alanine mutations on the phosphosites of these proteins resulted in defects of lumenogenesis during Ciona notochord development, demonstrating the crucial roles of phosphorylation of transmembrane transport-, vesicle trafficking-, and tight junction-related proteins in lumen formation. Overall, our study provides a valuable data resource for investigating notochord lumenogenesis and uncovers the molecular mechanisms of DYRK1-mediated notochord development and lumen inflation.

Keywords

• <i>Ciona</i> notochord
• phosphoproteomics
• proteomics
• lumen expansion
• vesicle trafficking
• transmembrane transport