Comprehensive Analysis of the Putative Substratome of FAM20C, the Master Serine Kinase of the Secretory Pathway

TL;DR

This study identifies a large set of proteins phosphorylated by FAM20C, a key kinase in the secretory pathway, and shows its role in important biological processes like blood clotting and immunity.

Contribution

The study provides the most comprehensive FAM20C substratome to date, revealing new substrates and biological functions.

Findings

FAM20C phosphorylates 443 phosphosites across 256 proteins, with 77% previously unlinked to FAM20C.

FAM20C is strongly associated with collagen fibril organization, complement activation, and blood coagulation.

The FAM20C consensus sequence is distinct from other acidophilic kinases, confirming its unique role in the secretory pathway.

Abstract

FAM20C, previously known as Golgi casein kinase (GCK), is a serine/threonine kinase localized to the Golgi apparatus and classified within the acidophilic kinase family. Its phosphorylation motif is characterized by a glutamic acid residue at the +2 position relative to the target site. Before its molecular identity was established, analysis of a limited number of phosphosites in secreted proteins showed that around 70% matched the GCK consensus sequence, suggesting that GCK is the principal kinase for secreted proteins. Following the identification of GCK as FAM20C, the generation of FAM20C knockout cell lines and phosphoproteomic data confirmed its role: approximately 80% of serine/threonine phosphosites in the secretome of two different human cell lines were shown to depend on FAM20C. In this study, comparative analysis of in vitro phosphorylation datasets from a broad panel of recombinant Ser/Thr kinases confirmed that the FAM20C consensus sequence is distinct from those of other acidophilic kinases. Examination of experimentally identified human phosphosites within the secretory pathway revealed strong conservation of the FAM20C consensus, firmly establishing this enzyme as the master Ser kinase of the entire pathway. From this dataset, we defined the putative FAM20C substratome, comprising 443 phosphosites across 256 proteins, ~77% of which had not been previously linked to FAM20C. This represents the most extensive FAM20C substratome to date and a valuable resource for functional studies. Notably, enrichment analysis highlights strong links between FAM20C and major extracellular pathways, including collagen fibril organization, complement activation, and blood coagulation, underscoring an underappreciated role for this kinase in regulating hemostasis and innate immunity.

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