Phosphorylation of secreted proteins by a new family of kinases

Abstract

Protein kinases are evolutionarily conserved enzymes that transfer a molecule of phosphate from ATP to protein substrates in a process known as phosphorylation. In 1883, the secreted milk protein casein was shown to contain phosphorous. In hindsight, this was the first indication for the existence of protein kinases that were ultimately discovered nearly one‐century later using casein as the substrate. We now know that protein phosphorylation is a universal mechanism that regulates nearly every aspect of cellular life. Despite the fact that casein was identified as the first phosphoprotein, the responsible kinase had remained obscure. The enzymes classically referred to as “casein kinases” do not mediate the physiological phosphorylation of casein because they are nuclear and cytosolic proteins and do not encounter casein within the secretory pathway. Numerous other secreted proteins and peptide hormones are phosphorylated; however, the molecular identities of the kinases responsible for these modifications were unknown as well. As a consequence, research on extracellular protein phosphorylation has been largely undeveloped.We discovered a novel family of atypical secretory pathway kinases that phosphorylate secreted proteins. This new kinase family is so different from canonical kinases that it was not included as a branch on the human “kinome” tree. One member of this new family, Fam20C, is the Golgi casein kinase, an enzyme that escaped identification for many years. Fam20C contains a signal peptide that directs it to the lumen of the secretory pathway where it phosphorylates proteins on S‐x‐E/pS motifs. The importance of this discovery is highlighted by the fact that ~75% of human serum, plasma and cerebrospinal fluid phosphoproteins are phosphorylated within this motif.Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing and mass spectrometry, we identify more than 100 phosphoproteins as Fam20C substrates. Functional annotations of Fam20C substrates suggest roles for the kinase in a broad spectrum of biological processes, including cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.