Mining of the literature and high-throughput mass spectrometry data from both healthy and diseased tissues and from body fluids reveals evidence that various extracellular proteins can exist in phosphorylated states. Extracellular kinases and phosphatases (ectokinases and ectophosphatases) are active in extracellular spaces during times of sufficiently high concentrations of adenosine triphosphate. There is evidence for a role of extracellular phosphorylation in various physiological functions, including blood coagulation, immune cell activation, and the formation of neuronal networks. Ectokinase activity is increased in some diseases, including cancer, Alzheimer's disease, and some microbial infections. We summarize the literature supporting the physiological and pathological roles of extracellularly localized protein kinases, protein phosphatases, and phosphorylated proteins and provide an analysis of the available mass spectrometry data to annotate potential extracellular phosphorylated proteins.

SummaryRecent advances in extracellular signaling suggest that extracellular protein phosphorylation is a regulatory mechanism outside the cell. The list of reported active extracellular protein kinases and phosphatases is growing, and phosphorylation of an increasing number of extracellular matrix molecules and extracellular domains of trans-membrane proteins is being documented. Here, we use public proteomic databases, collagens – the major components of the extracellular matrix, extracellular signaling molecules and proteolytic enzymes as examples to assess what the roles of extracellular protein phosphorylation may be in health and disease. We propose that novel tools be developed to help assess the role of extracellular protein phosphorylation and translate the findings for biomedical applications. Furthermore, we suggest that the phosphorylation state of extracellular matrix components as well as the presence of extracellular kinases be taken into account when designing translational medical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0482-4) contains supplementary material, which is available to authorized users.

While small-molecule kinase inhibitors became the most prominent anticancer drugs, novel combinatorial strategies need to be developed as the fight against cancer is not yet won. We review emerging literature showing that the release of several ectokinases is significantly upregulated in body fluids from cancer patients and that they leave behind their unique signatures on extracellular matrix (ECM) proteins. Our analysis of proteomic data reveals that fibronectin is heavily phosphorylated in cancer tissues particularly within its growth factor binding sites and on domains that regulate fibrillogenesis. We are thus making the case that cancer is not only a disease of cells but also of the ECM. Targeting extracellular kinases or the extracellular signatures they leave behind might thus create novel opportunities in cancer diagnosis as well as new avenues to interfere with cancer progression and malignancy.

A large number of extracellular matrix proteins have been found in phosphorylated states, yet little is known about how the phosphorylation of extracellular matrix proteins might affect cell functions. We thus tested the hypothesis whether the phosphorylation of fibronectin, a major adhesion protein, affects cell behavior. Controlled in vitro phosphorylation of fibronectin by a casein kinase II (CKII) significantly upregulated cell traction forces and total strain energy generated by fibroblasts on nanopillar arrays, and consequently other elementary cell functions including cell spreading and metabolic activity. Mass spectrometry of plasma fibronectin from healthy human donors then identified a constitutively phosphorylated site in the C-terminus, and numerous other residues that became phosphorylated by the CKII kinase in vitro. Our findings open up novel strategies for translational applications including targeting diseased ECM, or to develop assays that probe the phosphorylation state of the ECM or blood as potential cancer markers.