The collectrin-like part of the SARS-CoV-1 and -2 receptor ACE2 is shed by the metalloproteinases ADAM10 and ADAM17.

The transmembrane protease angiotensin converting enzyme 2 (ACE2) is a protective regulator within the renin angiotensin system and additionally represents the cellular receptor for SARS-CoV. The release of soluble ACE2 (sACE2) from the cell surface is hence believed to be a crucial part of its (patho)physiological functions, as both, ACE2 protease activity and SARS-CoV binding ability, are transferred from the cell membrane to body fluids. Yet, the molecular sources of sACE2 are still not completely investigated. In this study, we show different sources and prerequisites for the release of sACE2 from the cell membrane. By using inhibitors as well as CRISPR/Cas9-derived cells, we demonstrated that, in addition to the metalloprotease ADAM17, also ADAM10 is an important novel shedding protease of ACE2. Moreover, we observed that ACE2 can also be released in extracellular vesicles. The degree of either ADAM10- or ADAM17-mediated ACE2 shedding is dependent on stimulatory conditions and on the expression level of the pro-inflammatory ADAM17 regulator iRhom2. Finally, by using structural analysis and in vitro verification, we determined for the first time that the susceptibility to ADAM10- and ADAM17-mediated shedding is mediated by the collectrin-like part of ACE2. Overall, our findings give novel insights into sACE2 release by several independent molecular mechanisms.

Distance dependent shedding of IL-6R.

Proteolytic processing of membrane proteins by A disintegrin and metalloprotease-17 (ADAM17) is a key regulatory step in many physiological and pathophysiological processes. This so-called shedding is essential for development, regeneration and immune defense. An uncontrolled ADAM17 activity promotes cancer development, chronic inflammation and autoimmune diseases. Consequently, the ADAM17 activity is tightly regulated. As a final trigger for the shedding event a phosphatidylserine (PS) flip to the outer leaflet of the cell membrane was recently described. PS interacts with the extracellular part of ADAM17, which results in the shedding event by shifting the catalytic domain towards the membrane close to the cleavage sites within ADAM17 substrates. Our data indicate that the intrinsic proteolytic activity of the catalytic domain is prerequisite for the shedding activity and constantly present. However, the accessibility for substrate cleavage sites is controlled on several levels. In this report, we demonstrate that the positioning of the catalytic domain towards the cleavage sites is a crucial part of the shedding process. This finding contributes to the understanding of the complex and multilayered regulation of ADAM17 at the cell surface.

Splice variants of the dual specificity tyrosine phosphorylation-regulated kinase 4 (DYRK4) differ in their subcellular localization and catalytic activity.

Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.

Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes.

BACKGROUND: Although numerous non-radioactive methods are in use to measure the catalytic activity of protein kinases, most require specialized equipment and reagents and are not sufficiently sensitive for the detection of endogenous kinase activity in biological samples. Kinases of the DYRK family have important functions in developmental and pathophysiological processes in eukaryotic organisms including mammals. We aimed to develop a highly sensitive, low-tech assay suitable to determine the activity of DYRK family kinases in tissues or cells from diverse sources. RESULTS: Phosphorylation-site specific antibodies can be used to monitor the accumulation of the phosphorylated product in kinase assays. We present a modified configuration of an enzyme-linked immunosorbent assay (ELISA)-based kinase assay by using the phosphospecific antibody as the capture antibody. This assay format allowed the detection of small amounts of phosphopeptide in mixtures with an excess of the unphosphorylated substrate peptide (10 fmol phosphorylated peptide over a background of 50 pmol unphosphorylated peptide). Consequently, low substrate turnover rates can be determined. We applied this method to the measurement of endogenous DYRK1A activity in mouse heart tissue by immunocomplex kinase assay. Furthermore, we detected DYRK1-like kinase activity in Xenopus laevis oocytes and identified this kinase as a DYRK1 isoform distinct from the Xenopus DYRK1A ortholog. CONCLUSION: We present a non-radioactive and highly sensitive method for the measurement of endogenous activities of DYRKs in biological samples. Xenopus laevis oocytes contain an active DYRK1-related protein kinase more similar to mammalian DYRK1B than DYRK1A.