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The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na(+) transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na(+)-K(+)-2Cl(-) cotransporter, NKCC2.
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
A largely unilamellar epithelial layer lines body cavities and organ ducts such as the digestive tract and kidney tubules. This polarized epithelium is composed of biochemically and functionally separate apical and basolateral surfaces. The epidermal growth factor receptor (EGFR) signaling pathway is a critical regulator of epithelial homeostasis and is perturbed in a number of epithelial disorders. It is underappreciated that in vivo EGFR signaling is most often initiated by cell-surface delivery and processing of one of seven transmembrane ligands, resulting in release of the soluble form that binds EGFR. In polarized epithelial cells, EGFR is restricted largely to the basolateral surface, and apical or basolateral ligand delivery therefore has important biological consequences. In vitro approaches have been used to study the biosynthesis, cell-surface delivery, proteolytic processing, and release of soluble EGFR ligands in polarized epithelial cells. We review these results, discuss their relevance to normal physiology, and demonstrate the pathophysiological consequences of aberrant trafficking. These studies have uncovered a rich diversity of apico-basolateral trafficking mechanisms among the EGFR ligands, provided insights into the pathogenesis of an inherited magnesium-wasting disorder of the kidney (isolated renal hypomagnesemia), and identified a new mode of EGFR ligand signaling via exosomes.
Exposure to extensive ultraviolet (UV) rays is a major cause of skin cancer, which is thought to be initiated by DNA mutations. Members of the epidermal growth factor receptor (EGFR) family are important in various pathophysiologic processes like cancer and are shown to be phosphorylated upon UV exposure. Here we show that EGFR phosphorylation by modest UV doses is dependent on metalloprotease activity and resultant epidermal growth factor (EGF) family proligand shedding. This proligand cleavage releases the mature ligand, which then binds to and activates EGFR. We show that UV induced EGFR phosphorylation in transformed cell lines of melanocyte and keratinocyte origin, which was reduced upon preincubation with a broad-spectrum metalloprotease inhibitor, BB94. UV also activated EGFR downstream signaling via Erk and Akt pathways in a BB94-sensitive manner. Furthermore, using neutralizing antibodies we found that proligand amphiregulin was required for UV-induced EGFR activation in SCC-9 cells. Using RNAi this EGFR activation was further shown to depend on the metalloproteases ADAM9 and ADAM17 in SCC-9 cells. cDNA array hybridization and RT-PCR analysis showed overexpression of a Disintegrin and a Metalloproteases (ADAMs) and EGF family proligands in melanoma cell lines. Additionally, blocking EGFR signal transactivation by BB94 led to increased apoptosis in UV-irradiated cells. EGFR signal transactivation also led to increased stability of the DNA repair protein, PARP, under UV stress. Thus, both antiapoptotic and DNA repair pathways are activated simultaneously by EGFR signal transactivation. Together, our data provide novel insights into the mechanism of UV-induced EGFR activation, suggesting broad relevance of the UV-ADAM-proligand-EGFR-Erk/Akt pathway and its significance in skin cancer.
Copyright (c) 2008 Wiley-Liss, Inc.
Cytokines affect a variety of cellular functions, including regulation of cell numbers by suppression of programmed cell death. Suppression of apoptosis requires receptor signalling through the activation of Janus kinases and the subsequent regulation of members of the B-cell lymphoma 2 (Bcl-2) family. Here we demonstrate that a Bcl-2-family-related protein, Hax1, is required to suppress apoptosis in lymphocytes and neurons. Suppression requires the interaction of Hax1 with the mitochondrial proteases Parl (presenilin-associated, rhomboid-like) and HtrA2 (high-temperature-regulated A2, also known as Omi). These interactions allow Hax1 to present HtrA2 to Parl, and thereby facilitates the processing of HtrA2 to the active protease localized in the mitochondrial intermembrane space. In mouse lymphocytes, the presence of processed HtrA2 prevents the accumulation of mitochondrial-outer-membrane-associated activated Bax, an event that initiates apoptosis. Together, the results identify a previously unknown sequence of interactions involving a Bcl-2-family-related protein and mitochondrial proteases in the ability to resist the induction of apoptosis when cytokines are limiting.
Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. To investigate possible roles for mTLL-1 in mammalian development, we have used gene targeting in ES cells to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants were embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that were apparently confined solely to the heart. Constant features were incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression was specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c. embryos, respectively. Tll1 expression was also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, was not observed. Cardiac structures that were not affected in Tll1-/- embryos either showed no Tll1 expression (atrio-ventricular cushions) or showed overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum.