During cell-matrix adhesion, both tyrosine and serine/ threonine kinases are activated. Integrin ligation correlates with tyrosine phosphorylation, whereas the later stages of spreading and focal adhesion and stress fiber formation in primary fibroblasts requires interactions of cell surface proteoglycan with heparin-binding moieties. This correlates with protein kinase C (PKC) activation, and PKC␣ can become localized to focal adhesions in normal, but not transformed, cells. PKC activation has been thought to be downstream of initial receptor-ligand interactions. We now show, however, that syndecan-4 transmembrane heparan sulfate proteoglycan and PKC co-immunoprecipitate and co-patch in vivo. The core protein of syndecan-4 can directly bind the catalytic domain of PKC␣ and potentiate its activation by phospholipid mediators. It can also directly activate PKC␣ in the absence of other mediators. This activity resides in the sequence LGKKPIYKK in the center of the short cytoplasmic domain, and other syndecans lack this sequence and PKC regulatory properties. Syndecan-4 is a focal adhesion component, and this interaction may both localize PKC and amplify its activity at sites of forming adhesions. This represents the first report of direct transmembrane signaling through cell surface proteoglycans.In cell-matrix interactions, as with many cellular responses to ligands, both tyrosine and serine/threonine kinases participate. During adhesion to fibronectin, integrin clustering activates tyrosine kinases (1-3), but we, and others, have noted that full spreading (4) and the formation of stress fibers and focal adhesions requires additional signals (3,(5)(6)(7)(8)(9)(10)(11)(12). Adhesion to the "cell-binding" 105-kDa fragment of fibronectin through ligation of integrin ␣ 5 ␤ 1 is sufficient only for attachment and spreading in anchorage-dependent primary fibroblasts in the absence of serum and protein synthesis (8 -11). To drive the cytoskeletal and receptor clustering that accompany the formation of stress fibers and focal adhesions, an additional activation of protein kinase C (PKC) 1 is needed (2,8,9). This can be provided by addition of heparin-binding fibronectin moieties (8 -11), either of the whole heparin-binding domain of fibronectin or of a peptide PRARI from this domain. These agents appear to signal through a cell surface heparan sulfate proteoglycan, since treatment with heparinase prevents the response (11), and mutant cells lacking (13), or having undersulfated cell surface heparan sulfate proteoglycans (14), have reduced capacity to form focal adhesions or stress fibers. Similarly, addition of PRARI peptides to synovial fibroblasts prespread on substrates of 105-kDa fragment markedly increased the size of vinculin-positive adhesion plaques (12). The need for addition of heparin-binding agents can be circumvented by treatment of cells with active, but not inactive, phorbol esters (8). In addition, there is downstream activation of the RhoA G-protein (15), which may initiate a contractile response (3).Syn...

The nontransmembrane protein tyrosine phosphatase SHP-2 plays a critical role in growth factor and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate 1 (SHPS- Complex processes such as cell growth, differentiation, and migration require the integration of multiple types of extracellular signals, including those delivered by growth factors, cytokines, and hormones (soluble signals), and solid-state signals, transmitted by cell-cell and cell-extracellular matrix (ECM) interactions. Most of these signaling pathways involve changes in cellular tyrosyl phosphorylation. Tyrosyl phosphorylation is regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Although many PTKs are implicated in signaling pathways for both soluble and solid-state signals, the roles of specific PTPs are less well defined.1Multiple reverse-genetic studies suggest that the nontransmembrane PTP SHP-2 is a required positive (i.e., signalenhancing) component of growth factor and cytokine signal transduction pathways (for reviews, see references 35, 56, and 63). Consistent with these studies, fibroblasts from mice containing a deletion of SHP-2 exon 3 (hereafter referred to as SHP-2 mutant mice), which express low levels of a defective SHP-2 protein that lacks its N-terminal SH2 domain (48), exhibit impaired mitogen-activated protein kinase (MAPK) activation in response to fibroblast growth factor (FGF), epidermal growth factor, and insulin-like growth factor I (48, 55). SHP-2 functions similarly in lower organisms. Dominant-negative SHP-2 blocks FGF-induced mesoderm induction in Xenopus ectodermal explants and completion of gastrulation in early embryos, leading to severe tail truncations (38, 57). Likewise, corkscrew (csw), the Drosophila SHP-2 homolog (39, 40), is required for multiple receptor tyrosine kinase (RTK) pathways involved in early development (e.g., Torso, Sevenless, Breathless, and Drosophila EGF receptor) (1,39,40), and the recently described Caenorhabditis elegans homolog, Ptp-2, is a component of the Let-23 pathway (13).The precise mechanism by which SHP-2 orthologs function, as well as their specific target(s), has remained unclear. SHP-2 binds directly to and may dephosphorylate some growth factor and cytokine receptors (for reviews, see references 35 and 63). In other pathways, however, SHP-2 binds to distinct signaling intermediates. One class of SHP-2 binding proteins, exemplified by the Drosophila daughter of sevenless (dos) gene product, consists of an N-terminal pleckstrin homology domain and multiple proline-rich stretches and potential tyrosyl phosphorylation sites (42). Dos is essential for Sevenless signaling (15,42) and may be a direct substrate for Csw (15). Mammalian cells express several groups of molecules with overall topology similar to that of Dos (for a review, see reference 63). including insulin receptor substrate family members (for a review, see reference 64), ...

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.

Non-alcoholic fatty liver disease (NAFLD) is currently one of the most common types of chronic liver injury. Elevated serum uric acid is a strong predictor of the development of fatty liver as well as metabolic syndrome. Here we demonstrate that uric acid induces triglyceride accumulation by SREBP-1c activation via induction of endoplasmic reticulum (ER) stress in hepatocytes. Uric acid-induced ER stress resulted in an increase of glucose-regulated protein (GRP78/94), splicing of the X-box-binding protein-1 (XBP-1), the phosphorylation of protein kinase RNA-like ER kinase (PERK), and eukaryotic translation initiation factor-2a (eIF-2a) in cultured hepatocytes. Uric acid promoted hepatic lipogenesis through overexpression of the lipogenic enzyme, acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1) via activation of SREBP-1c, which was blocked by probenecid, an organic anion transport blocker in HepG2 cells and primary hepatocytes. A blocker of ER stress, tauroursodeoxycholic acid (TUDCA), and an inhibitor of SREBP-1c, metformin, blocked hepatic fat accumulation, suggesting that uric acid promoted fat synthesis in hepatocytes via ER stress-induced activation of SREBP-1c. Uric acid-induced activation of NADPH oxidase preceded ER stress, which further induced mitochondrial ROS production in hepatocytes. These studies provide new insights into the mechanisms by which uric acid stimulates fat accumulation in the liver.

The transmembrane proteoglycan syndecan-4, which is a coreceptor with integrins in cytoskeleton-matrix interactions, appears to be multimerized in vivo. Both purified and recombinant core proteins form sodium dodecyl sulfate-resistant oligomers, and we now report that a synthetic peptide corresponding to the central region of syndecan-4 cytoplasmic domain (4V) also oligomerizes. The degree of oligomerization correlates with the previously reported ability to bind protein kinase C (PKC) and regulate its activity. Only multimeric recombinant syndecan-4 core protein, but not the monomeric protein, potentiated the activity of PKC␣, and only oligomeric syndecan-4 cytoplasmic peptides were active. Changes in peptide sequence caused parallel loss of stable oligomeric status and ability to regulate a mixture of PKC␣␤␥ activity. A synthetic peptide encompassing the whole cytoplasmic domain of syndecan-4 (4L) containing a membrane-proximal basic sequence did not form higher order oligomers and could not regulate the activity of PKC␣␤␥ unless induced to aggregate by phosphatidylinositol 4,5-bisphosphate. Oligomerization and PKC regulatory activity of the 4V peptide were both increased by addition of N-terminal cysteine and reduced by phosphorylation of the cysteine thiol group. Concentration of syndecan-4 at sites of focal adhesion formation may enhance multimerization and both localize PKC and potentiate its activity to induce stable complex formation.Extracellular matrix molecules such as fibronectin regulate many cellular processes through information encoded in the ligand-receptor interaction. Fibronectin has at least two distinct classes of cell-surface receptors: integrins and heparan sulfate proteoglycans. Integrins bind at several sites, but adhesion for many cell types is primarily through the classical RGD sequence in the tenth type III repeat of the molecule (1, 2). Clustering of specific integrins by either immobilized extracellular molecules or anti-integrin antibodies has many biological effects (reviewed in Refs. 3-6). It stimulates tyrosine phosphorylation (7, 8), elevates intracellular calcium (9), activates the Na ϩ /H ϩ antiporter (10), and activates phosphatidylinositol 4-phosphate 5-kinase (11) with cytoskeletal rearrangement (3, 12). Ligand-induced dimerization or oligomerization is a key event in transmembrane signaling by hormone or growth factor receptors with tyrosine kinase activity. This leads to an increase in tyrosine kinase activity, autophosphorylation of receptors, and the induction of diverse biological responses (13,14). Although integrins have no intrinsic tyrosine kinase activity, it is clear that their clustering is needed prior to subsequent tyrosine phosphorylation events (reviewed in Refs. 3-6). In addition, integrin-associated kinase(s) has been identified (15,16).Interactions between integrins and the cell-binding domain of fibronectin are only sufficient for attachment and spreading in normal primary fibroblasts (17), and the integrin ␣5␤1, which is the primary ligand for adhesio...

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is involved in the organization of the actin cytoskeleton by regulating actin-associated proteins. The transmembrane heparan sulfate proteoglycan syndecan-4 also plays a critical role in protein kinase C (PKC) signaling in the formation of focal adhesions and actin stress fibers. The cytoplasmic domain of syndecan-4 core protein directly interacts with and potentiates PKC␣ activity, and it can directly interact with the phosphoinositide PIP 2 . We, therefore, investigated whether the interaction of inositol phosphates and inositol phospholipids with syndecan-4 could regulate PKC activity. Data from in vitro kinase assays using purified PKC␣␤␥ show that in the absence of phosphatidylserine and diolein, PIP 2 increased the extent of autophosphorylation of PKC␣␤␥ and partially activated it to phosphorylate both histone III-S and an epidermal growth factor receptor peptide. This activity was dose-dependent, and its calcium dependence varied with PKC isotype/source. Addition of the cytoplasmic syndecan-4 peptide, but not equivalent syndecan-1 or syndecan-2 peptides, potentiated the partial activation of PKC␣␤␥ by PIP 2 , resulting in activity greater than that observed with phosphatidylserine, diolein, and calcium. This study indicates that syndecan-4 cytoplasmic domain may bind both PIP 2 and PKC␣, localize them to forming focal adhesions, and potentiate PKC␣ activity there.The control of cellular adhesion status is complex, involving several signaling mechanisms (1-4). Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 plays important roles in the organization of the actin cytoskeleton. PIP 2 may control actin polymerization by regulating the binding of actin-binding proteins such as profilin and gelsolin to actin (5, 6). PIP 2 may also interact with ␣-actinin and vinculin (7) and regulate their association with the cytoskeleton (8). The level of PIP 2 decreases upon detachment of cells from the substratum and increases upon reattachment to fibronectin (1). The difference in the levels of PIP 2 is probably due to different rates of phosphorylation of phosphatidyl 4-phosphate to PIP 2 by phosphatidylinositol 4-phosphate 5-kinase. Phosphatidylinositol 4-phosphate 5-kinase is stimulated 3-4-fold by adhesion of cells to fibronectin (1), probably through interactions with the small GTP-binding proteins Rac and Rho, the latter of which has also been implicated in the regulation of assembly of actin stress fibers and focal adhesions (9 -13).PIP 2 may enter several different pathways in signal transduction. It can be hydrolyzed by phospholipase C␥ to generate two intracellular messengers: inositol 1,4,5-triphosphate, which mobilizes Ca 2ϩ , and diacylglycerol, which is a physiological activator of protein kinase C (PKC). It can be further phosphorylated by phosphatidylinositol 3-kinase to generate phosphatidylinositol 3,4,5-triphosphate (PIP 3 ), which has been proposed to regulate numerous activities including cytoskeletal organization (14) and vesicle trafficking (15). PIP 2 c...

Objectives “Second victims” are defined as healthcare professionals whose wellness is influenced by adverse clinical events. The Second Victim Experience and Support Tool (SVEST) was used to measure the second-victim experience and quality of support resources. Although the reliability and validity of the original SVEST have been validated, those for the Korean tool have not been validated. The aim of the study was to evaluate the psychometric properties of the Korean version of the SVEST. Methods The study included 305 clinical nurses as participants. The SVEST was translated into Korean via back translation. Content validity was assessed by seven experts, and test-retest reliability was evaluated by 30 clinicians. Internal consistency and construct validity were assessed via confirmatory factor analysis. The analyses were performed using SPSS 23.0 and STATA 13.0 software. Results The content validity index value demonstrated validity; item- and scale-level content validity index values were both 0.95. Test-retest reliability and internal consistency reliability were satisfactory: the intraclass consistent coefficient was 0.71, and Cronbach α values ranged from 0.59 to 0.87. The CFA showed a significantly good fit for an eight-factor structure (χ2 = 578.21, df = 303, comparative fit index = 0.92, Tucker-Lewis index = 0.90, root mean square error of approximation = 0.05). Conclusions The K-SVEST demonstrated good psychometric properties and adequate validity and reliability. The results showed that the Korean version of SVEST demonstrated the extent of second victimhood and support resources in Korean healthcare workers and could aid in the development of support programs and evaluation of their effectiveness.

We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R2 and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R2 motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.