The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using timelapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.The dynamic regulation of integrin-mediated adhesions is important for cell migration in both normal and pathological processes, including tumor invasion and metastasis. Integrinmediated adhesions assemble at the leading edge of cells and establish a structural link between the extracellular matrix and the actin cytoskeleton. The correct spatial and temporal control of adhesion formation and disassembly at both the leading and trailing edge of cells is important for efficient cell migration. However, the mechanisms that regulate the turnover of adhesions in motile cells are still not well understood.Paxillin is an adaptor and phospho-protein that localizes to focal adhesions. Paxillin serves an important scaffolding function at focal adhesions by recruiting and binding to many signaling and structural proteins (1). First identified as a 68-kDa protein that was heavily phosphorylated in Src-transformed fibroblasts (2), Src-mediated phosphorylation of paxillin at Tyr-31 and Tyr-118 is important for focal adhesion disassembly at the leading edge of migrating cells (3). Paxillin expression and function is highly regulated by both alternative splicing and posttranslational modifications (4). An internal, alternative translation start site in paxillin is conserved in all species and produces the paxillin isoform known as paxillin delta (4), which functions as a paxillin antagonist that impairs cell migration (5).Paxillin can also be modified by calpain-mediated proteolysis (6, 7). Calpains are a family of calcium-dependent, intracellular cysteine proteases tha...
. Natural resistance to liver cold ischemia-reperfusion injury associated with the hibernation phenotype. Am J Physiol Gastrointest Liver Physiol 288: G473-G480, 2005; doi:10.1152/ajpgi.00223.2004.-The success of liver grafts is currently limited by the length of time organs are cold preserved before transplant. Novel insights to improve viability of cold-stored organs may emerge from studies with animals that naturally experience low body temperatures (T b) for extended periods. In this study, we tested whether livers from hibernating ground squirrels tolerate cold ischemia-warm reperfusion (cold I/R) for longer times and with better quality than livers from rats or summer squirrels. Hibernators were used when torpid (T b Ͻ 10°C) or aroused (Tb ϭ 37°C). Livers were stored at 4°C in University of Wisconsin solution for 0 -72 h and then reperfused with 37°C buffer in vitro. Lactate dehydrogenase (LDH) release after 60 min was increased 37-fold in rat livers after 72 h cold I/R but only 10-fold in summer livers and approximately three-to sixfold in torpid and aroused hibernator livers, despite twofold higher total LDH content in livers from hibernators compared with rats or summer squirrels. Reperfusion for up to 240 min had the least effect on LDH release in livers from hibernators and the greatest effect in rats. Compared with rats or summer squirrels, livers from hibernators after 72 h cold I/R showed better maintenance of mitochondrial respiration, bile production, and sinusoidal lining cell viability, as well as lower vascular resistance and Kupffer cell phagocytosis. These results demonstrate that the hibernation phenotype in ground squirrels confers superior resistance to liver cold I/R injury compared with rats and summer squirrels. Because hibernation-induced protection is not dependent on animals being in the torpid state, the mechanisms responsible for this effect may provide new strategies for liver preservation in humans. transplantation; cold storage; Kupffer cells; sinusoidal lining cells DESPITE MAJOR ADVANCES IN hypothermic preservation of organs before transplant, problems associated with extended cold ischemia-reperfusion (cold I/R) injury still limit the optimal use of transplantation as a therapeutic intervention for organ failure. For example, human livers can be successfully stored at 4°C for up to 12 h in University of Wisconsin (UW) solution, but graft failure is greatly increased after extended storage periods (14,33,34). In the rat liver transplant model, when livers are cold stored in UW for up to 24 h, 100% of the recipients survive (42); in contrast, without nutritional manipulation, none survive after receiving livers stored for 48 h (39).The damaging effects of cold I/R are evident on organ reperfusion with warmed, oxygenated blood, with damage increasing with the length of the cold ischemia period (10,35,40).A variety of pharmacological strategies has been explored to improve graft function after cold I/R to extend cold storage times and provide more flexibility in organ distribut...
a b s t r a c tMethods that do not require animal sacrifice to detect botulinum neurotoxins (BoNTs) are critical for BoNT antagonist discovery and the advancement of quantitative assays for biodefense and pharmaceutical applications. Here we describe the development and optimization of fluorogenic reporters that detect the proteolytic activity of BoNT/A, B, D, E, F, and G serotypes in real time with femtomolar to picomolar sensitivity. Notably, the reporters can detect femtomolar concentrations of BoNT/A in 4 h and BoNT/E in 20 h, sensitivity that equals that of animal-based methods. The reporters can be used to determine the specific activity of BoNT preparations with intra-and inter-assay coefficients of variation of approximately 10%. Finally, we find that the greater sensitivity of our reporters compared with those used in other commercially available assays makes the former attractive candidates for high-throughput screening of BoNT antagonists.Ó 2011 Elsevier Inc. All rights reserved.Botulinum neurotoxins (BoNTs), 1 produced by the bacteria of the genus Clostridium, are the most lethal substances known. The zinc-dependent endopeptidases act by entering neurons and cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and, thus, compromise the protein machinery responsible for neurotransmitter release [1][2][3][4][5][6][7][8]. Failure to promptly treat a victim of BoNT poisoning can result in flaccid paralysis, respiratory failure, or death [9]. The combination of extreme potency and a lack of medical treatments other than antitoxin administration and intensive care has made BoNT a biodefense priority requiring the discovery and development of assays to quantify toxins and to identify antagonists to counteract intoxication [10][11][12][13]. Despite their lethality, BoNTs are widely used for cosmetic and pharmaceutical applications due in part to their exquisite specificity for the neuromuscular junction. BoNTs provide relief of muscle tension and pain by inhibiting neurons that cause excessive muscle contractions. Therapeutic preparations of BoNT/A and B serotypes are Food and Drug Administration (FDA) approved for treating glabellar lines, strabismus, cervical dystonia, blepharospasm, cranial nerve VII disorders, and primary axillary hyperhidrosis. Dozens of ''off-label'' BoNT clinical applications have also been documented [14][15][16][17].The mouse bioassay, or lethality test, has been the standard for testing BoNT-containing samples for the past 30 years [18][19][20]. Government agencies use this method for testing food and serum samples for the presence of BoNT, whereas the pharmaceutical industry uses it for quality control and to quantify ''for human use'' BoNT preparations. The test is carried out by injecting mice intraperitoneally with approximately 0.5 ml of sample per mouse and recording the number of deaths over a 1-to 7-day period. The assay is very sensitive, with a detection limit of 5-10 pg for BoNT/A [21,22]. Results for the mouse bioassay are reported in...
Despite the growing body of evidence supporting prolactin (PRL) actions in human breast cancer, little is known regarding PRL regulation of its own receptor in these cells. Ligand-initiated endocytosis is a key process in the regulation of receptor availability and signaling cascades that may lead to oncogenic actions. Although exposure to exogenous PRL accelerates degradation of the long isoform of the PRL receptor (lPRLR), neither the signals initiated by PRL that lead to lPRLR internalization and subsequent down-regulation, nor the relationship to downstream pathways are understood in breast cancer cells. In this study, we showed that PRL-induced down-regulation of the lPRLR was reduced by inhibition of src family kinases (SFKs), but not Janus kinase 2, in MCF-7 cells. Inhibition of SFKs also resulted in accumulation of a PRL-induced PRLR fragment containing the extracellular domain, which appeared to be generated from newly synthesized PRLR. lPRLR was constitutively associated with SFKs in lipid rafts. PRL-induced SFK activation led to recruitment of the guanosine triphosphatase, dynamin-2, to an internalization complex, resulting in endocytosis. Inhibition of endocytosis by small interfering RNA-mediated knockdown of dynamin-2 blocked PRL-induced down-regulation of lPRLR, confirming that internalization is essential for this process. Endocytosis also was required for optimal phosphorylation of ERK1/2 and Akt, but not for Janus kinase 2 or signal transducer and activator of transcription 5, indicating that internalization selectively modulates signaling cascades. Together, these data indicate that SFKs are key mediators of ligand-initiated lPRLR internalization, down-regulation, and signal transduction in breast cancer cells, and underscore the importance of target cell context in receptor trafficking and signal transduction.
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