SummaryWnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding -barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.

The type II transmembrane multidomain serine proteinase MT-SP1/matriptase is highly expressed in many human cancer-derived cell lines and has been implicated in extracellular matrix re-modeling, tumor growth, and metastasis. We have expressed the catalytic domain of MT-SP1 and solved the crystal structures of complexes with benzamidine at 1.3 Å and bovine pancreatic trypsin inhibitor at 2.9 Å. MT-SP1 exhibits a trypsinlike serine proteinase fold, featuring a unique nine-residue 60-insertion loop that influences interactions with protein substrates. The structure discloses a trypsinlike S1 pocket, a small hydrophobic S2 subsite, and an open negatively charged S4 cavity that favors the binding of basic P3/P4 residues. A complementary charge pattern on the surface opposite the active site cleft suggests a distinct docking of the preceding low density lipoprotein receptor class A domain. The benzamidine crystals possess a freely accessible active site and are hence well suited for soaking small molecules, facilitating the improvement of inhibitors. The crystal structure of the MT-SP1 complex with bovine pancreatic trypsin inhibitor serves as a model for hepatocyte growth factor activator inhibitor 1, the physiological inhibitor of MT-SP1, and suggests determinants for the substrate specificity.The activity of proteolytic enzymes is required at multiple stages during the growth, invasion, and progression of human tumors (for a review, see Ref. 1). For example, these complex processes entail extensive re-modeling of the extracellular matrix as well as the activation of latent growth factors and pro-angiogenic proteins. Consequently, the high level expression of particular proteinases often correlates with poor patient survival for several different cancers (see, for instance, Ref. The enzyme was initially assigned as a gelatinase, because of its gelatinolytic properties and gelatinase-like molecular weight. However, isolation and sequencing of the cDNA revealed a 683-residue multidomain proteinase with a C-terminal serine proteinase domain. The enzyme was then named matriptase to emphasize its matrix degrading properties and trypsin-like specificity (5). Independently, Takeuchi and coworkers (6) cloned and characterized a type-II membranebound trypsin-like serine proteinase from a human prostatic cancer cell line, which they called membrane-type serine proteinase 1, MT-SP1. This 855-residue proteinase contained two tandem repeats of the complement component C1r/s domain (CUB, derived from complement factor/1R-urchin embryonic growth factor/bone morphogenetic protein) and four tandem repeats of the low density lipoprotein receptor (LDLR) class A domain between the N-terminal transmembrane signal anchor and the C-terminal catalytic domain (5, 6). Because the matriptase sequence reported by Lin turned out to be part of the translated MT-SP1 cDNA sequence, matriptase is likely to be a form of MT-SP1 produced by ectodomain shedding (7). Alternatively, the two cDNAs may result from alternative splicing. MT-SP1 is highly exp...

The Wnt/b-catenin signaling pathway is critical in both cellular proliferation and organismal development. However, how the b-catenin degradation complex is inhibited upon Wnt activation remains unclear. Using a directed RNAi screen we find that protein phosphatase 1 (PP1), a ubiquitous serine/threonine phosphatase, is a novel potent positive physiologic regulator of the Wnt/bcatenin signaling pathway. PP1 expression synergistically activates, and inhibition of PP1 inhibits, Wnt/b-catenin signaling in Drosophila and mammalian cells as well as in Xenopus embryos. The data suggest that PP1 controls Wnt signaling through interaction with, and regulated dephosphorylation of, axin. Inhibition of PP1 leads to enhanced phosphorylation of specific sites on axin by casein kinase I. Axin phosphorylation markedly enhances the binding of glycogen synthase kinase 3, leading to a more active b-catenin destruction complex. Wnt-regulated changes in axin phosphorylation, mediated by PP1, may therefore determine b-catenin transcriptional activity. Specific inhibition of PP1 in this pathway may offer therapeutic approaches to disorders with increased b-catenin signaling.

Human urokinase type plasminogen activator (u-PA) is a member of the chymotrypsin family of serine proteases that can play important roles in both health and disease. We have used substrate phage display techniques to characterize the specificity of this enzyme in detail and to identify peptides that are cleaved 840 -5300 times more efficiently by u-PA than peptides containing the physiological target sequence of the enzyme. In addition, unlike peptides containing the physiological target sequence, the peptide substrates selected in this study were cleaved as much as 120 times more efficiently by u-PA than by tissue type plasminogen activator (t-PA), an intimately related enzyme. Analysis of the selected peptide substrates strongly suggested that the primary sequence SGRSA, from position P3 to P2, represents optimal subsite occupancy for substrates of u-PA. Insights gained in these investigations were used to design a variant of plasminogen activator inhibitor type 1, the primary physiological inhibitor of both u-PA and t-PA, that inhibited u-PA approximately 70 times more rapidly than it inhibited t-PA. These observations provide a solid foundation for the design of highly selective, high affinity inhibitors of u-PA and, consequently, may facilitate the development of novel therapeutic agents to inhibit the initiation and/or progression of selected human tumors.

The role of subsite interactions in defining the stringent substrate specificity of tissue-type plasminogen activator (t-PA) has been examined by using an fd phage library that displayed random hexapeptide sequences and contained 2 x 108 independent recombinants. Forty-four individual hexapeptides were isolated and identified as improved substrates for t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide that contained the primary sequence of the actual cleavage site in plasminogen. These results suggest that small peptides can mimic determinants that mediate specific proteolysis, emphasize the importance of subsite interactions in determining protease specificity, and have important implications for the evolution of protease cascades.plasminogen during catalysis, either to induce a conformational change in t-PA or to properly position a suboptimal sequence in the active site of t-PA. A third hypothesis, which does not necessarily require secondary sites of interaction between t-PA and plasminogen, is that t-PA recognizes an unusual, constrained conformation of the plasminogen target sequence that cannot be significantly populated by small, linear peptides. Only the second of these three hypotheses unequivocally predicts the existence of small peptides that could be efficiently cleaved by t-PA. Consequently, to distinguish among these hypotheses, we screened a library of random hexamers for peptide sequences that could be hydrolyzed by t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide containing the primary sequence of the actual cleavage site in plasminogen.A wide variety of critical biological processes (1-3) depend on specific cleavage of individual target proteins by serine proteases. Enzymes capable of catalyzing this uniquely selective proteolysis have evolved while retaining high homology to related nonselective proteases. For example, tissue-type plasminogen activator (t-PA), a trypsin-like enzyme that catalyzes the rate-limiting step of the endogenous fibrinolytic cascade, has only one known substrate in vivo, a single bond (Arg560 -Val561) in the proenzyme plasminogen (4). Part of the specificity of t-PA for plasminogen is mediated by formation of a ternary complex between t-PA, plasminogen, and the cofactor fibrin (5). Even in the absence of fibrin, however, t-PA remains specific for cleavage of plasminogen. This stringent specificity is an inherent property of the protease domain of t-PA (6), in spite of its high homology to trypsin, an archetypal nonselective protease (7).Trypsin activates native plasminogen poorly, exhibiting only 10-30% of the catalytic efficiency of t-PA (6, 8). By contrast, trypsin cleaves small peptides containing the primary sequence of the activation site in plasminogen 29,000-200,000 times more rapidly than t-PA. The relative specificity of these two related enzymes for the same primary sequence, in two distinc...

PTEN, a phosphoinositide-3-phosphatase, serves dual roles as a tumor suppressor and regulator of cellular anabolic/catabolic metabolism. Adaptation of a redox-sensitive cysteinyl thiol in PTEN for signal transduction by hydrogen peroxide may have superimposed a vulnerability to other mediators of oxidative stress and inflammation, especially reactive carbonyl species, which are commonly occurring by-products of arachidonic acid peroxidation. Using MCF7 and HEK-293 cells, we report that several reactive aldehydes and ketones, e.g. electrophilic α,β-enals (acrolein, 4-hydroxy-2-nonenal) and α,β-enones (prostaglandin A2, Δ12-prostaglandin J2 and 15-deoxy-Δ-12,14-prostaglandin J2) covalently modify and inactivate cellular PTEN, with ensuing activation of PKB/Akt kinase; phosphorylation of Akt substrates; increased cell proliferation; and increased nuclear β-catenin signaling. Alkylation of PTEN by α,β-enals/enones and interference with its restraint of cellular PKB/Akt signaling may accentuate hyperplastic and neoplastic disorders associated with chronic inflammation, oxidative stress, or aging.

The importance of secondary subsites in defining both the specificity and efficiency of cleavage suggests that substrate recognition by PSA is mediated by an extended binding site. Elucidation of preferred subsite occupancy allowed refinement of the structural model of PSA and should facilitate the development of more sensitive activity-based assays and the design of potent inhibitors.

Tissue-type plasminogen activator (t-PA) is a remarkably specific protease: the only known substrate of this enzyme in vivo is a single peptide bond (Arg560-Val561) within the proenzyme plasminogen. Part of the substrate specificity of t-PA is due to a ternary interaction between fibrin, t-PA and plasminogen which reduces the Km of t-PA for plasminogen by a factor of 440. However, even in the absence of fibrin, t-PA continues to hydrolyze plasminogen more rapidly than does trypsin, a homologous serine protease. We have measured the extent of the specificity of t-PA for plasminogen by assaying t-PA and trypsin toward substrates modeled after the peptide sequence in plasminogen surrounding Arg560-Val561. Surprisingly, t-PA hydrolyzes these substrates with kcat/Km values which are 28,000-210,000-fold lower than those obtained using trypsin. Both the high activity toward plasminogen and the low activity toward peptides are also exhibited by the isolated protease domain. This suggests that the protease domain, in spite of its high homology to the nonspecific enzyme trypsin, is inherently specific for recognition of one or more structural features displayed by native plasminogen.