Cytoskeletal Protein ABP-280 Directs the Intracellular Trafficking of Furin and Modulates Proprotein Processing in the Endocytic Pathway

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.     

BMP-4 is proteolytically activated by furin and/or PC6 during vertebrate embryonic development.

Bone morphogenetic protein-4 (BMP-4) is a multifunctional developmental regulator. BMP-4 is synthesized as an inactive precursor that is proteolytically activated by cleavage following the amino acid motif -Arg-Ser-Lys-Arg-. Very little is known about processing and secretion of BMPs. The proprotein convertases (PCs) are a family of seven structurally related serine endoproteases, at least one of which, furin, cleaves after the amino acid motif -Arg-X-Arg/Lys-Arg-. To examine potential roles of PCs during embryonic development we have misexpressed a potent protein inhibitor of furin, alpha1-antitrypsin Portland (alpha1-PDX) in early Xenopus embryos. Ectopic expression of alpha1-PDX phenocopies the effect of blocking endogenous BMP activity, leading to dorsalization of mesoderm and direct neural induction. alpha1-PDX-mediated neural induction can be reversed by co-expression of downstream components of the BMP-4 signaling pathway. Thus, alpha1-PDX can block BMP activity upstream of receptor binding, suggesting that it inhibits an endogenous BMP-4 convertase(s). Consistent with this hypothesis, alpha1-PDX prevents cleavage of BMP-4 in an oocyte translation assay. Using an in vitro digestion assay, we demonstrate that four members of the PC family have the ability to cleave BMP-4, but of these, only furin and PC6B are sensitive to alpha1-PDX. These studies provide the first in vivo evidence that furin and/or PC6 proteolytically activate BMP-4 during vertebrate embryogenesis.     

Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases

Membrane type-1 matrix metalloproteinase (MT1-MMP) is the prototypical member of a subgroup of membrane-anchored proteinases that belong to the matrix metalloproteinase family. Although synthesized as a zymogen, MT1-MMP plays an essential role in extracellular matrix remodeling after an undefined process that unmasks its catalytic domain. We now report the existence of a proprotein convertase-MT1-MMP axis that regulates the processing and functional activity of the metalloproteinase. Two sets of basic motifs in the propeptide region of MT1-MMP are identified that potentially can be recognized by the proprotein convertase family of subtilisin-like proteases. Processing of proMT1-MMP as well as the expression of its proteolytic activity were blocked by mutating these recognition motifs or by inhibiting the proprotein convertases furin and PC6 with the serpin-based inhibitor alpha(1) antitrypsin Portland. Furthermore, both furin-dependent and furin-independent MT1-MMP processing pathways are identified that require tethering of the metalloproteinase to the cell surface. These findings demonstrate the existence of a proprotein convertase-MT1-MMP axis that can regulate extracellular matrix remodeling.     

Carboxy-terminal proteolytic processing at a consensus furin cleavage site is a prerequisite event for quail ZPC secretion

In avian species, a glycoprotein homologous to mammalian ZPC is synthesized in the granulosa cells of developing follicles. We have previously reported that the newly synthesized ZPC (proZPC) in granulosa cells is cleaved at a consensus furin cleavage site to generate mature ZPC prior to secretion. In the present study, we examined the effect of the proteolytic cleavage of proZPC on ZPC secretion by using a specific inhibitor of furin endoprotease and site-directed mutagenesis of the furin cleavage site. Western blot analysis demonstrated that the furin inhibitor efficiently blocked both the proteolytic cleavage of proZPC and the subsequent ZPC secretion. A site-directed mutant that possessed a mutated sequence for furin cleavage was not secreted from the cells. The immunocytochemical observations indicated that proZPC produced in the presence of a furin inhibitor or those produced by the site-directed mutant of the furin cleavage site had accumulated in the endoplasmic reticulum. These results indicate that proZPC is proteolytically cleaved at the consensus furin cleavage site with furin-like protease, and the failure of this cleavage results in its accumulation in the endoplasmic reticulum. Therefore, the C-terminal proteolytic processing of proZPC at the consensus furin cleavage site is a prerequisite event for quail ZPC secretion.     

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.     

Development of selectivity of alpha1-antitrypsin variant by mutagenesis in its reactive site loop against proprotein convertase. A crucial role of the P4 arginine in PACE4 inhibition

PACE4, furin and PC6 are Ca2+-dependent serine endoproteases that belong to the subtilisin-like proprotein convertase (SPC) family. Recent reports have supported the involvement of these enzymes in processing of growth/differentiation factors, viral replication, activation of bacterial toxins and tumorigenesis, indicating that these enzymes are a fascinating target for therapeutic agents. In this work, we evaluated the sensitivity and selectivity of three rat alpha1-antitrypsin variants which contained RVPR352, AVRR352 and RVRR352, respectively, within their reactive site loop using both inhibition of enzyme activity toward a fluorogenic substrate in vitro and formation of a SDS-stable protease/inhibitor complex ex vivo. The RVPR variant showed relatively broad selectivity, whereas the AVRR and RVRR variants were more selective than the RVPR variant. The AVRR variant inhibited furin and PC6 but not PACE4. This selectivity was further confirmed by complex formation and inhibition of pro-complement C3 processing. On the other hand, although the RVRR variant inhibited both PACE4 and furin effectively, it needed a 600-fold higher concentration than the RVPR variant to inhibit PC6 in vitro. These inhibitors will be useful tools in helping us to understand the roles of PACE4, furin and PC6.     

Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.

Activation of furin requires autoproteolytic cleavage of its 83-amino acid propeptide at the consensus furin site, Arg-Thr-Lys-Arg107/. This RER-localized cleavage is necessary, but not sufficient, for enzyme activation. Rather, full activation of furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of furin. Exposure of membrane preparations containing an inactive RER-localized soluble furin construct to either: (i) an acidic and calcium-containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a furin inhibitor. Consistent with these findings, following cleavage in the RER, the furin propeptide remains associated with the enzyme and functions as a potent inhibitor of the endoprotease. Co-immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (-Arg70-Gly-Val-Thr-Lys-Arg75/-). The significance of this cleavage in regulating the compartment-specific activation of furin, and the relationship of the furin activation pathway to those of other serine endoproteases are discussed.     

Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs 9 (ADAMTS9) is a highly conserved metalloprotease that has been identified as a tumor suppressor gene and is required for normal mouse development. The secreted ADAMTS9 zymogen undergoes proteolytic excision of its N-terminal propeptide by the proprotein convertase furin. However, in contrast to other metalloproteases, propeptide excision occurs at the cell surface and leads to decreased activity of the zymogen. Here, we investigated the potential cellular mechanisms regulating ADAMTS9 biosynthesis and cell-surface processing by analysis of molecular complexes formed by a construct containing the propeptide and catalytic domain of pro-ADAMTS9 (Pro-Cat) in HEK293F cells. Cross-linking of cellular proteins bound to Pro-Cat followed by mass spectrometric analysis identified UDP-glucose:glycoprotein glucosyltransferase I, heat shock protein gp96 (GRP94), BiP (GRP78), and ERdj3 (Hsp40 homolog) as associated proteins. gp96 and BiP were present at the cell surface in an immunoprecipitable complex with pro-ADAMTS9 and furin. Treatment with geldanamycin, an inhibitor of the HSP90α family (including gp96), led to decreased furin processing of pro-ADAMTS9 and accumulation of the unprocessed pro-ADAMTS9 at the cell surface. gp96 siRNA down-regulated the levels of cell-surface pro-ADAMTS9 and furin, whereas the levels of cell-surface pro-ADAMTS9, but not of cell-surface furin, were decreased upon treatment with BiP siRNA. These data identify for the first time the cellular chaperones associated with secretion of an ADAMTS protease and suggest a role for gp96 in modulating pro-ADAMTS9 processing.     

Furin Is the Major Proprotein Convertase Required for KISS1-to-Kisspeptin Processing

KISS1 is a broadly functional secreted proprotein that is then processed into small peptides, termed kisspeptins (KP). Since sequence analysis showed cleavage at KR or RR dibasic sites of the nascent protein, it was hypothesized that enzyme(s) belonging to the proprotein convertase family of proteases process KISS1 to generate KP. To this end, cell lines over-expressing KISS1 were treated with the proprotein convertase inhibitors, Dec-RVKR-CMK and α1-PDX, and KISS1 processing was completely inhibited. To identify the specific enzyme(s) responsible for KISS1 processing, mRNA expression was systematically analyzed for six proprotein convertases found in secretory pathways. Consistent expression of the three proteases – furin, PCSK5 and PCSK7 – were potentially implicated in KISS1 processing. However, shRNA-mediated knockdown of furin – but not PCSK5 or PCSK7 – blocked KISS1 processing. Thus, furin appears to be the essential enzyme for the generation of kisspeptins.     

The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct trans-Golgi network/endosomal compartments

The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A-dispersible, BaCl(2)-responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosine-based motif (Tyr(1802)GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane-distal AC (AC2) promotes a localization characteristic of the full-length PC6B-cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo.     

The Paired Basic Amino Acid-cleaving Enzyme 4 (PACE4) Is Involved in the Maturation of Insulin Receptor Isoform B: AN OPPORTUNITY TO REDUCE THE SPECIFIC INSULIN RECEPTOR-DEPENDENT EFFECTS OF INSULIN-LIKE GROWTH FACTOR 2 (IGF2)*

Gaining the full activity of the insulin receptor (IR) requires the proteolytic cleavage of its proform by intra-Golgi furin-like activity. In mammalian cells, IR is expressed as two isoforms (IRB and IRA) that are responsible for insulin action. However, only IRA transmits the growth-promoting and mitogenic effects of insulin-like growth factor 2. Here we demonstrate that the two IR isoforms are similarly cleaved by furin, but when this furin-dependent maturation is inefficient, IR proforms move to the cell surface where the proprotein convertase PACE4 selectively supports IRB maturation. Therefore, in situations of impaired furin activity, the proteolytic maturation of IRB is greater than that of IRA, and accordingly, the amount of phosphorylated IRB is also greater than that of IRA. We highlight the ability of a particular proprotein convertase inhibitor to effectively reduce the maturation of IRA and its associated mitogenic signaling without altering the signals emanating from IRB. In conclusion, the selective PACE4-dependent maturation of IRB occurs when furin activity is reduced; accordingly, the pharmacological inhibition of furin reduces IRA maturation and its mitogenic potential without altering the insulin effects.     

Maturation of HIV envelope glycoprotein precursors by cellular endoproteases

The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection. The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process. The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells. To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases. In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow). The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins. Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage. Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins. In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins. Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing. Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented. Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases. Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.     

Regulation of ADAMTS9 secretion and enzymatic activity by its propeptide

ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg74 downward arrow and Arg287 downward arrow) the ADAMTS9 propeptide was also furin-processed at Arg209. Substitution of Ala for Arg74, Arg209, and Arg287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface.     

Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases: IMPLICATIONS FOR THE REGULATION OF ADRENAL STEROIDOGENESIS*

Group X secretory phospholipase A₂ (GX sPLA₂) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA₂ is produced as a pro-enzyme (pro-GX sPLA₂) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA₂ activation have not been identified. We previously reported that GX sPLA₂ negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA₂ expression construct (FLAG-pro-GX sPLA₂), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA₂ processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA₂ processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA₂ processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA₂ to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA₂ processing and sPLA₂ activity in Y1 cells, and it significantly attenuated GX sPLA₂-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA₂ is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.     

Basolateral sorting of furin in MDCK cells requires a phenylalanine-isoleucine motif together with an acidic amino acid cluster

Furin is a subtilisin-related endoprotease which processes a wide range of bioactive proteins. Furin is concentrated in the trans-Golgi network (TGN), where proteolytic activation of many precursor proteins takes place. A significant fraction of furin, however, cycles among the TGN, the plasma membrane, and endosomes, indicating that the accumulation in the TGN reflects a dynamic localization process. The cytosolic domain of furin is necessary and sufficient for TGN localization, and two signals are responsible for retrieval of furin to the TGN. A tyrosine-based (YKGL) motif mediates internalization of furin from the cell surface into endosomes. An acidic cluster that is part of two casein kinase II phosphorylation sites (SDSEEDE) is then responsible for retrieval of furin from endosomes to the TGN. In addition, the acidic EEDE sequence also mediates endocytic activity. Here, we analyzed the sorting of furin in polarized epithelial cells. We show that furin is delivered to the basolateral surface of MDCK cells, from where a significant fraction of the protein can return to the TGN. A phenylalanine-isoleucine motif together with the acidic EEDE cluster is required for basolateral sorting and constitutes a novel signal regulating intracellular traffic of furin.     

Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail.

Human furin catalyzes the proteolytic maturation of many proproteins in the exocytic and endocytic secretory pathways by cleavage at the C-terminal side of the consensus sequence-ArgXaaLys/ArgArg decreases -. Both the trans-Golgi network (TGN) concentration and intracellular routing of furin require sequences in its 56 amino acid cytoplasmic tail. Here, we show that the furin cytoplasmic tail contains multiple trafficking signals. Localization to the TGN requires a cluster of acidic amino acids that, together with a pair of serine residues, forms a casein kinase II (CK II) phosphorylation site. We show that CK II efficiently phosphorylates these serines in vitro, and using a permeabilized cell system we provide evidence that CK II is the in vivo furin kinase. Analysis by mass spectrometry shows that, in vivo, furin exists as di-, mono- and non-phosphorylated forms. Finally, employing (i) furin constructs that mimic either non-phosphorylated or phosphorylated furin and (ii) the phosphatase inhibitor tautomycin, we show that the phosphorylation state of the furin cytoplasmic tail modulates retrieval of the endoprotease to the TGN. Thus, routing of furin is a two-tiered process combining a set of trafficking signals comprised of the primary amino acid sequence of the tail with its phosphorylation state.