Endoplasmic reticulum-associated degradation of the mouse PC1/3-N222D hypomorph and human PCSK1 mutations contributes to obesity.

BACKGROUND: The proprotein convertase 1/3 (PC1/3), encoded by proprotein convertase subtilisin/kexin type 1 (PCSK1), cleaves and hence activates several orexigenic and anorexigenic proproteins. Congenital inactivation of PCSK1 leads to obesity in human but not in mice. However, a mouse model harboring the hypomorphic mutation N222D is obese. It is not clear why the mouse models differ in phenotype. METHODS: Gene expression analysis was performed with pancreatic islets from Pcsk1(N222D/N222D) mice. Subsequently, biosynthesis, maturation, degradation and activity were studied in islets, pituitary, hypothalamus and cell lines. Coimmunoprecipitation of PC1/3-N222D and human PC1/3 variants associated with obesity with the endoplasmic reticulum (ER) chaperone BiP was studied in cell lines. RESULTS: Gene expression analysis of islets of Pcsk1(N222D/N222D) mice showed enrichment of gene sets related to the proteasome and the unfolded protein response. Steady-state levels of PC1/3-N222D and in particular the carboxy-terminally processed form were strongly reduced in islets, pituitary and hypothalamus. However, impairment of substrate cleavage was tissue dependent. Proinsulin processing was drastically reduced, while processing of proopiomelanocortin (POMC) to adrenocorticotropic hormone (ACTH) in pituitary was only mildly impaired. Growth hormone expression and IGF-1 levels were normal, indicating near-normal processing of hypothalamic proGHRH. PC1/3-N222D binds to BiP and is rapidly degraded by the proteasome. Analysis of human PC1/3 obesity-associated mutations showed increased binding to BiP and prolonged intracellular retention for all investigated mutations, in particular for PC1/3-T175M, PC1/3-G226R and PC1/3-G593R. CONCLUSIONS: This study demonstrates that the hypomorphic mutation in Pcsk1(N222D) mice has an effect on catalytic activity in pancreatic islets, pituitary and hypothalamus. Reduced substrate processing activity in Pcsk1(N222D/N222D) mice is due to enhanced degradation in addition to reduced catalytic activity of the mutant. PC1/3-N222D binds to BiP, suggesting impaired folding and reduced stability. Enhanced BiP binding is also observed in several human obesity-associated PC1/3 variants, suggesting a common mechanism.

A nonsense loss-of-function mutation in PCSK1 contributes to dominantly inherited human obesity.

BACKGROUND: A significant proportion of severe familial forms of obesity remain genetically elusive. Taking advantage of our unique cohort of multigenerational obese families, we aimed to assess the contribution of rare mutations in 29 common obesity-associated genes to familial obesity, and to evaluate in these families the putative presence of nine known monogenic forms of obesity. METHODS: Through next-generation sequencing, we sequenced the coding regions of 34 genes involved in polygenic and/or monogenic forms of obesity in 201 participants (75 normal weight individuals, 54 overweight individuals and 72 individuals with obesity class I, II or III) from 13 French families. In vitro functional analyses were performed to investigate the mutation PCSK1-p.Arg80* which was identified in a family. RESULTS: A novel heterozygous nonsense variant in PCSK1 (p.Arg80*), encoding a propeptide truncated to less than two exons (out of 14), was found to co-segregate with obesity in a three-generation family. We demonstrated that this mutation inhibits PCSK1 enzyme activity and that this inhibition most likely does not involve a strong physical interaction. Furthermore, both mutations PCSK1-p.Asn180Ser and POMC-p.Phe144Leu, which had previously been reported to be associated with severe obesity, were also identified in this study, but did not co-segregate with obesity. Finally, we did not identify any rare mutations co-segregating with obesity in common obesity susceptibility genes, except for CADM2 and QPCTL, where we found two novel variants (p.Arg81His and p.Leu98Pro, respectively) in three obese individuals. CONCLUSIONS: We showed for the first time that a nonsense mutation in PCSK1 was likely to cause dominantly inherited human obesity, due to the inhibiting properties of the propeptide fragment encoded by the null allele. Furthermore, the present family sequencing design challenged the contribution of previously reported mutations to monogenic or at least severe obesity.

Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene.

Human obesity has an inherited component, but in contrast to rodent obesity, precise genetic defects have yet to be defined. A mutation of carboxypeptidase E (CPE), an enzyme active in the processing and sorting of prohormones, causes obesity in the fat/fat mouse. We have previously described a women with extreme childhood obesity (Fig. 1), abnormal glucose homeostasis, hypogonadotrophic hypogonadism, hypocortisolism and elevated plasma proinsulin and pro-opiomelanocortin (POMC) concentrations but a very low insulin level, suggestive of a defective prohormone processing by the endopeptidase, prohormone convertase 1 (PC1; ref. 4). We now report this proband to be a compound heterozygote for mutations in PC1. Gly-->Arg483 prevents processing of proPC1 and leads to its retention in the endoplasmic reticulum (ER). A-->C+4 of the intro-5 donor splice site causes skipping of exon 5 leading to loss of 26 residues, a frameshift and creation of a premature stop codon within the catalytic domain. PC1 acts proximally to CPE in the pathway of post-translational processing of prohormones and neuropeptides. In view of the similarity between the proband and the fat/fat mouse phenotype, we infer that molecular defects in prohormone conversion may represent a generic mechanism for obesity, common to humans and rodents.

Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice.

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8(-/-)) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8(-/-) beta cells and were replaced by immature, pale insulin "progranules," which were larger than in ZnT8(+/+) islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8(-/-) mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8(-/-) genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome.

BACKGROUND: Hypotonia-cystinuria syndrome (HCS) and 2p21 deletion syndrome are two recessive contiguous gene deletion syndromes associated with cystinuria type I. The deletions differ in size and the number of genes involved. In HCS patients, only SLC3A1 and PREPL are disrupted. In the 2p21 deletion syndrome, two additional genes (C2orf34 and PPM1B) are lost. OBJECTIVE: Clinical and molecular analysis of two siblings who presented with an atypical HCS phenotype. METHODS: Molecular analysis of the SLC3A1/PREPL locus was performed in the patients using quantitative polymerase chain reaction (PCR) methods. RESULTS: HCS in both siblings was confirmed with the deletion screen of the SLC3A1/PREPL locus. Fine mapping of the breakpoint revealed a deletion of 77.4 kb, including three genes: SLC3A1, PREPL and C2orf34. Features not present in classical HCS were a mild/moderate mental retardation and a respiratory chain complex IV deficiency documented in patient 2. CONCLUSIONS: We report the first patients with a deletion of SLC3A1, PREPL and C2orf34. They present with a phenotype intermediate between HCS and 2p21 deletion syndrome. These patients facilitate the elucidation of the contribution of each gene to the phenotype in the different 2p21 deletion syndromes.

Prenatal diagnosis of cerebral lesions in Tuberous sclerosis complex (TSC). Case report and review of the literature.

Tuberous sclerosis complex (TSC) is an autosomal-dominant neurocutaneous disorder with multi-organ involvement. The diagnosis is suspected at fetal ultrasound on the discovery of multiple cardiac rhabdomyomas (CRs). They typically develop in utero and undergo spontaneous regression during the first years of live. With developing neuroradiological methods more light is shed on antenatal cerebral lesions like cortical tubers or giant cell astrocytomas. Unfortunately these do not regress, but instead are in principle progressive in size and number, correlated with epilepsy, mental retardation and behavioral problems. It is unknown whether fetal cerebral lesions, are always correlated with a poor neurological outcome or a progressive course of disease. This makes prenatal counseling extremely difficult. We report one case of de novo TSC with first detection of cortical tubers on fetal ultrasound, later developing multiple CRs. The pregnancy was continued and the child is developing well during 16 months of follow-up. Minor motor seizures from the 10th month onwards are successfully treated with Valproate. The published cases with antenatal diagnosis of TSC are revised, trying to get more insight into the postnatal course of prenatally diagnosed TSC. This is crucial, either when termination of pregnancy (TOP) is considered, but even more for proper postnatal care and follow-up.

[Alpha-foetoprotein assessment in amniotic fluid for the detection of neural tube defects: limited added value beyond week 20 ultrasound; retrospective study]. / Alfafoetoproteïnebepaling in vruchtwater voor de detectie van neuralebuisdefecten: beperkte meerwaarde boven de 20-wekenecho; retrospectiefonderzoek.

OBJECTIVE: To evaluate the diagnostic additional value of routine alpha-foetoprotein (AFP) assessment in amniotic fluid for the detection of neural tube defects (NTDs), compared with week 20 ultrasonographic examination. DESIGN: Retrospective. METHOD: We retrospectively determined AFP concentrations in amniotic fluid obtained from 7981 women who had undergone amniocentesis for karyotyping and AFP assessment. An AFP concentration greater than 2.5 times the median was considered abnormal. Women were categorised into 4 groups based on the indication for invasive prenatal diagnostic assessment: advanced maternal age (group I; n = 6179), increased risk of foetal NTDs (group II; n = 258), ultrasonographically confirmed foetal NTDs (group III; n = 55) or other indications (group IV; n = 1489). RESULTS: In group I, 18 of 6179 samples had increased AFP levels (0.3%), 2 of which were associated with NTDs. In group II, 2 of 258 samples had increased AFP levels (0.8%); both were associated with NTDs. Increased AFP levels were found in 44 of 55 samples from group III (80%), and 223 of 1489 samples from group IV (15.0%). CONCLUSION: Routine assessment of AFP in amniotic fluid based on advanced maternal age provides little additional value in the detection of NTDs beyond that of week 20 ultrasound.

Human lactase-phlorizin hydrolase is not processed by furin, PC1/PC3, PC2, PACE4 and PC5/PC6A of the family of subtilisin-like proprotein processing proteases.

Human lactase-phlorizin hydrolase (LPH, EC 3.2.1.23/62) is synthesized as a single-chain precursor glycoprotein (pro-LPH) with a relative molecular mass of just over 200 kDa. Maturation to the mature enzyme (m-LPH, 160 kDa) occurs after passage of pro-LPH through the Golgi complex and involves the proteolytic removal of a 849 amino acid propeptide. The role of this propeptide as well as its removal is not fully understood and the proteolytic enzyme or enzymes involved are unknown. We studied the potential role of five different members of the family of subtilisin-like proprotein processing proteases in the maturation process of human LPH using a vaccinia virus based coexpression system in pig kidney PK(15) cells. Infected/transfected PK(15) cells expressed full-length pro-LPH but no maturation to m-LPH was observed. Coexpression of human pro-LPH with human furin, human PC1/PC3, human PC2, human PACE4 and mouse PC6A in PK(15) cells did not result in maturation of the enzyme. Cleavage and secretion of von Willebrand factor precursor (pro-vWF) was used as a positive control. None of the five proprotein processing proteases tested were capable of cleaving human pro-LPH, strongly suggesting that they are not involved in the maturation of this enzyme.

Amyloid precursor protein is not processed by furin, PACE 4, PC1/3, PC2, PC4 and PC5/6 of the furin family of proprotein processing enzymes.

Proteolytic cleavage of the amyloid precursor protein (APP) has previously been shown to release its extracellular domain into the medium. The identification of the responsible proteinase(s), termed secretase(s), is a high priority in ongoing Alzheimer research. This is hampered by the unusual characteristics of these enzyme(s) and by the fact that they cleave only membrane associated APP. We report here, using a vaccinia virus based expression system, that pig kidney PK(15) cells express full-length, membrane bound APP695, but that secretion of APP is low. This heterologous expression system allows to assay candidate secretases in a cellular context by simple co-transfection of the APP and candidate secretase cDNA containing plasmids. Eight different members of the mouse and human furin family of proprotein processing enzymes were tested in this assay, but none of them enhanced the secretion of APP. Secretion of von Willebrand's factor was used as a positive control.

Expression in human lung tumor cells of the proprotein processing enzyme PC1/PC3. Cloning and primary sequence of a 5 kb cDNA.

Northern blot analysis of human lung tumors indicated that the gene, which encodes the subtilisin-like proprotein processing enzyme PC1/PC3, was highly expressed in almost all carcinoid tumors tested. In small cell lung carcinomas (SCLCs), expression varied. In non-SCLCs and normal lung, no expression was found. Analysis of SCLC cell lines revealed that expression was restricted preferentially to cell lines of the classical type. In lung tumor cells expressing the PC1/PC3 gene, transcripts of 3 kb and 5 kb were detected, the 5 kb mRNA always being the most abundant species. We isolated a cDNA corresponding to the 5 kb human PC1/PC3 transcript, determined the nucleotide sequence of it and deduced the amino acid sequence of the corresponding protein. Furthermore, we conclude that the two PC1/PC3 transcripts have 3' non-coding regions of different size and encode the same protein.

Proprotein processing activity and cleavage site selectivity of the Kex2-like endoprotease PACE4.

Proprotein processing activity of the Kex2-like mammalian endoprotease PACE4 and its cleavage selectivity for sites with basic amino acid residues were determined. Using a recombinant vaccinia virus-based expression system, PACE4 was expressed in pig kidney PK(15) cells and, like two other Kex2-like endoproteases furin and PC6A, shown to correctly process the precursor of von Willebrand factor (pro-vWF). Furthermore, characteristics of the cleavage site selectivity of PACE4 were compared to those of furin and PC6A using the vWF cleavage site mutants vWFR-1G, vWFK-2A, and vWFR-4A as substrates. Cleavage site selectivity of PACE4 and PC6A appeared to be similar but they differed from that of furin.

Furin-mediated proprotein processing activity: involvement of negatively charged amino acid residues in the substrate binding region.

Furin, which is encoded by the recently discovered FUR gene, appears to be the first known mammalian member of the subtilisin family of serine proteases with cleavage selectivity for paired or multiple basic residues. A consensus cleavage sequence, Arg-X-Lys/Arg-Arg has been proposed. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Homology modelling of the catalytic domain of this protein suggested that negatively charged amino acid residues near or in the substrate binding region might contribute to the observed specificity for substrate segments with paired and multiple basic amino acid residues. To investigate this hypothesis, furin mutants were generated in which negatively charged residues, predicted to be located near or in the substrate binding pockets and involved in interactions with basic residues of the substrate, were replaced by neutral residues. Analysis of processing by these furin mutants of wild-type and cleavage mutants of pro-von Willebrand factor (pro-vWF) revealed that particular negatively charged residues are critical for specific cleavage activity.

The Dfur2 gene of Drosophila melanogaster: genetic organization, expression during embryogenesis, and pro-protein processing activity of its translational product Dfurin2.

The gene structure and expression of the Dfur2 gene of Drosophila melanogaster, which encodes the subtilisin-like serine endoprotease Dfurin2, was studied. The Dfur2 gene is very compact in contrast to the related Dfur1 gene, which has an estimated size of over 100 kbp. The 6-kb Dfur2 mRNA is encoded by 16 exons dispersed over a genomic region of about 9 kbp. The exon/intron organization shows conservation of intron positions not only in comparison with Dfur1, but also with the related mammalian genes FUR, PC1/PC3, PC2, and PC4. This conservation supports the hypothesis that all genes belonging to the family of subtilisin-like pro-protein processing enzymes are evolutionary related by descent from a common ancestral gene. In primer extension experiments, Dfur2 transcription initiation sites were identified in the presumed Dfur2 promoter region. This region was found to contain general RNA polymerase II promoter elements like a potential TATA box, a potential CAP signal, and several potential CCAAT boxes. Also, several sequence motifs putatively corresponding to binding sites for Drosophila transcription factors like zeste, bicoid, and engrailed were found to be present. RNA in situ hybridization experiments on Drosophila embryos revealed presumably maternal Dfur2 expression until the syncytial blastoderm (stage 5 of embryogenesis), no expression during gastrulation (stage 9), transient expression in a subset of neurons in the central nervous system of stage 12-13 embryos, and, from stage 13 onwards, expression in the developing tracheal tree. In a vaccinia expression system, the endoprotease Dfurin2 not only cleaved wild-type precursor of von Willebrand factor (pro-vWF) with pro-region cleavage site R-S-K-R decreases, but also, although to a lesser extent, pro-vWF mutants in which the P2 (vWFK-2A) or P4 (vWFR-4A) basic residue with respect to the pro-region cleavage site had been mutated. This cleavage specificity resembles that of human furin. The cleavage of pro-vWF by Dfurin2 shows that the previously reported lack of cleavage of the precursor of the beta A-chain of activin-A by Dfurin2 in this vaccinia expression system is substrate determined.

Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin.

Monoclonal antibodies were raised against the recently discovered subtilisin-like proprotein processing enzyme furin. As immunogen, a bacterially expressed hybrid protein was used which consisted of glutathione S-transferase fused to almost the entire human furin protein. Ten monoclonal antibodies were obtained and these could be divided into four categories on the basis of their reactivity towards a number of bacterially expressed hybrid proteins, each of which contained a different portion of human furin. Four of the monoclonal antibodies did not recognize mouse furin. All monoclonal antibodies were tested for their applicability in Western blot and immunofluorescence analysis. Western blot analysis was performed with COS-1 cells in which biologically active forms of human and mouse furin were expressed transiently under control of the SV40 late promoter. This approach was necessary, since physiological levels of fur gene encoded proteins appeared to be very low. In cells transfected with human or mouse fur cDNA, a protein of about 100 kDa and a doublet of about 90 kDa could be detected with most of the monoclonal antibodies. Some of these antibodies appeared to be also reactive in immunofluorescence analysis of transfected COS-1 cells.

Limitations of inhibitory activities of polyphenols on furin-mediated substrate processing.

Recently, selected polyphenols were reported to exert proprotein convertase (PC) inhibitory activities on in vitro cleavage of a fluorogenic peptide substrate and it was concluded that this anti-protease activity might be responsible for the reported anti-cancer properties of these polyphenols. This prompted investigations to identify PC inhibiting polyphenols that could affect IGF-1R-mediated tumorigenesis since pro-IGF-1R is bioactivated by PCs like furin. Initial screening of polyphenols for their impact on in vitro cleavage of fluorogenic peptide substrate Pyr-RTKR-AMC by human furin (hfurin(573)) indeed revealed varying inhibitory effects. (-)EGCG, chrysin, and quercetin, were subsequently evaluated using uncleaved diphtheria toxin as substrate in vitro. However, none displayed any inhibitory impact on processing. Binding of (-)EGCG to both furin and the diphtheria toxin protein was demonstrated. Subsequently, it was found that for seven polyphenols tested, addition of casein or gamma globulin led to reduction or even annihilation of in vitro Pyr- RTKR-AMC cleavage inhibition. No such effect was seen with the furin inhibitor nona-D-arginine. Western blot studies to investigate possible effects of selected polyphenols on processing in cells of the tumorigenesis-linked proproteins pro-IGF-1R and pro-GPC3 also revealed no inhibitory effects. In conclusion, our results confirm the reported PC inhibitory effects of polyphenols on fluorogenic peptide substrate cleavage in vitro. However, the data show that polyphenolic inhibitory effects on hfurin(573)-mediated in vitro fluorogenic peptide substrate cleavage cannot be extrapolated to similar effects on processing of genuine proproteins, whether in vitro or in cells. This undermines the anti-protease rationale for the reported polyphenolic anti-cancer properties.

The autism candidate gene Neurobeachin encodes a scaffolding protein implicated in membrane trafficking and signaling.

Autism is a developmental disorder of the central nervous system characterized by impairments in social interaction, communication and restricted repetitive and stereotyped behavior. It is generally assumed that in most cases autism has a polygenic cause, but the pathogenesis is still unknown. Neurobeachin (NBEA) has recently been identified as a candidate gene for autism in a patient with a de novo chromosomal translocation and three patients with a monoallelic deletion. This multidomain scaffolding protein has been suggested to be involved in neuronal post-Golgi membrane traffic. Knockout of Nbea in two independent mouse models has demonstrated a role in neurotransmitter release and synaptic functioning. Knockdown in a cell line has shown a role as negative regulator of secretion of large dense-core vesicles (LDCVs) and haploinsufficiency in blood platelets results in dense granules with an aberrant morphology. A potential role in vesicle transport is further supported by a study of SEL-2, the C.elegans homologue of NBEA. This protein was identified as a negative regulator of LIN-12/Notch activity, probably due to defects in endosomal trafficking. Members of the Notch pathway have also been shown to be modifiers of the NBEA homologue in Drosophila, rugose. These new insights in the function of NBEA may help identifying novel pathways affected in autistic patients. In particular, it suggests that impaired functionality of LDCVs, which contain neurotrophins, neuropeptides and monoamines, might contribute to the pathogenesis of autism in at least a subgroup of patients.

Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome.

Hypotonia-cystinuria syndrome (HCS) and 2p21 deletion syndrome are two recessive contiguous gene deletion syndromes associated with cystinuria type I. In HCS patients, only SLC3A1 and PREPL are disrupted. In the 2p21 deletion syndrome, two additional genes (C2orf34 and PPM1B) are lost. Molecular analysis of the SLC3A1/PREPL locus was performed in the patients using quantitative polymerase chain reaction (PCR) methods. HCS in both siblings was confirmed with the deletion screen of the SLC3A1/PREPL locus. Fine mapping of the breakpoint revealed a deletion of 77.4 kb, including three genes: SLC3A1, PREPL and C2orf34. Features not present in classical HCS were a mild/moderate mental retardation and a respiratory chain complex IV deficiency. We report the first patients with a deletion of SLC3A1, PREPL and C2orf34. They present with a phenotype intermediate between HCS and 2p21 deletion syndrome.

Molecular and cellular regulation of prohormone processing.

The processing of prohormones involves cleavage at specific basic amino acids by members of the subtilisin-like serine endoprotease family, followed by trimming of the COOH terminus by carboxypeptidase E. The enzymes are regulated by the intra-organelle ionic environment, through post-translational processing and by interaction with endogenous inhibitors. Much has been learned about their catalytic function and cell biology from in vitro gene transfer experiments using chimeric molecules and by site-directed mutagenesis. Further insight into their molecular properties and physiological function has been gained recently from the study of in vivo mutants.

Furin: the prototype mammalian subtilisin-like proprotein-processing enzyme. Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway.

Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.

Lefty proteins exhibit unique processing and activate the MAPK pathway.

Lefty polypeptides, novel members of the transforming growth factor-beta (TGF-beta) superfamily, are involved in the formation of embryonic lateral patterning. Members of the TGF-beta superfamily require processing for their activation, suggesting cleavage to be an essential step for lefty activation. Transfection of different cell lines with lefty resulted in expression of a 42-kDa protein, which was proteolytically processed to release two polypeptides of 34 and 28 kDa. Since members of the proprotein convertase (PC) family cleave different TGF-beta factors and are involved in the establishment of embryonic laterality, we studied their role in lefty processing. Cotransfection analysis showed that PC5A processed the lefty precursor to the 34-kDa form in vivo, whereas furin, PACE4, PC5B, and PC7 had a limited activity. None of these PCs showed activity in the processing of the lefty polypeptide to the 28-kDa lefty form. The mutation of the consensus sequences for PC cleavage in the lefty protein allowed the lefty cleavage sites to be identified. Mutations of the sequence RGKR to GGKG (amino acids 74-77) and of RHGR to GHGR (amino acids 132-135) prevented the proteolytic processing of the lefty precursor to the 34- and 28-kDa forms, respectively. To identify the biologically active form of lefty, we studied the effect of lefty treatment on pluripotent P19 cells. Lefty did not induce Smad2 or Smad5 phosphorylation, Smad2/Smad4 heterodimerization, or nuclear translocation of Smad2 or Smad4, but activated the MAPK pathway in a time- and dose-dependent fashion. Further analysis showed the 28-kDa (but not the 34-kDa) polypeptide to induce MAPK activity. Surprisingly, the 42-kDa lefty protein was also capable of inducing MAPK activity, indicating that the lefty precursor is biologically active. The data support a molecular model of processing as a mechanism for regulation of lefty signaling.