Identification of a DEF-type docking domain for extracellular signal-regulated kinases 1/2 that directs phosphorylation and turnover of the BH3-only protein BimEL

The BH3-only protein, Bim, exists as three splice variants (Bim(S), Bim(L), and Bim(EL)) of differing pro-apoptotic potency. Bim(EL), the least effective killer, is degraded by the proteasome in response to phosphorylation by extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2-dependent phosphorylation correlates with the presence of a domain unique to the Bim(EL) splice variant that includes the major ERK1/2 phosphorylation site Ser(65). However, efficient phosphorylation by ERK1/2, c-Jun N-terminal kinase, or p38 requires the presence in the substrate of a discrete kinase-docking domain as well as the phosphoacceptor site. Here we show that the region unique to Bim(EL) (amino acids 41-97) harbors two potential DEF-type ERK1/2 kinase-docking domains, DEF1 and DEF2. Peptide competition assays revealed that the DEF2 peptide could act autonomously to bind active ERK1/2, whereas the DEF1 peptide did not. Truncation analysis identified a minimal region, residues 80-97, containing the DEF2 motif as sufficient for ERK1/2 binding. Mutation of key residues in the DEF2 motif abolished the interaction of ERK1/2 and Bim(EL) and also abolished ERK1/2-dependent phosphorylation of Bim(EL) in vivo, thereby stabilizing the protein and enhancing cytotoxicity. Our results identify a new physiologically relevant functional motif in Bim(EL) that may account for the distinct biological properties of this splice variant.     

Role of FcαR EC2 region in extracellular membrane localization

The FcαR receptor (CD89) binds to the constant region of Immunoglobulin (Ig) A to mediate mucosal immunity [1–2]. FcαR consist of five exons: two that code for the signal peptide regions S1 & S2, two for the extracellular regions EC1 and EC2, and the final exon for the transmembrane/cytoplasmic tail region [3]. Previously, we reported that the EC1 region plays an essential role for extracellular membrane localization of the receptor [4], where the absence of EC1 would prevent the variants from localizing to the cell surface, even with a full signal peptide. In the case of FcαR Variant 4 (lacking the S2 region only), there was some “leakiness” to membrane surface localization.     

MDM2 splice variants predominantly localize to the nucleoplasm mediated by a COOH-terminal nuclear localization signal

Of the >40 alternative and aberrant splice variants of MDM2 that have been described to date, the majority has lost both the well-characterized nuclear localization signal (NLS1) and the nuclear export signal (NES) sequence. Because cellular localization of proteins provides insight regarding their potential function, we determined the localization of three different MDM2 splice variants. The splice variants chosen were the common variants MDM2-A and MDM2-B. In addition, MDM2-FB26 was chosen because it is one of the few variants described that contains the complete p53-binding site. All three splice variants predominantly localized to the nucleus. Nuclear localization of MDM2-A and MDM2-B was controlled by a previously uncharacterized nuclear localization signal (NLS2), whereas nucleoplasmic localization of MDM2-FB26 was mediated by NLS1. p53 and full-length MDM2 colocalized with the splice variants in the nucleus. MDM2-A and MDM2-B both contain a COOH-terminal RING finger domain, and interaction with full-length MDM2 through this domain was confirmed. MDM2-FB26 was the only splice variant evaluated that contained a p53-binding domain; however, interaction between MDM2-FB26 and p53 could not be shown. p14(ARF) did not colocalize with the splice variants and was predominantly expressed within the nucleoli. In summary, nuclear localization signals responsible for the nucleoplasmic distribution of MDM2 splice variants have been characterized. Colocalization and interaction of MDM2-A and MDM2-B with full-length MDM2 in the nucleus have important physiologic consequences, for example, deregulation of p53 activity.     

Alternative splicing of RyR1 alters the efficacy of skeletal EC coupling

Alternative splicing of ASI residues (Ala³⁴⁸¹–Gln³⁴⁸⁵) in the skeletal muscle ryanodine receptor (RyR1) is developmentally regulated: the residues are present in adult ASI(+)RyR1, but absent in the juvenile ASI(?)RyR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RyR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr³⁴⁷¹–Gly³⁵⁰⁰) in the splice variants. Depolarisation-dependent Ca²⁺ release was enhanced by >50% in myotubes expressing ASI(?)RyR1 compared with ASI(+)RyR1, although DHPR L-type currents and SR Ca²⁺ content were unaltered, while ASI(?)RyR1 channel function was actually depressed. The effect on EC coupling did not depend on changes in ASI domain secondary structure. Probing RyR1 function with peptides possessing the ASI domain sequence indicated that the domain contributes to an inhibitory module in RyR1. The action of the peptide depended on a sequence of basic residues and their alignment in an α-helix adjacent to the ASI splice site. This is the first evidence that the ASI residues contribute to an inhibitory module in RyR1 that influences EC coupling. Implications for development and DM1 are discussed.     

Identification and differential expression of a novel alternative splice isoform of the beta A4 amyloid precursor protein (APP) mRNA in leukocytes and brain microglial cells.

The gene for the beta A4-amyloid precursor protein (APP) consists of 19 exons which code for a typical N- and O-glycosylated transmembrane protein with four extracellular domains followed by the transmembrane domain and a short cytoplasmic domain. The beta A4-amyloid sequence is part of exons 16 and 17. Several APP isoforms can be generated by alternative splicing of exons 7 and 8, encoding domains with homologies to Kunitz-type protease inhibitors and the MRC OX-2 antigen, respectively. The mechanism by which the pathological beta A4 is generated is unknown, it is however a critical event in Alzheimer's disease and is distinct from the normally occurring cleavage and secretion of APPs within the beta A4 sequence. We report here for the first time considerable APP mRNA expression by rat brain microglial cells. In addition we showed by S1 nuclease protection and polymerase chain reaction analysis of reverse transcribed RNA (RT-PCR) that T-lymphocytes, macrophages, and microglial cells expressed a new APP isoform by selection of a novel alternative splice site and exclusion of exon 15 of the APP gene. This leads to a transmembrane, beta A4 sequence containing APP variant, lacking 18 amino acid residues close to the amyloidogenic region. The use of this novel alternative splice site alters the structure of APP in close proximity to the beta A4 region and thus may determine a variant, potentially pathogenic processing of leukocyte-derived APP in brain.     

Fusion of a cleavable signal peptide to the ectodomain of neutral endopeptidase (EC 3.4.24.11) results in the secretion of an active enzyme in COS-1 cells

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types and is especially abundant at the apical "brush border" membrane of the kidney proximal tubules. The enzyme consists of a short amino-terminal cytosolic domain of 27 amino acids, a single hydrophobic sequence which is believed to be responsible for anchoring the enzyme in the plasma membrane, and a large extracellular domain containing the active site. This model is consistent with the proposed function of neutral endopeptidase, which is believed to play an important role in the inactivation of small regulatory peptides at the cell surface. Site-directed mutagenesis has allowed the identification of 1 glutamic acid and 2 histidine residues essential for catalysis. All are located near the COOH terminus of the protein. We do not know, however, whether other segments of the protein are involved in the structure of the active site. The exact role of the cytosolic and transmembrane domains is also unknown. In this report, we have induced the secretion of a soluble form of recombinant neutral endopeptidase in COS-1 cells by fusing in-frame, the cDNA encoding the signal peptide of a secreted protein (pro-opiomelanocortin) to the cDNA sequences of the complete ectodomain of neutral endopeptidase. Characterization of the secreted recombinant protein indicated that it has the same catalytic properties as the membrane-bound recombinant enzyme or as the enzyme extracted from kidney brush border membranes. Thus the extracellular domain alone is sufficient for conferring full catalytic activity to neutral endopeptidase.     

Regulation and secretion of an extracellular esterase from Streptomyces scabies.

Production of a heat-stable, extracellular esterase by Streptomyces scabies is regulated by zinc ions. The esterase-encoding gene (est) from S. scabies was cloned and expressed in Streptomyces lividans. In S. lividans, expression of the est gene is also regulated by Zn2+, and the esterase is efficiently secreted in this organism. The sequence of the est gene suggests that a 39-amino acid signal peptide is removed during secretion of this protein. Deletion analysis has indicated that the hydrophobic domain of the signal peptide is required for secretion. Gel retardation assays and DNaseI footprinting using an S-30 protein extract from S. scabies have previously identified a specific 23-bp protein-binding site upstream from the est coding sequence. Deletion of this protein-binding sequence significantly decreased expression of the est gene.     

Molecular dissection of the agonist binding site of an AMPA receptor.

Two discontinuous segments (S1 and S2), separated by membrane-associated domains, in ionotropic glutamate receptor (GluR) subunits show sequence similarity to bacterial periplasmic amino acid-binding proteins, suggesting an evolutionary and structural relationship. Experimental evidence arguing for and against the inferred extracellular location of the S1 and S2 domains in GluRs has been presented. Here, we report that an extracellularly expressed fusion protein consisting of the S1 and S2 domains of alpha-amino-5-methyl-3-hydroxyisoxazolone-4-propionate (AMPA)-selective glutamate receptor GluR-D joined together via a hydrophilic linker peptide specifically reproduces the AMPA-binding properties of GluR-D, whereas the separately expressed segments do not bind ligand. This provides direct evidence that the S1 and S2 segments of GluR-D contain the structural determinants necessary and sufficient for selective agonist binding. Dissection of a functional neurotransmitter binding site as a soluble protein separate from the integral membrane channel will facilitate new approaches to analyse the structure of GluRs.     

The Heparin-Binding Activity of Secreted Modular Calcium-Binding Protein 1 (SMOC-1) Modulates Its Cell Adhesion Properties

Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.     

A DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing

Signal-dependent alternative splicing is important for regulating gene expression in eukaryotes, yet our understanding of how signals impact splicing mechanisms is limited. A model to address this issue is alternative splicing of Drosophila TAF1 pre-mRNA in response to camptothecin (CPT)-induced DNA damage signals. CPT treatment of Drosophila S2 cells causes increased inclusion of TAF1 alternative cassette exons 12a and 13a through an ATR signaling pathway. To evaluate the role of TAF1 pre-mRNA sequences in the alternative splicing mechanism, we developed a TAF1 minigene (miniTAF1) and an S2 cell splicing assay that recapitulated key aspects of CPT-induced alternative splicing of endogenous TAF1. Analysis of miniTAF1 indicated that splice site strength underlies independent and distinct mechanisms that control exon 12a and 13a inclusion. Mutation of the exon 13a weak 5' splice site or weak 3' splice site to a consensus sequence was sufficient for constitutive exon 13a inclusion. In contrast, mutation of the exon 12a strong 5' splice site or moderate 3' splice site to a consensus sequence was only sufficient for constitutive exon 12a inclusion in the presence of CPT-induced signals. Analogous studies of the exon 13 3' splice site suggest that exon 12a inclusion involves signal-dependent pairing between constitutive and alternative splice sites. Finally, intronic elements identified by evolutionary conservation were necessary for full repression of exon 12a inclusion or full activation of exon 13a inclusion and may be targets of CPT-induced signals. In summary, this work defines the role of sequence elements in the regulation of TAF1 alternative splicing in response to a DNA damage signal.     

Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1

In this paper, we report the identification and molecular characterization of a splice variant of human Mnk1 which has been named as Mnk1b. Human Mnk1b mRNA is homologous to human Mnk1 mRNA but lacking a region corresponding to exon 19, which causes a change in the reading frame generating a stop codon. The resulting protein lacks the last 89 amino acids at the C-terminal region that are replaced by 12 amino acids with an entirely new sequence. The C-terminal end in Mnk1 corresponds to the extracellular signal-regulated kinase (ERK1/2) binding site. Although Mnk1b lacks this domain and, consequently, is not phosphorylated by ERK1/2, it is able, however, to phosphorylate eIF4E in vitro and in vivo in a mitogen-activated protein kinases-independent manner. This result suggests that Mnk1b may play a key role in regulating protein translation in the absence of stimuli. Interestingly, a significant population of cells shows Mnk1b within the nucleus whereas Mnk1 is always detected in the cytoplasm. This fact may be explained because Mnk1b maintains the nuclear localization signal (NLS) but lacks the nuclear export sequence (NES).     

Cloning of two new splice variants of Siglec-10 and mapping of the interaction between Siglec-10 and SHP-1

Using a three-hybrid strategy in yeast, we have cloned a new splice variant of Siglec-10, called Siglec-10 Sv3. This splice variant lacks part of exon 3, but keeps the reading frame, as well as the crucial regions for interaction with Sias and the motifs for intracellular signaling. The expression of Siglec-10 Sv3 in T- and B-cells was detected by RT-PCR. Moreover, cDNA of another new splicing form of Siglec-10, named Siglec-10 Sv4, was identified by RT-PCR. One common characteristic of all Siglec-10 splice forms (except for Siglec-10 Sv2) is their cytoplasmic tail with two ITIMs and one CD150-like sequence. We confirmed the recruitment of SHP-1 to the Siglec-10 cytoplasmic tail by Western blot analysis and demonstrated that this interaction depends on tyrosine phosphorylation. Mutational analyses showed that ITIM Y609 of Siglec-10 and the N-terminal SH2 domain of SHP-1 play a pivotal role in the interaction between Siglec-10 and SHP-1. Finally, we demonstrated that Siglec-10 was not able to bind SAP/SH2d1A, indicating that the so-called CD150-like motif in Siglec-10 might be a docking site for other signal transduction mediators.     

The C-terminal tail domain of metavinculin, vinculin's splice variant, severs actin filaments

Vinculin and its splice variant, metavinculin (MV), are key elements of multiple protein assemblies linking the extracellular matrix to the actin cytoskeleton. Vinculin is expressed ubiquitously, whereas MV is mainly expressed in smooth and cardiac muscle tissue. The only difference in amino acid sequence between the isoforms is a 68-residue insert in the C-terminal tail domain of MV (MVt). Although the functional role of this insert remains elusive, its importance is exemplified by point mutations that are associated with dilated and hypertrophic cardiomyopathy. In vinculin, the actin binding site resides in the tail domain. In this paper, we show that MVt binds actin filaments similarly to the vinculin tail domain. Unlike its splice variant, MVt did not bundle actin filaments. Instead, MVt promoted severing of actin filaments, most efficiently at substoichiometric concentrations. This surprising and seemingly contradictory alteration of vinculin function by the 68-residue insert may be essential for modulating compliance of vinculin-induced actin bundles when exposed to rapidly increasing external forces.     

Cyclooxygenase variants: the role of alternative splicing

Alternative splicing of cellular pre-mRNA is responsible for production of multiple mRNAs from individual genes. Splice variants are expressed in cell- and tissue-specific contexts that are important in development and physiology. Alternative splicing can serve as a regulatory mechanism whereby developmental programming and environmental factors/stimuli affect biological activities of translated proteins. Cyclooxygenase (COX)-1 and -2 genes produce splice variants whose biological expression, relevance, and activities have been of significant interest. COX variants are produced by a variety of splicing mechanisms. Four structural domains of the COX proteins (the amino terminal signal peptide, membrane-binding domain, dimerization domain, and catalytic domain) are defined by specific COX exons. COX splice variants may, therefore, result in potential changes in protein subcellular localization, dimerization, and activity. COX variant proteins may act in roles which diverge from those of COX-1 and -2.     

A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1

Gamma-secretase-like proteolysis at site 3 (S3), within the transmembrane domain, releases the Notch intracellular domain (NICD) and activates CSL-mediated Notch signaling. S3 processing occurs only in response to ligand binding; however, the molecular basis of this regulation is unknown. Here we demonstrate that ligand binding facilitates cleavage at a novel site (S2), within the extracellular juxtamembrane region, which serves to release ectodomain repression of NICD production. Cleavage at S2 generates a transient intermediate peptide termed NEXT (Notch extracellular truncation). NEXT accumulates when NICD production is blocked by point mutations or gamma-secretase inhibitors or by loss of presenilin 1, and inhibition of NEXT eliminates NICD production. Our data demonstrate that S2 cleavage is a ligand-regulated step in the proteolytic cascade leading to Notch activation.     

Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.