Analysis of the site occupancy constraints of primary amino acid sequences in the motif directing palmitylation of the vaccinia virus 37-kDa envelope protein

Vaccinia virus (VV) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine residues 185186 of the 372-amino acid protein. We have previously reported on a loosely conserved consensus motif. Further analysis has identified a conserved consensus sequence, Hydro*AAC(C)A (Hydro* represents a hydrophobic portion of a protein determined by any one of the following: a hydrophobic sequence, a transmembrane domain 1-12 amino acids away from the modification site, or the prior addition of a hydrophobic molecule; C, palmitate acceptor cysteines; A, aliphatic residue) that is responsible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstreamdownstream of the palmitate acceptor residues in p37 by site-directed mutagenesistransient expression of mutated proteins in VV-infected cells. The two aliphatic alanines naturally found at positions 183184 of the wild-type p37 allow for efficient palmitylation. In contrast, the replacement of leucine at position 187 with glycine increases palmitylation efficiency. The 10 amino acids immediately upstream of the palmitate acceptor site are absolutely necessary while the downstream 10 amino acids are dispensable. These results together with previous data suggests that the Hydro*AAC(C)A motif is required for efficient palmitylation of p37.     

Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.     

A conserved functional domain of Drosophila coracle is required for localization at the septate junction and has membrane-organizing activity

The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members.     

The 30-kD domain of protein 4.1 mediates its binding to the carboxyl terminus of pICln, a protein involved in cellular volume regulation

Erythrocyte protein 4.1 (P4.1) is an 80-kD cytoskeletal protein that is important for the maintenance of the structural integrity and flexibility of the red blood cell membrane. Limited chymotryptic digestion of erythroid P4.1 yields 4 structural domains corresponding to the 30-, 16-, 10-, and 22/24-kD domains. Using a yeast two-hybrid system, we isolated cDNA clones encoding pICln that specifically interacts with the 30-kD domain of P4.1. In this report, we show that the carboxyl-terminus (amino acid residues 103-237) of pICln binds to the 30-kD domain of P4.1 in a yeast two-hybrid system. The direct association between the 30-kD domain of P4.1 and pICln was further confirmed by the following findings: (1) the S35-methione-labeled pICln specifically bound to both GST/P4.1-80 (80 kD) and GST/P4.1-30 (30 kD) fusion proteins, but not to the proteins that lack the 30-kD domain; (2) coimmunoprecipitation analysis of the cell extracts from transfected SiHa cells showed that pICln and P4.1 associate in transfected cells. It was reported that pICln can form a complex with actin and may play a role involved in cellular volume regulation. The direct association between P4.1 and pICln suggests that pICln may link P4.1-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel.     

Identification of conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit critical for function. Evidence for formation of a heterodimeric receptor complex prior to ligand binding

A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues, a tryptophan-serine motif (WSXWS) in the extracellular domain, and a proline-rich cytoplasmic domain. The high affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (hGM-CSFR) consists of two subunits, alpha (hGM-CSFR alpha) and beta (hGM-CSFR beta), both of which are members of the receptor superfamily. In this study, we prepared mutations in conserved amino acids of the receptor subunit necessary for GM-CSF binding (hGM-CSFR alpha) and analyzed mutant receptors for low affinity binding, internalization, and high affinity binding when complexed with the beta subunit. Mutations in the cytoplasmic domain did not affect GM-CSF binding or receptor internalization. Mutation of a single conserved serine residue within the WSXWS motif diminishes cell surface receptor expression but not ligand binding. Mutation of either the second or third conserved cysteine residue of hGM-CSFR alpha resulted in complete loss of low affinity binding; however, co-expression of the cysteine 2 mutant with hGM-CSFR beta yielded a high affinity receptor complex. Since neither the cysteine 2 mutant nor the beta subunit can bind ligand alone, this result suggests that hGM-CSFR alpha and hGM-CSFR beta exist in a preformed heterodimeric protein complex on the plasma membrane.     

Identification of a novel cortactin SH3 domain-binding protein and its localization to growth cones of cultured neurons

Cortactin is an actin-binding protein that contains several potential signaling motifs including a Src homology 3 (SH3) domain at the distal C terminus. Translocation of cortactin to specific cortical actin structures and hyperphosphorylation of cortactin on tyrosine have been associated with the cortical cytoskeleton reorganization induced by a variety of cellular stimuli. The function of cortactin in these processes is largely unknown in part due to the lack of information about cellular binding partners for cortactin. Here we report the identification of a novel cortactin-binding protein of approximately 180 kDa by yeast two-hybrid interaction screening. The interaction of cortactin with this 180-kDa protein was confirmed by both in vitro and in vivo methods, and the SH3 domain of cortactin was found to direct this interaction. Since this protein represents the first reported natural ligand for the cortactin SH3 domain, we designated it CortBP1 for cortactin-binding protein 1. CortBP1 contains two recognizable sequence motifs within its C-terminal region, including a consensus sequence for cortactin SH3 domain-binding peptides and a sterile alpha motif. Northern and Western blot analysis indicated that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies revealed colocalization of CortBP1 with cortactin and cortical actin filaments in lamellipodia and membrane ruffles in fibroblasts expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin was also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during neurite outgrowth.     

Role of a BRCT domain in the interaction of DNA ligase III-alpha with the DNA repair protein XRCC1

The BRCT domain (for BRCA1 carboxyl terminus) is a protein motif of unknown function, comprising approximately 100 amino acids in five conserved blocks denoted A-E. BRCT domains are present in the tumour suppressor protein BRCA1 [1-3], and the domain is found in over 40 other proteins, defining a superfamily that includes DNA ligase III-alpha and the essential human DNA repair protein XRCC1. DNA ligase III-alpha and XRCC1 interact via their carboxyl termini, close to or within regions that contain a BRCT domain [4]. To examine whether the primary role of the carboxy-terminal BRCT domain of XRCC1 (denoted BRCT II) is to mediate the interaction with DNA ligase III-alpha, we identified the regions of the domain that are required and sufficient for the interaction. An XRCC1 protein in which the conserved D-block tryptophan was disrupted by point mutation retained the ability to interact with DNA ligase III-alpha, so this tryptophan must mediate a different, although conserved, role. XRCC1 in which the weakly conserved C-block was mutated lost the ability to interact with DNA ligase III-alpha. Moreover, 20 amino acids spanning the C-block of BRCT II conferred full DNA ligase III-alpha binding activity upon an unrelated polypeptide. An XRCC1 protein in which this 20mer was deleted could not maintain normal levels of DNA ligase III-alpha in transfected rodent cells, a phenotype associated with defective repair [5]. In summary, these data demonstrate that a BRCT domain can mediate a biologically important protein-protein interaction, and support the existence of additional roles.     

Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase

The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of approximately 200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.     

Neurobehavioral deficits in mice lacking the erythrocyte membrane cytoskeletal protein 4.1

The erythrocyte membrane cytoskeletal protein 4.1 (4.1R) is a structural protein that confers stability and flexibility to erythrocytes via interactions with the cytoskeletal proteins spectrin and F-actin and with the band 3 and glycophorin C membrane proteins. Mutations in 4.1R can cause hereditary elliptocytosis, a disease characterized by a loss of the normal discoid morphology of erythrocytes, resulting in hemolytic anemia [1]. Different isoforms of the 4.1 protein have been identified in a wide variety of nonerythroid tissues by immunological methods [2-5]. The variation in molecular weight of these different 4.1 isoforms, which range from 30 to 210 kDa [6], has been attributed to complex alternative splicing of the 4.1R gene [7]. We recently identified two new 4.1 genes: one is generally expressed throughout the body (4. 1G) [8] and the other is expressed in central and peripheral neurons (4.1N) [9]. Here, we examined 4.1R expression by in situ hybridization analysis and found that 4.1R was selectively expressed in hematopoietic tissues and in specific neuronal populations. In the brain, high levels of 4.1R were discretely localized to granule cells in the cerebellum and dentate gyrus. We generated mice that lacked 4.1R expression; these mice had deficits in movement, coordination, balance and learning, in addition to the predicted hematological abnormalities. The neurobehavioral findings are consistent with the distribution of 4.1R in the brain, suggesting that 4.1R performs specific functions in the central nervous system.     

Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography

Receptor-associated protein (RAP) is an endoplasmic reticulum/Golgi protein involved in the processing of receptors of the low density lipoprotein receptor family. A approximately 95-kDa membrane glycoprotein, designated gp95/sortilin, was purified from human brain extracts by RAP affinity chromatography and cloned in a human cDNA library. The gene maps to chromosome 1p and encodes an 833-amino acid type I receptor containing an N-terminal furin cleavage site immediately preceding the N terminus determined in the purified protein. Gp95/sortilin is expressed in several tissues including brain, spinal cord, and testis. Gp95/sortilin is not related to the low density lipoprotein receptor family but shows intriguing homologies to established sorting receptors: a 140-amino acid lumenal segment of sortilin representing a hitherto unrecognized type of extracellular module shows extensive homology to corresponding segments in each of the two lumenal domains of yeast Vps10p, and the extreme C terminus of the cytoplasmic tail of sortilin contains the casein kinase phosphorylation consensus site and an adjacent dileucine sorting motif that mediate assembly protein-1 binding and lysosomal sorting of the mannose-6-phosphate receptors. Expression of a chimeric receptor containing the cytoplasmic tail of gp95/sortilin demonstrates evidence that the tail conveys colocalization with the cation-independent mannose6-phosphate receptor in endosomes and the Golgi compartment.     

Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5

Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. We have purified human calnexin in association with the human interferon-gamma receptor and cloned calnexin cDNA from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind 45Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the "E-F hand" motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. We have also determined the cDNA sequences of mouse and rat calnexins. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35.     

In vitro assembly of the mouse U14 snoRNP core complex and identification of a 65-kDa box C/D-binding protein

The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) that have been categorized into two major families based on evolutionarily conserved sequence elements. U14 snoRNA is a member of the larger, box C/D snoRNA family and possesses nucleotide box C and D consensus sequences. In previous studies, we have defined a U14 box C/D core motif that is essential for intronic U14 snoRNA processing. These studies also revealed that nuclear proteins that recognize boxes C/D are required. We have now established an in vitro U14 snoRNP assembly system to characterize protein binding. Electrophoretic mobility-shift analysis demonstrated that all the sequences and structures of the box C/D core motif required for U14 processing are also necessary for protein binding and snoRNP assembly. These required elements include a base paired 5',3' terminal stem and the phylogenetically conserved nucleotides of boxes C and D. The ability of other box C/D snoRNAs to compete for protein binding demonstrated that the box C/D core motif-binding proteins are common to this family of snoRNAs. UV crosslinking of nuclear proteins bound to the U14 core motif identified a 65-kDa mouse snoRNP protein that requires boxes C and D for binding. Two additional core motif proteins of 55 and 50 kDa were also identified by biochemical fractionation of the in vitro-assembled U14 snoRNP complex. Thus, the U14 snoRNP core complex is a multiprotein particle whose assembly requires nucleotide boxes C and D.     

Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinase activities

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We and others have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting signaling molecules within caveolae membranes. In this regard, it has been shown that a 20-amino acid membrane-proximal region of the cytosolic NH2-terminal domain of caveolin is sufficient to mediate the interaction of caveolin with signaling proteins, namely G-proteins, Src-like kinases, eNOS, and H-Ras. This caveolin-derived protein domain has been termed the caveolin-scaffolding domain. Binding of the caveolin-scaffolding domain functionally suppresses the activity of G-protein alpha subunits, eNOS, and Src-like kinases, suggesting that caveolin binding may also play a negative regulatory role in signal transduction. Here, we report the direct interaction of caveolin with a growth factor receptor, EGF-R, a known caveolae-associated receptor tyrosine kinase. Two consensus caveolin binding motifs have been previously defined using phage display technology. One of these motifs is present within the conserved kinase domains of most known receptor tyrosine kinases (termed region IX). We now show that this caveolin binding motif within the kinase domain of the EGF-R can mediate the interaction of the EGF-R with the scaffolding domains of caveolins 1 and 3 but not with caveolin 2. In addition, the scaffolding domains of caveolins 1 and 3 both functionally inhibit the autophosphorylation of the EGF-R kinase in vitro. Importantly, this caveolin-mediated inhibition of the EGF-R kinase could be prevented by the addition of an EGF-R-derived peptide that (i) contains a well conserved caveolin binding motif and (ii) is located within the kinase domain of the EGF-R and most known receptor tyrosine kinases. Similar results were obtained with protein kinase C, a serine/threonine kinase, suggesting that caveolin may function as a general kinase inhibitor. The implications of our results are discussed within the context of caveolae-mediated signal transduction. In this regard, caveolae-coupled signaling might explain how linear signaling pathways can branch and interconnect extensively, forming a signaling module or network.     

Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma

Previously we identified four proteins in seminal plasma that were associated with bull fertility. The purpose of this study was to identify the 55-kDa protein prevalent in seminal plasma of higher-fertility males. The 55-kDa protein was quantified by video densitometry in two-dimensional electrophoresis gels of seminal plasma from 26 bulls of known fertility. Relative density of the 55-kDa protein was positively correlated (r = 0.48) with bull fertility. The 55-kDa (pI 4.5) fertility-associated protein spot was isolated by electroelution after two-dimensional PAGE separation of seminal plasma of 36 bulls. N-terminal sequence analysis of the pure protein yielded a 15-amino acid sequence (VKPXSSGXSEEKQLN) that was 86% homologous to bovine osteopontin-k precursor. Polyclonal antiserum generated against the 55-kDa protein reacted with a single spot in two-dimensional PAGE Western blots of seminal plasma. Western blot analyses using polyclonal antisera generated against the amino terminus (LF123) and carboxyl terminus (LF124) of human recombinant osteopontin confirmed that the 55-kDa polypeptide was osteopontin. Partially purified 55-kDa protein was obtained by HPLC-MonoQ column chromatography. Protein characterization revealed that the 55-kDa protein was glycosylated, but not phosphorylated, consistent with the identity of the 55-kDa protein as osteopontin.     

Yrb2p is a nuclear protein that interacts with Prp20p, a yeast Rcc1 homologue

A conserved family of Ran binding proteins (RBPs) has been defined by their ability to bind to the Ran GTPase and the presence of a common region of approximately 100 amino acids (the Ran binding domain). The yeast Saccharomyces cerevisiae genome predicts only three proteins with canonical Ran binding domains. Mutation of one of these, YRB1, results in defects in transport of macromolecules across the nuclear envelope (Schlenstedt, G., Wong, D. H., Koepp, D. M., and Silver, P. A. (1995) EMBO J. 14, 5367-5378). The second one, encoded by YRB2, is a 327-amino acid protein with a Ran binding domain at its C terminus and an internal cluster of FXFG and FG repeats conserved in nucleoporins. Yrb2p is located inside the nucleus, and this localization relies on the N terminus. Results of both genetic and biochemical analyses show interactions of Yrb2p with the Ran nucleotide exchanger Prp20p/Rcc1. Yrb2p binding to Gsp1p (yeast Ran) as well as to a novel 150-kDa GTP-binding protein is also detected. The Ran binding domain of Yrb2p is essential for function and for its association with Prp20p and the GTP-binding proteins. Taken together, we suggest that Yrb2p may play a role in the Ran GTPase cycle distinct from nuclear transport.     

Complete deficiency of glycophorin A in red blood cells from mice with targeted inactivation of the band 3 (AE1) gene

Glycophorin A is the major transmembrane sialoglycoprotein of red blood cells. It has been shown to contribute to the expression of the MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane. Several lines of evidence suggest a close interaction between glycophorin A and band 3 during their biosynthesis. Recently, we have generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell membrane proteins. In this report, we show that the band 3 -/- red blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin C. In contrast, the band 3 -/- red blood cells are completely devoid of glycophorin A (GPA), as assessed by Western blot and immunocytochemistry techniques, whereas the polymerase chain reaction (PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase experiments show that GPA is not incorporated in the membrane and is rapidly degraded in the cytoplasm. Based on these findings and other published evidence, we propose that band 3 plays a chaperone-like role, which is necessary for the recruitment of GPA to the red blood cell plasma membrane.     

Mutational analysis of the Mu transposase. Contributions of two distinct regions of domain II to recombination

Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH2-terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the Asp-Asp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH2-terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.