Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains. Roles of the CGLCG motif in the D1- and D3-domains

Plasmids encoding the amino-terminal region of porcine submaxillary mucin were modified by site-specific mutagenesis to assess the roles of individual half-cystine residues in the assembly of disulfide-linked multimers of mucin. COS-7 cells with the plasmid containing C1199A expressed primarily monomers, suggesting that half-cystine 1199 in the D3-domain is involved in forming mucin multimers. This residue is in the sequence C1199SWRYEPCG, which is highly conserved in the D3-domain of other secreted mucins and human prepro-von Willebrand factor. In contrast, cells with the plasmid containing C1276A expressed trimers like those with unmutated plasmid, suggesting that half-cystine 1276 is not involved in formation of disulfide-bonded multimers. The roles of the half-cystines in the CGLCG motifs in the assembly of disulfide-bonded multimers of mucin were also assessed. Cells with plasmids in which both half-cystines in the motif in the D1- or D3-domain of mucin are replaced by alanine expressed proteins that were poorly secreted, suggesting that these mutations impair normal folding of the expressed proteins. A plasmid with a mutant D1-domain motif expressed monomers, whereas one with a mutant D3-domain motif expressed monomers and trimers. However, the trimers expressed by the latter plasmid were assembled in non-acidic compartments, as judged by expression studies in the presence of monensin, which inhibits trimer formation by unmutated plasmid, but not by the mutant plasmid. These results suggest that the CGLCG motif in the D1-domain is required for multimerization in the trans-Golgi complex. However, the CGLCG motif in the D3-domain appears to prevent formation of mucin multimers in non-acidic compartments of the cell. Plasmids encoding the D1- and D2-domains, the D1- and D3-domains, or only the D3-domain also expressed oligomers in the presence of monensin, suggesting that the three D-domains must be contiguous to avoid multimerization in non-acidic compartments. It is possible that these motifs in mucins are engaged in the thiol-disulfide interchange reactions during the assembly of disulfide-bonded multimers of mucin.     

The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase

The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues. We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.     

An epidemiological study of diagnostic and therapeutic strategies in office practice patients with subacute or chronic pain in the thoracic or low back. Comparison of practices in primary care and rheumatology settings

Mucin was purified by the gel filtration method on columns with high porous molecular sives in buffers with SDS and proteinase inhibitors. The addition of proteinase inhibitors distinctly inhibited proteolytic activity. It was found that the obtained mucin, after disulphide-bound reduction, is dissociated to mucin subunits and N-glycosylated glycoprotein of molecular weight about 75 kDa. This protein has carbohydrate and amino acid composition different from high molecular fraction. The 75 kDa protein is strongly associated with high molecular mass mucin subunits and can be separated either during electrophoresis or fractionation in buffers with 2-mercaptoethanol.     

Assembly of amino-terminal fibronectin dimers into the extracellular matrix

Fibronectin is a dimeric adhesion molecule that consists of three types of repeating modules. Adherent cells bind soluble fibronectin and incorporate it into insoluble fibrils in the extracellular matrix. The amino-terminal 70-kDa portion of fibronectin mediates binding to the cell surface, but amino-terminal fragments do not accumulate in the extracellular matrix. The ninth type I and first type III modules, the cell adhesion region, and the cysteines that form the interchain disulfide bonds have also been implicated in matrix assembly. To further define which regions of fibronectin are essential for matrix assembly, we generated a dimeric protein (d70 kDa) in which the 70-kDa amino terminus is directly linked to the last 51 amino acids of fibronectin, which contain the cysteines involved in interchain disulfide bonding. d70 kDa bound to cells and accumulated in the extracellular matrix. Incorporation of d70 kDa into the extracellular matrix was dependent upon protein synthesis; in cycloheximide-treated cultures that lacked a pre-existing matrix, d70 kDa accumulated in the extracellular matrix only in the presence of intact fibronectin. Monomeric 70-kDa protein was not incorporated into the matrix in the presence or absence of cycloheximide. These data indicate that fibronectin molecules containing only the amino-terminal 70-kDa region and the carboxyl-terminal 51 amino acids can become assembled into the extracellular matrix.     

Involvement of the endoplasmic reticulum in the assembly and envelopment of African swine fever virus

African swine fever (ASF) virus is a large enveloped DNA virus assembled in the cytoplasm of cells. In this study, the membrane compartments involved in the envelopment of ASF virus were investigated. A monoclonal antibody recognizing p73, the major structural protein of ASF virus, was generated to analyze the binding of p73 to membranes during the assembly of the virus. Approximately 50% of the intracellular pool of p73 associated with membranes as a peripheral membrane protein. Binding was rapid and complete within 15 min of synthesis. Subcellular membrane fractionation showed that newly synthesized p73 molecules cosedimented with endoplasmic reticulum (ER) membranes and remained associated with the ER during a 2-h chase. A similar distribution on gradients was recorded for p17, a structural membrane protein of ASF virus. The results suggested that the ER was involved in the assembly of ASF virus. A protease protection assay demonstrated a time-dependent envelopment of the membrane bound, but not cytosolic, pool of p73. Envelopment of p73 took place 1 h after binding to membranes and was completed 1 h before the first detection of p73 in virions secreted from cells. Envelopment was unaffected by brefeldin A and monensin, drugs that block membrane transport between the ER and Golgi. Taken together the results provide evidence for the binding of ASF virus structural proteins to a specific membrane compartment and implicate a role for the ER in the assembly and envelopment of ASF virus.     

The carboxyl-terminal region of protein C is essential for its secretion

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.     

Cloning and deduced amino acid sequence of a novel cartilage protein (CILP) identifies a proform including a nucleotide pyrophosphohydrolase

The cDNA cloning and expression in vitro and in eukaryotic cells of a novel protein isolated from human articular cartilage, cartilage intermediate layer protein (CILP) is described. A single 4. 2-kilobase mRNA detected in human articular cartilage encodes a polypeptide of 1184 amino acids with a calculated molecular mass of 132.5 kDa. The protein has a putative signal peptide of 21 amino acids, and is a proform of two polypeptides. The amino-terminal half corresponds to CILP (molecular mass of 78.5 kDa, not including post-translational modifications) and the carboxyl-terminal half corresponds to a protein homologous to a porcine nucleotide pyrophosphohydrolase, NTPPHase (molecular mass of 51.8 kDa, not including post-translational modifications). CILP has 30 cysteines and six putative N-glycosylation sites. The human homolog of porcine NTPPHase described here contains 10 cysteine residues and two putative N-glycosylation sites. In the precursor protein the NTPPHase region is immediately preceded by a tetrapeptide conforming to a furin proteinase cleavage consensus sequence. Expression of the full-length cDNA in a cell-free translation system and in COS-7 or EBNA cells indicates that the precursor protein is synthesized as a single polypeptide chain that is processed, possibly by a furin-like protease, into two polypeptides upon or preceding secretion.     

Short-term and long-term cytokine release by mouse bone marrow mast cells and the differentiated KU-812 cell line are inhibited by brefeldin A

Mast cells and basophils produce a wide range of cytokines, including large amounts of both IL-6 and granulocyte-macrophage CSF (GM-CSF). However, the route by which cytokines are secreted is poorly understood. In the current study, we used two inhibitors of vesicular transport, brefeldin A and monensin, to examine the routes of secretion of IL-6 and GM-CSF in the differentiated KU812 human cell line and cultured mouse bone marrow mast cells (mBMMC). Studies of cytokine production over 6 to 24 h demonstrated that IL-6 and GM-CSF release from both cell types were inhibited by brefeldin A (BFA) following activation with calcium ionophore, A23187. Monensin had similar inhibitory effects to that of BFA on the initial and ongoing IL-6 release from KU812 cells. In contrast, the amount of each cytokine remaining within the cells was significantly enhanced. Similar results were obtained following IgE-mediated activation of mBMMC. BFA significantly inhibited both the constitutive secretion of IL-6 and the immediate ionophore-induced increase in IL-6 release from KU812 cells at 20 min postactivation. However, treatment with these agents did not alter the release of histamine and beta-hexaminidase from either mBMMC or KU812 cells. These studies suggest that both the initial 20-min release of IL-6 and secretion of IL-6 and GM-CSF over up to 24 h by mBMMC and differentiated KU-812 cells occur predominately through a vesicular transport-dependent mechanism, and that little, if any, IL-6 and GM-CSF is released through degranulation.     

Apical sorting of bovine enteropeptidase does not involve detergent-resistant association with sphingolipid-cholesterol rafts

Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts.     

Atypical porcine type I interferon. Biochemical and biological characterization of the recombinant protein expressed in insect cells

A recombinant baculovirus was designed to express short porcine type I interferon (spI interferon), a novel and atypical type I interferon that was recently described as the product of a gene transcribed in pig trophoblast at the time of implantation in the uterus [Lefèvre, F. & Boulay, V.C. (1993) J. Biol. Chem. 268, 19,760-19,768]. The recombinant protein, secreted into the culture medium of Sf9 cells at 3 days post infection (60,000 IU/ml), was purified by ion-exchange and reverse-phase HPLC. N-terminal sequencing confirmed the predicted signal peptide cleavage site and therefore the size of the mature protein (149 amino acids), the shortest of all reported type I interferons. Purified spI interferon, with a specific antiviral activity using Madin-Darby bovine kidney cells of 3.7 x 10(7) IU/mg, is an N-glycosylated monomer of 19 kDa that possesses several physicochemical characteristics of interferons: (a) disulfide bonds are necessary for bioactivity; spI interferon is thermolabile, stable at pH 2, and able to renature after complete denaturation (1% 2-mercaptoethanol, 1% SDS, and 5 M urea); (b) the carbohydrate chain is not essential for bioactivity since no loss of antiviral activity is observed following complete deglycosylation. In this study, antiviral and anti-proliferation activities of spI interferon in cell culture were compared with those of other interferons, especially with porcine type 1 interferon-alpha. A major difference with porcine type 1 interferon-alpha was that spI interferon was not active on human cells in either test, and it was relatively more active on pig cells compared to bovine cells than porcine type 1 interferon-alpha. Serological cross-neutralization results obtained with anti-(spI interferon) serum confirmed that several members of interferon families are not antigenically related to spI interferon, in agreement with previous observations; this provides further evidence that spI interferon could represent a new family of type I interferon.     

Apolipoprotein-mediated cellular cholesterol and phospholipid efflux depend on a functional Golgi apparatus

Several studies have demonstrated that lipid-free apolipoproteins can promote cholesterol and phospholipid efflux from cells; however, the mechanisms and the role of cell-mediated pathways involved remain incompletely elucidated. We have recently demonstrated that brefeldin A or monensin, agents that disrupt Golgi apparatus structure and function, inhibit intracellular cholesterol efflux from cells to high density lipoproteins. In the present study we examined the effects of those agents on cell cholesterol and phospholipid efflux to purified apolipoprotein A-I (apoA-I) and apolipoprotein-depleted acceptors from cholesterol-loaded fibroblasts. Brefeldin A or monensin treatment of cells during incubation with apoA-I inhibited efflux of cellular cholesterol by greater than 80% compared with control cells, measured by changes in cellular cholesterol radioactivity, mass, and the substrate pool of cholesterol available for esterification by acyl coenzyme A:cholesterol acyltransferase. Inhibition of cholesterol efflux by these agents could not be overcome by increasing the apoA-I concentration and persisted during incubations up to 24 h. Similarly, brefeldin A and monensin inhibited up to 80% of apoA-I-mediated efflux of labeled phospholipids from cholesterol-loaded cells relative to controls. In contrast, lipid efflux mediated by apolipoprotein-depleted acceptors (trypsin-modified HDL and sonicated phospholipid vesicles) was not sensitive to these drugs. On the basis the known effects of brefeldin A and monensin on Golgi apparatus structure and function, these results are consistent with the notion that efflux of cell lipids by apolipoprotein-dependent mechanisms, but not by apolipoprotein-independent mechanisms, require active cellular processes involving an intact and functional Golgi apparatus.     

Identification of the region of alpha-catenin that plays an essential role in cadherin-mediated cell adhesion

alpha-Catenin is an intrinsic component of the cadherin adhesion complex and is a 102-kDa protein with multiple interaction sites, including homodimerization sites, and binding sites for beta- and gamma-catenin (plakoglobin), alpha-actinin, and actin. Besides the binding to beta- or gamma-catenin, it is unknown, however, which interaction is critical for the function of cadherins. By expressing a series of E-cadherin-alpha-catenin chimeric molecules on leukemia cells (K562), we have identified the region of alpha-catenin that confers aggregation inducing activity to nonfunctional tail-less E-cadherin. The region has been mapped to the carboxyl-terminal 295 amino acids of alpha-catenin. Consistent with this result, expression in alpha-catenin-deficient cells (DLD-1/Delta alpha) of a mutant alpha-catenin molecule consisting of the amino-terminal beta-/gamma-catenin-binding site and the carboxyl-terminal cell adhesion region identified in the above experiments induced E-cadherin-mediated cell aggregation and compaction. Cells expressing E-cadherin chimeric molecules with the homologous carboxyl-terminal region of vinculin, which contains the actin-binding site of vinculin, did not, however, aggregate as strongly as ones expressing E-cadherin-alpha-catenin chimeric molecules.     

Simian sarcoma-associated virus fails to infect Chinese hamster cells despite the presence of functional gibbon ape leukemia virus receptors

We have sequenced the envelope genes from each of the five members of the gibbon ape leukemia virus (GALV) family of type C retroviruses. Four of the GALVs, including GALV strain SEATO (GALV-S), were originally isolated from gibbon apes, whereas the fifth member of this family, simian sarcoma-associated virus (SSAV), was isolated from a woolly monkey and shares 78% amino acid identity with GALV-S. To determine whether these viruses have identical host ranges, we evaluated the susceptibility of several cell lines to either GALV-S or SSAV infection. GALV-S and SSAV have the same host range with the exception of Chinese hamster lung E36 cells, which are susceptible to GALV-S but not SSAV. We used retroviral vectors that differ only in their envelope composition (e.g., they contain either SSAV or GALV-S envelope protein) to show that the envelope of SSAV restricts entry into E36 cells. Although unable to infect E36 cells, SSAV infects GALV-resistant murine cells expressing the E36-derived viral receptor, HaPit2. These results suggest that the receptors present on E36 cells function for SSAV. We have constructed several vectors containing GALV-S/SSAV chimeric envelope proteins to map the region of the SSAV envelope that blocks infection of E36 cells. Vectors bearing chimeric envelopes comprised of the N-terminal region of the GALV-S SU protein and the C-terminal region of SSAV infect E36 cells, whereas vectors containing the N-terminal portion of the SSAV SU protein and C-terminal portion of GALV-S fail to infect E36 cells. This finding indicates that the region of the SSAV envelope protein responsible for restricting SSAV infection of E36 cells lies within its amino-terminal region.     

The C terminus of the hepatitis B virus e antigen precursor is required for a tunicamycin-sensitive step that promotes efficient secretion of the antigen

The Hepatitis B virus encodes the secreted e antigen (HBe) whose function in the viral life cycle is unknown. HBe derives from a 25-kDa precursor that is directed to the secretory pathway. After cleavage of the signal sequence, the resulting 22-kDa protein (P22) is processed in a post-endoplasmic reticulum compartment to mature HBe by removal of the 34-amino acid C-terminal domain. The efficiency of HBe secretion is specifically decreased in cells grown in the presence of tunicamycin, an inhibitor of N-glycosylation. Inasmuch as HBe precursor is not N-glycosylated, our data suggest that a cellular tunicamycin-sensitive protein increases the intracellular transport through the HBe secretory pathway. The study of the secretion of HBe derived from C-terminal-truncated precursors demonstrates that the tunicamycin-sensitive secretion absolutely requires a part of the C-terminal region that is removed to form mature HBe, indicating that the cellular tunicamycin-sensitive protein increases the efficiency of the intracellular transport of P22. We have also shown that the Escherichia coli beta-galactosidase can be secreted when fused to the HBe precursor signal sequence and that the P22 C-terminal domain renders the secretion of this reporter protein also tunicamycin-sensitive.     

A 50-kDa variant form of human surfactant protein D

The dominant form of human surfactant protein D (SP-D) is a multimeric collagenous glycoprotein composed of monomeric subunits that have a molecular mass of 43 kDa under reducing conditions. However, in evaluating monoclonal antibodies to human SP-D, an additional monomeric subunit was identified with a reduced molecular mass of 50 kDa. This 50-kDa variant was detected in approximately half of the samples evaluated and was found in lavage fluid from normal subjects, patients with alveolar proteinosis or idiopathic pulmonary fibrosis and in amniotic fluid. This 50-kDa variant had the same amino-terminal sequence, amino acid composition and apparent size of the carboxy-terminal collagenase-resistant fragment (20 kDa) as the 43-kDa subunit. The major difference was in the amino-terminal portion of the molecule and was due to altered glycosylation, as determined by carbohydrate staining, chemical deglycosylation, treatment with N-glycanase and neuraminidase and reduced signals for threonine at positions 5, 9 and 10 during amino-terminal sequencing. After gel filtration chromatography, the 50-kDa form was not present in the high molecular weight fraction, which is commonly used in purification of SP-D, but was found only in the smaller molecular weight fraction of monomers and trimers of SP-D. In conclusion, the 50 kDa-form of surfactant protein D is produced by post-translational glycosylation and does not form higher ordered oligomers, but its precise physiological function remains to be determined.     

Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-beta, the TGF-beta propeptide, and the latent TGF-beta binding protein (LTBP). To interact with its cell surface receptors, TGF-beta must be released from the latent complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-beta. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S-transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in cross-linking and formation of TGF-beta by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-beta generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (deltaN293) or 441 (deltaN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that deltaN293 LTBP-1S was matrix associated via a transglutaminase-dependent reaction, whereas deltaN441 LTBP-1S was not. This suggests that residues 294-441 are critical to the transglutaminase reactivity of LTBP-1S.     

Protein tyrosine phosphorylation in signalling pathways leading to the activation of gelatinase A: activation of gelatinase A by treatment with the protein tyrosine phosphatase inhibitor sodium orthovanadate

Fibroblasts in monolayer culture secrete gelatinase A (MMP2; 72 kDa type IV collagenase) only in its proenzyme form. Unlike other secreted matrix metalloproteinases, progelatinase A is refractory to activation by serine proteinases. Disparate agents, including monensin, cytochalasin D, and concanavalin A, have been found to mediate the activation of gelatinase A zymogen secreted by fibroblast monolayers. Our finding that monensin-mediated activation can be reversed by the protein tyrosine kinase inhibitor genistein (Li et al., Experimental Cell Research 232 (1997) 332) prompted us to investigate the effect of the specific inhibitor of protein tyrosine phosphatases, sodium orthovanadate, on progelatinase A activation. Treatment of fibroblast monolayers with orthovanadate also results in the secretion of activated gelatinase A. This activation is dose- and time-dependent, requires protein synthesis, and is associated with cell membranes. Vanadate-mediated activation does not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. As with progelatinase activation mediated by monensin, concanavalin A, and cytochalasin D, orthovanadate treatment results in increased synthesis of the membrane proteinase MT1-MMP, that can catalyze the activation of progelatinase A. Protein tyrosine kinase inhibitors are able to prevent the increase of MT1-MMP mRNA, as shown by Northern blot and RT-PCR. In addition, orthovanadate potentiates the effects of monensin and concanavalin A. While treatment with monensin or concanavalin A result only in an increase of the putative activator MT1-MMP, orthovanadate also reduces the production of the specific inhibitor TIMP-2. These experiments implicate protein tyrosine phosphorylation in the signal transduction pathways which lead to the activation of progelatinase A.     

Lower extremity arterial occlusions in young patients with Crohn''s colitis and premature atherosclerosis: report of six cases

AlphaKAP is a protein produced from a gene within the gene of the a isoform of calmodulin-dependent protein kinase II (CaM-kinase IIa). It consists of the association domain of CaM-kinase IIa and a highly hydrophobic amino-terminal stretch consisting of 25 amino acids which is absent from CaM-kinase IIalpha. We previously demonstrated that alphaKAP is an integral membrane protein by subcellular fractionation analysis [Sugai, R., Takeuchi, M., Okuno, S., and Fujisawa, H. (1996) J. Biochem. 120, 773-779], but the exact subcellular localization of alphaKAP was not well understood. Here we demonstrate that alphaKAP is localized on the nuclear membrane of COS-7 cells transiently expressing alphaKAP. The nuclear membrane and perinuclear small vesicles were immunostained with an antibody against a synthetic peptide corresponding to the carboxyl-terminal 15 amino acids of alphaKAP. In contrast to the intact alphaKAP, the mutant alphaKAP, from which the hydrophobic amino-terminal segment had been deleted, accumulated within nuclei. Thus, alphaKAP may function as an anchoring protein for CaM-kinase II and/or other proteins in the perinuclear membrane.