Mutagenesis of cardiac troponin I. Role of the unique NH2-terminal peptide in myofilament activation

Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Yet, how the NH2-terminal extension may itself affect thin filament Ca2+ signaling is unknown. To approach this question we have used molecular cloning, mutagenesis, and bacterial synthesis of a full-length cTnI and a truncated mutant (cTnI/NH2) missing the 32 amino acids. Using reconstituted preparations we could show no differences between cTnI and cTnI/NH2 either in inhibition of actomyosin ATPase activity, in Ca(2+)-reversible inhibitory activity, or in the relation between pCa and Ca2+ binding to the regulatory site of cTnC at either pH 7.0 or 6.5. There were also no significant differences at either pH in the pCa-MgATPase activity relation of myofibrils into which the various species of TnI has been exchanged. Our results indicate: 1) that phosphorylation most likely induces a new state of TnI activity rather than altering an intrinsic effect of the NH2-terminal peptide on Ca2+ activation; and 2) that domains outside the NH2-terminal extension are important with regard to differences in effects of acidic pH on Ca2+ activation on cardiac and skeletal myofilaments.     

The Shp-2 tyrosine phosphatase has opposite effects in mediating the activation of extracellular signal-regulated and c-Jun NH2-terminal mitogen-activated protein kinases

Shp-2 is a widely expressed cytoplasmic tyrosine phosphatase with two SH2 domains. A targeted mutant allele of the Shp-2 gene with a deletion of 65 amino acids in the NH2-terminal SH2 domain was created that leads to embryonic lethality at mid-gestation in homozygous mutant mice. To define the Shp-2 function in cell signaling, we have established mutant fibroblast cell lines, and have examined the effect of the Shp-2 mutation on extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. Insulin-like growth factor (IGF)-I-induced ERK activation was completely abolished, while ERK activity upon platelet-derived growth factor and epidermal growth factor stimulation was significantly reduced and shortened in mutant cells. Stimulation of ERK by phorbol 12-myristate 13-acetate was not affected in mutant cells, but the phorbol 12-myristate 13-acetate-induced ERK activity decayed much faster compared with that in wild-type cells. In contrast, JNK activation upon heat shock was significantly enhanced in Shp-2 mutant cells. Based on these results, we conclude that Shp-2 plays differential positive regulatory roles in various mitogenic signaling pathways leading to ERK activation, and that Shp-2 is a negative effector in JNK activation by cellular stress. This is the first evidence that a tyrosine phosphatase has opposite effects in mediating the activation of ERK and JNK MAP kinases.     

Mapping of the complement regulatory domains in the human factor H-like protein 1 and in factor H1

The human factor H-like protein 1 (FHL-1) is composed of seven repetitive domains (short consensus repeats; SCRs) that are identical in sequence to the seven NH2-terminal SCRs of the complement regulatory protein factor H. We have identified the native FHL-1 protein as a 42-kDa human plasma protein by immunoblotting and by comparing the mobility to that of a recombinant FHL-1 protein. Here, we demonstrate the existence of two distinct co-migrating human plasma proteins that represent the 42-kDa FHL-1 protein and the previously identified 43-kDa factor H-related 1 beta protein. Similar to factor H, the recombinant FHL-1 protein displays cofactor activity in factor I-mediated cleavage of C3b. To identify relevant SCRs of factor H and FHL-1, we recombinantly expressed the domains shared between the two proteins in the baculovirus expression system. Recombinant FHL-1 and all truncated forms that include SCRs 1 to 4 displayed cofactor activity. All four NH2-terminal SCRs are essential, as deletion mutants composed of SCR 1 and 4 only; of SCRs 1, 2, and 4 only, or of SCRs 1, 3, and 4 only were functionally inactive. Similarly, the distance between these individually folding domains is critical for function, as a recombinant protein that had two and four amino acids inserted between SCRs 1 and 2 or between SCRs 3 and 4, respectively, had no activity. These results demonstrate that all four NH2-terminal SCRs of FHL-1 (and of factor H) are required for cofactor activity in factor I-mediated cleavage of C3b, and that the distance between these SCRs is essential.     

NH2-terminal structural motifs in staphylokinase required for plasminogen activation

Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys10-Lys11 peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH2-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH2-terminal region Lys11-Gly-Asp-Asp-Ala-Ser16-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis. Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH2-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH2-terminal amino acid substituted with Lys-generated derivatives from which the NH2-terminal initiation Met was no longer removed, yielding inactive (50%) Sak42DDeltaN14(M), A15K and Sak42DDeltaN15(M),S16K, and inactive Sak42DDeltaN16(M),Y17K. Lys variants without NH2-terminal Met, generated from fusion proteins in which a His6 tag and a factor Xa recognition sequence were linked to the NH2 terminus of the Sak variants, were indistinguishable from their NH2-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DDeltaN11(M),G12K could be restored by additional substitution of both Asp13 and Asp14 with Asn, yielding active Sak42DDeltaN11(M),G12K, D13N, D14N, whereas substitution in Sak42DDeltaN16(M),Y17K of Phe18 and Glu19 with Asn yielded inactive Sak42DDeltaN16(M),Y17K,F18N,E19N. These data, in combination with the recent finding that the 20 NH2-terminal amino acids of Sak lack secondary structure, suggest that the NH2-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH2-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.     

Antibodies to an NH2-terminal fragment of betaS globin. I. Preparation and radioimmunoassay

Polypeptide fragments corresponding to the NH2-terminal 55 amino acids of betaS and betaA globins were prepared by cyanogen bromide treatment of globin and isolated by gel filtration on Sephadex G-50. Sheep were immunized with the isolated NH2-terminal fragments, and one of these sheep produced precipitating antibodies to the NH2-terminal fragment of betaS globin. These antibodies also reacted with betaA and betaS globin and hemoglobins A and S, as shown by immunodiffusion and quantitative precipitation studies. A radioimmunoassay was developed using the radioiodinated NH2-terminal fragment as tracer, and dextran-coated charcoal for separating bound and free peptide. The radioimmunoassay was used to characterize the interaction of the antibodies and the NH2-terminal fragment of betaS globin.     

The MKK7 gene encodes a group of c-Jun NH2-terminal kinase kinases

The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (alpha, beta, and gamma isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7alpha isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7alpha isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7beta and MKK7gamma isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.     

Monoclonal antibodies against human collagenase and stromelysin

Mouse monoclonal antibodies against recombinant human fibroblast procollagenase and prostromelysin have been generated and characterized. The epitope-containing domains for the antibodies have been assigned based on their immunoreactivities against recombinant proenzymes, mature enzymes, truncated collagenases, proteolytic fragments of stromelysin, and chimeric molecules constructed from different domains of the two enzymes. These antibodies can be divided into four groups: (1) antibodies that recognize the truncated 19-kDa NH2-terminal collagenase, (2) antibodies that recognize the C-terminal domain of collagenase and stromelysin, (3) antibodies that recognize a 31-kDa NH2-terminal collagenase fragment, and (4) antibodies that recognize the 19-kDa NH2-fragment of stromelysin. The prostromelysin-specific antibody 11N13 is of particular interest; it neutralizes stromelysin activity in a stromelysin peptide substrate assay, with an IC50 value of 75 nM. MAb 11N13 may be useful for in vivo and in vitro studies to validate the roles of stromelysin in tumor cell invasion, metastasis, and connective tissue disorders.     

MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function

The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.     

Alternate amino terminal processing of surfactant protein A results in cysteinyl isoforms required for multimer formation

The biological functions of rat surfactant protein A (SP-A), an oligomer composed of 18 polypeptide subunits derived from a single gene, are dependent on intact disulfide bonds. Reducible and collagenase-reversible covalent linkages of as many as six or more subunits in the molecule indicate the presence of at least two NH2-terminal interchain disulfide bonds. However, the reported primary structure of rat SP-A predicts that only Cys6 in this region is available for interchain disulfide formation. Direct evidence for a second disulfide bridge was obtained by analyses of a set of three mutant SP-As with telescoping deletions from the reported NH2-terminus. Two of the truncated recombinant proteins formed reducible dimers despite deletion of the domain containing Cys6. Edman degradation revealed that each mutant protein was a mixture of two isoforms with and without an isoleucine-lysine-cysteine (IKC) extension at the NH2-terminus, which was derived from the COOH-terminal end of the reported signal peptide. Large variations in the abundance of the IKC isoforms between truncated SP-As suggested that the amino acid sequences located downstream from the signal peptide modulated alternate-site cleavage by signal peptidase. Elution of the newly identified cysteine in the position of DiPTH-Cys indicated participation in disulfide linkage, which was interchain based on the direct correlation between prevalence of the IKC variant and the extent of dimerization for each truncated protein. Sequencing of both native rat SP-A and human SP-A also revealed isoforms with disulfide-forming NH2-terminal extensions. The extended rat SP-A isoforms were enriched in the more fully glycosylated and multimeric SP-A species separated on SDS-PAGE gels. Thus, a novel post translational modification results in naturally occurring cysteinyl isoforms of rat SP-A, which are essential for multimer formation.     

SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines

Activation of the matrix metalloproteinase 2 (MMP-2) has been shown to play a major role in the proteolysis of extracellular matrix (ECM) associated with tumor invasion. Although the precise mechanism of this activation remains elusive, levels of the membrane type 1-MMP (MT1-MMP) at the cell surface and of the tissue inhibitor of MMP-2 (TIMP-2) appear to be two important determinants. Induction of MMP-2 activation in cells cultivated on collagen type I gels indicated that the ECM is important in the regulation of this process. In this study, we show that SPARC/osteonectin, a small ECM-associated matricellular glycoprotein, can induce MMP-2 activation in two invasive breast cancer cell lines (MDA-MB-231 and BT549) but not in a noninvasive counterpart (MCF-7), which lacks MT1-MMP. Using a set of peptides from different regions of SPARC, we found that peptide 1.1 (corresponding to the NH2-terminal region of the protein) contained the activity that induced MMP-2 activation. Despite the requirement for MT1-MMP, seen in MCF-7 cells transfected with MT1-MMP, the activation of MMP-2 by SPARC peptide 1.1 was not associated with increased steady-state levels of MT1-MMP mRNA or protein in either MT1-MMP-transfected MCF-7 cells or constitutively expressing MDA-MB-231 and BT549 cells. We did, however, detect decreased levels of TIMP-2 protein in the media of cells incubated with peptide 1.1 or recombinant SPARC; thus, the induction of MMP-2 activation by SPARC might be due in part to a diminution of TIMP-2 protein. We conclude that SPARC, and specifically its NH2-terminal domain, regulates the activation of MMP-2 at the cell surface and is therefore likely to contribute to the proteolytic pathways associated with tumor invasion.     

Structure-activity relation of NH2-terminal human parathyroid hormone fragments

Human parathyroid hormone (hPTH) is involved in the regulation of the calcium level in blood. This hormone function is located in the NH2-terminal 34 amino acids of the 84-amino acid peptide hormone and is transduced via the adenylate cyclase and the phosphatidylinositol signaling pathways. It is well known that truncation of the two NH2-terminal amino acids of the hormone leads to complete loss of in vivo normocalcemic function. To correlate loss of calcium level regulatory activity after stepwise NH2-terminal truncation and solution structure, we studied the conformations of fragments hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37) in comparison to hPTH-(1-37) in aqueous buffer solution under near physiological conditions by circular dichroism spectroscopy, two-dimensional nuclear magnetic resonance spectroscopy, and restrained molecular dynamics calculations. All peptides show helical structures and hydrophobic interactions between Leu-15 and Trp-23 that lead to a defined loop region from His-14 to Ser-17. A COOH-terminal helix from Met-18 to at least Leu-28 was found for all peptides. The helical structure in the NH2-terminal part of the peptides was lost in parallel with the NH2-terminal truncation and can be correlated with the loss of calcium regulatory activity.     

Identification of bile canalicular cell surface antigen HAM.4 as dipeptidyl peptidase IV (DPPIV) and characterization of its role in hepatic regeneration after partial hepatectomy in rats

Dipeptidyl peptidase IV (DPPIV) has been implicated in the control of cell growth and differentiation. A rat hepatocyte membrane antigen recognized by a monoclonal antibody (HAM.4) has now been shown to be identical to DPPIV by immunoblot analysis and amino acid sequencing. The amounts of DPPIV immunoreactive protein and enzymatic activity in serum increased in a manner independent of de novo protein synthesis, and without any biochemical or immunohistochemical changes in hepatic DPPIV, during liver regeneration after partial hepatectomy in rats. DPPIV purified from serum by HAM.4 antibody-based affinity chromatography lacked the NH2-terminal 36 amino acids of the membrane-bound enzyme, suggesting that proteolytic cleavage may mediate the release of DPPIV into serum. No significant differences in the restoration of liver mass or in hepatic DNA synthesis were apparent between DPPIV-deficient and normal rats after partial hepatectomy, suggesting that DPPIV may not be essential for hepatic regeneration.     

Expression and circular dichroism studies of the extracellular domain of the alpha subunit of the nicotinic acetylcholine receptor

To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle alpha subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (alpha210GPI) consisting of the 210 NH2-terminal amino acids of the alpha subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface alpha-bungarotoxin binding in oocytes. Coexpression of alpha210GPI with an analogous construct made from the delta subunit showed no evidence of heterodimer formation. The alpha210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The alpha210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded alpha subunit. Circular dichroism studies of alpha210GPI suggest that this region of the receptor includes considerable beta-sheet secondary structure, with a small proportion of alpha-helix.     

Expression, purification, and properties of the aldehyde dehydrogenase homologous carboxyl-terminal domain of rat 10-formyltetrahydrofolate dehydrogenase

We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.