Selective effects of CAPP1-calmodulin on its target proteins

Occupancy of one of the two phenothiazine-binding sites on calmodulin does not significantly decrease the affinity of calmodulin for its target proteins; however, it does affect the ability of calmodulin to activate some enzymes. Previously we demonstrated that a covalent adduct of calmodulin with one molecule of phenothiazine (CAPP1-calmodulin) is an antagonist for the calmodulin-dependent enzymes, cAMP phosphodiesterase and myosin kinase, and a partial agonist for calcineurin. We now show that CAPP1-calmodulin is a full agonist for glycogen synthase kinase and phosphorylase kinase. Unlike phenothiazines, CAPP1-calmodulin is specific for calmodulin-regulated proteins; it has no effect on protein kinase C. With the exception of phosphorylase kinase, occupancy of two phenothiazine-binding sites completely eliminates the ability of calmodulin to activate these proteins. Thus, the study of the interaction of CAPP1-calmodulin with calmodulin target proteins demonstrates that calmodulin interacts differently with different proteins. This is confirmed by studies of the effect of calmodulin fragments, 1-77 and 78-148, on calmodulin-regulated enzymes.     

Inhibition of mitosis in PtK2 cells by CAPP1-calmodulin

Various indirect evidence has indicated that calcium ions and the calcium-binding regulator protein, calmodulin, may regulate mitosis in higher eukaryotes. We have used the competitive antagonist, CAPP1-calmodulin, to antagonize intracellular calmodulin and test the hypothesis that calmodulin serves as a regulator of mitosis. We find that CAPP1-calmodulin inhibits the transit of cells through metaphase at estimated intracellular concentrations up to that of native calmodulin; beyond that level, the inhibition of mitosis vanishes. The membrane-permeant anticalmodulin agents, W7 and calmidazolium, also inhibit the progress of cells through metaphase. The similarity of the inhibitory curves for CAPP1-calmodulin, W7, and calmidazolium suggests that all these agents inhibit mitosis by antagonizing intracellular calmodulin. In order to test whether this inhibition of metaphase transit is due to an effect of the agents on intracellular free calcium, we used the calcium indicator Fura-2 to measure intracellular calcium levels after CAPP1-calmodulin injection or during calmidazolium treatment. We found that, while intracellular calcium levels are modestly elevated during calmidazolium treatment, they were unaffected by CAPP1-calmodulin, a result suggesting that mitosis inhibition was not due to an effect on intracellular free calcium. The reasons for the anomalous dose-response behavior of these drugs are not known; however, the behavior of cells at drug levels below the point of anomaly supports the hypothesis that calmodulin acts as a regulator of mitosis in these cells.     

Replication origins, factors and attachment sites

The initiation of eukaryotic DNA synthesis occurs at specific sites determined by both cis- and trans-acting elements. Here I review advances in the characterization of yeast origins, origin-binding proteins and the relationship of DNA replication to nuclear substructure in yeast.     

Animal glycolipids as attachment sites for microbes

The abundance of carbohydrate at the animal cell surface may explain why microbes have selected primarily carbohydrates as essential attachment sites for colonization or infection. Of the various surface glycoconjugates of interest, primary attention has been given to glycolipids, due in part to an efficient binding assay based on a thin-layer chromatogram with separated glycolipids. In this way the general character of carbohydrate recognition by microbes is being mapped. Mainly two examples are briefly described to illustrate some generalizations: lactosylceramide-recognition by several bacteria, and Gal alpha l----4Gal-binding by Escherichia coli and the Shiga toxin. The unique recognition of internally placed sequences, the often low-affinity binding, and the preference of certain sequences before others are interpreted to be of decisive biological value. The binding to internal parts makes it technically possible to approximate the binding epitope on a receptor glycolipid. For this the binding preferences to glycolipids carrying the binding site in different saccharide environments (isoreceptors) are compared with the computer-calculated preferred conformations (definition of steric hindrances to epitope access). Several binding epitopes dissected with this approach have a common surface character: a nonpolar area of ring hydrogens over one or two sugars, surrounded by polar oxygens or amide. This is in agreement with the recent Lemieux concept for antibody-carbohydrate interaction. This information facilitates a rational synthesis of receptor analogues for potential applications. An outline is finally given of an improved general approach for receptor analysis.     

Chromosomal sites of DNA-membrane attachment in Escherichia coli

Evidence is presented to show that both the chromosomal replication point and the chromosomal origin in Escherichia coli are associated with a structure possessing the sedimentation properties and enzyme sensitivities characteristic of membrane. The data suggest that both newly synthesized DNA strands and template DNA strands are bound at this replication point and that both strands are also bound at the origin.     

Characterization of the bilin attachment sites in R-phycoerythrin

The amino acid sequence around the sites of attachment of all the bilin prosthetic groups of Gastroclonium coulteri R-phycoerythrin, (alpha beta)6 gamma, have been determined. The sequences of tryptic peptides derived from the alpha and beta subunits are (Formula: see text) where the designations alpha and beta refer to the subunits from which the peptides derived. Cysteinyl residues involved in bilin attachment are indicated with an asterisk. Each peptide carries a single bilin, either phycoerythrobilin (PEB) or phycourobilin (PUB). Spectroscopic studies on the gamma subunit indicate the presence of one PEB and three PUB groups. However, five unique tryptic peptides, gamma-A through gamma-E, were characterized, indicating that Gastroclonium R-phycoerythrin is a mixture of at least two species, (alpha beta)6 gamma and (alpha beta)6 gamma', with gamma subunits differing in amino acid sequence. The sequences of the gamma subunit bilin peptides (see below) were not homologous to those from alpha and beta subunits of any biliprotein. (Formula: see text) The bilins in all these peptides are attached through single linkages to a cysteinyl residue, except for the phycourobilin on peptide beta-3 which is attached through two thioether linkages to cysteinyl residues 10 amino acids apart. The availability of small bilin peptides was exploited to obtain more accurate molar extinction coefficients for peptide-linked PEB and PUB groups. Application of these extinction coefficients in the calculation of the bilin content of R-, B-, and C-phycoerythrins shows that there are 5 bilins/alpha beta in each of these three biliprotein types.     

Different usage of the glycosaminoglycan attachment sites of biglycan

Biglycan is a member of the small leucine-rich proteoglycan family. Its core protein comprises two chondroitin/dermatan sulfate attachment sites on serine 42 and serine 47, respectively, which are the fifth and tenth amino acid residues, respectively, after removal of the prepro peptide. Because the regulation of glycosaminoglycan chain assembly is not fully understood and because of the in vivo existence of monoglycanated biglycan, mutant core proteins were stably expressed in human 293 and Chinese hamster ovary cells in which i) either one or both serine residues were converted into alanine or threonine residues, ii) the number of acidic amino acids N-terminal of the respective serine residues was altered, and iii) a hexapeptide was inserted between the mutated site 1 and the unaltered site 2. Labeling experiments with [(35)S]sulfate and [(35)S]methionine indicated that serine 42 was almost fully used as the glycosaminoglycan attachment site regardless of whether site 2 was available or not for chain assembly. In contrast, substitution of site 2 was greatly influenced by the presence or absence of serine 42, although additional mutations demonstrated a direct influence of the amino acid sequence between the two sites. When site 2 was not substituted with a glycosaminoglycan chain, there was also no assembly of the linkage region. These results indicate that xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan chain assembly and implicate a cooperative effect on the xylosyl transfer to site 2 by xylosylation of site 1, which probably becomes manifest before the removal of the propeptide. It is shown additionally that biglycan expressed in 293 cells may still contain the propeptide sequence and may carry heparan sulfate chains as well as sulfated N-linked oligosaccharides.     

Multiple tRNA attachment sites in prothymosin alpha.

A covalent complex formed by bacterial tRNAs and prothymosin alpha, an abundant acidic nuclear protein involved in proliferation of mammalian cells, upon production of the recombinant rat protein in Escherichia coli cells was studied. Several tRNA attachment sites were identified in the prothymosin alpha molecule using a combination of deletion analysis of prothymosin alpha and site-specific fragmentation of the protein moiety of the prothymosin alpha-tRNA complex. The electrophoretic mobilities of the tRNA-linked prothymosin alpha and its derivatives are consistent with one tRNA molecule attached to one prothymosin alpha molecule, thus suggesting that alternative tRNA linking to one of several available attachment sites occurs. The possible effect of tRNA attachment on the nuclear uptake of prothymosin alpha is discussed.     

Fluorescent localization of calcium at sites of cell attachment and spreading

Chlorotetracycline (CTC), a calcium-chelating fluorescent probe identifies and localizes calcium when applied to cultured neural crest cells. The fluorescence occurs at specific membrane regions involved in cell attachment and spreading as well as with the formation of cytoskeletal stress fibers (actin microfilament bundles). The observed CTC reaction indicates that calcium sequestration and the development of these membrane-cytoskeletal features share a temporal and spatial pattern. Thus, the selective availability of calcium may be an important determinant of cell morphogenesis.     

Expression of collagen XVIII and localization of its glycosaminoglycan attachment sites

Collagen XVIII is the only currently known collagen that carries heparan sulfate glycosaminoglycan side chains. The number and location of the glycosaminoglycan attachment sites in the core protein were determined by eukaryotic expression of full-length chick collagen XVIII and site-directed mutagenesis. Three Ser-Gly consensus sequences carrying glycosaminoglycan side chains were detected in the middle and N-terminal part of the core protein. One of the Ser-Gly consensus sequences carried a heparan sulfate side chain, and the remaining two had mixed chondroitin and heparan sulfate side chains; thus, recombinant collagen XVIII was a hybrid of heparan sulfate and chondroitin proteoglycan. In contrast, collagen XVIII from all chick tissues so far assayed have exclusively heparan sulfate side chains, indicating that the posttranslational modification of proteins expressed in vitro is not entirely identical to the processing that occurs in a living embryo. Incubating the various mutated collagen XVIIIs with retinal basement membranes showed that the heparan sulfate glycosaminoglycan side chains mediate the binding of collagen XVIII to basement membranes.     

The alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin

This study was undertaken to determine the roles of individual alpha/beta 1 integrin heterodimers in promoting cellular interactions with the different attachment-promoting domains of laminin (LN). To do this, antibodies to the integrin beta 1 subunit or to specific integrin alpha subunits were tested for effects on cell attachment to LN, to elastase fragments E1-4 and E1, derived from the short arms and core of LN's cruciform structure, and to fragment E8 derived from the long arm of this structure. The human JAR choriocarcinoma cells used in this study attached to LN and to fragments E1 and E8. Attachment to E1-4 required a much higher substrate coating concentration, suggesting that it is a poor substrate for JAR cell attachment. The ability of cells to attach to different LN domains suggested the presence of more than one LN receptor. These multiple LN receptors were shown to be beta 1 integrin heterodimers because antibodies to the integrin beta 1 subunit inhibited attachment of JAR cells to LN and its three fragments. To identify the individual integrin alpha/beta 1 heterodimers that mediate interactions with these LN domains, mAbs specific for individual beta 1 heterodimers in human cells were used to study JAR cell interactions with LN and its fragments. An anti-alpha 6/beta 1-specific mAb, GoH3, virtually eliminated cell attachment to E8 and partially inhibited attachment to E1 and intact LN. Thus the major alpha 6/beta 1 attachment domain is present in fragment E8. An alpha 1/beta 1-specific mAb (S2G3) strongly inhibited cell attachment to collagen IV and partially inhibited JAR attachment to LN fragment E1. Thus, the alpha 1/beta 1 heterodimer is a dual receptor for collagen IV and LN, interacting with LN at a site in fragment E1. In combination, the anti-alpha 1- and anti-alpha 6-specific antibodies completely inhibited JAR cell attachment to LN and fragment E1. Thus, the alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers each function as LN receptors and act together to mediate the interactions of human JAR choriocarcinoma cells with LN.     

The distribution of fibronectin in attachment sites of chick fibroblasts

Primary chick heart fibroblasts were cultured on glass coverslips and examined by reflection contrast microscopy and then by indirect immunofluorescence microscopy using affinity purified antifibronectin antibody. Fibronectin was found to be primarily localized in areas of ‘close’ contact and in intensely staining fibrous webs that were usually external to the cytoplasmic matrix. Focal contacts, in which there is intimate contact between the cell and the substratum by localized attachment, failed to react with the anti-fibronectin antibody despite a variety of extraction procedures designed to increase the accessibility of antibody to this type of attachment site. It is concluded that in chick fibroblasts, fibronectin participates in cell attachment at areas of close contact and fibrillar attachment sites, but is often excluded from the attachment pads of focal contacts.     

Protein:DNA interactions at chromosomal loop attachment sites

We have recently identified an evolutionarily conserved class of sequences that organize chromosomal loops in the interphase nucleus, which we have termed "matrix association regions" (MARs). MARs are about 200 bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs, often reside near cis-acting regulatory sequences, and their binding sites are abundant (greater than 10,000 per mammalian nucleus). Here we demonstrate that the interactions between the mouse kappa immunoglobulin gene MAR and topoisomerase II or the "nuclear matrix" occur between multiple and sometimes overlapping binding sites. Interestingly, the sites most susceptible to topoisomerase II cleavage are localized near the breakpoints of a previously described illegitimate recombination event. The presence of multiple binding sites within single MARs may allow DNA and RNA polymerase passage without disrupting primary loop organization.     

Structural analysis of staphylococcal bacteriophage phi 11 attachment sites.

The lysogenization of bacteriophage phi 11 in Staphylococcus aureus occurs by site-specific recombination. The DNA segments containing the attachment sites on the host chromosome, the phage genome, and the two junctions created by insertion of the prophage were cloned, and the nucleotide sequences were determined. The attachment sites share a very short common sequence of 10 base pairs.     

Identification of the keratan sulfate attachment sites on bovine fibromodulin

The small keratan sulfate-substituted proteoglycan (fibromodulin) from articular cartilage was shown to contain keratan sulfate linked to the core protein through N-glycosidic linkages to residues Asn-109, Asn-147, Asn-182, and Asn-272. Biosynthetic experiments with articular chondrocytes in the presence of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, demonstrated a specific inhibition of [35S]SO4 incorporation into fibromodulin. Under the same conditions no effect on the addition of keratan sulfate to the large aggregating proteoglycan was detected. Fibromodulin substituted with keratan sulfate was purified from bovine articular cartilage extracts by density gradient centrifugation, ion-exchange chromatography, and gel-permeation chromatography. Isolation of glycosylated peptides from tryptic digests of fibromodulin by ion-exchange chromatography and reversed-phase high performance liquid chromatography revealed four separate hexosamine-rich species, that were also immunoreactive with monoclonal antibody 5D4. Sequence analysis of these glycopeptides gave blank cycles at positions which corresponded to Asn followed by X-Ser/Thr in the sequence derived from cDNA (Oldberg, A., Antonsson, P., Lindblom, K., and Heinegard, D. (1989) EMBO J. 8, 2601-2604). Hence, all four Asn residues in the leucine-rich region of the fibromodulin core protein can serve as acceptor sites for keratan sulfate addition.     

Physical map of the chromosomal region of Corynebacterium diphtheriae containing corynephage attachment sites attB1 and attB2.

The chromosome of Corynebacterium diphtheriae C7 was recently shown to contain two equivalent attachment sites (attB1 and attB2) for lysogenization by corynephages (R. Rappuoli, J.L. Michel, and J.R. Murphy, J. Bacteriol. 153:1202-1210, 1983). Portions of bacterial chromosome containing each attB site, as well as a 3.5-kilobase (kb) EcoRI fragment containing both attB1 and attB2 sites, were cloned in the pUC8 plasmid vector. Restriction endonuclease mapping and Southern blot hybridization analysis of restriction endonuclease fragments showed that attB1 and attB2 are 2.25 kb apart on the chromosome. Furthermore, a 0.85-kb HincII-EcoRI restriction endonuclease fragment containing attB1, a 0.77-kb HincII-BamHI fragment containing attB2, and a 1.2-kb EcoRI-BamHI fragment containing attP share short homologous regions. No homology was detected between the sequences flanking the two attB sites. The isolation of a segregant which had lost the entire chromosomal segment contained between attB1 and attB2 suggests that this region is not essential for growth.     

Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites

A major goal in the study of vertebrate mitosis is to identify proteins that create the kinetochore-microtubule attachment site. Attachment sites within the kinetochore outer plate generate microtubule dependent forces for chromosome movement and regulate spindle checkpoint protein assembly at the kinetochore. The Ndc80 complex, comprised of Ndc80 (Hec1), Nuf2, Spc24, and Spc25, is essential for metaphase chromosome alignment and anaphase chromosome segregation. It has also been suggested to have roles in kinetochore microtubule formation, production of kinetochore tension, and the spindle checkpoint. Here we show that Nuf2 and Hec1 localize throughout the outer plate, and not the corona, of the vertebrate kinetochore. They are part of a stable "core" region whose assembly dynamics are distinct from other outer domain spindle checkpoint and motor proteins. Furthermore, Nuf2 and Hec1 are required for formation and/or maintenance of the outer plate structure itself. Fluorescence light microscopy, live cell imaging, and electron microscopy provide quantitative data demonstrating that Nuf2 and Hec1 are essential for normal kinetochore microtubule attachment. Our results indicate that Nuf2 and Hec1 are required for organization of stable microtubule plus-end binding sites in the outer plate that are needed for the sustained poleward forces required for biorientation at kinetochores.     

Microfilaments and actin-associated proteins at sites of membrane-substrate attachment within acetylcholine receptor clusters

Rat myotubes in tissue culture form broad areas of close contact with the substrate. These areas often display two distinct, interdigitating sets of membrane domains. One, the "contact domain", is close to the substrate; the other, termed the "AChR domain", is further from the substrate and is rich in acetylcholine receptors (AChR). We have used fluorescence techniques to study the organization of the cytoskeleton in these areas. Substrate-apposed membrane of the myotubes was exposed either by shearing or by permeabilizing the cells with a neutral detergent. Phalloidin derivatives and affinity-purified polyclonal or monoclonal antibodies specific for cytoskeletal proteins were then applied to the samples. Sheared samples were observed by epifluorescence microscopy; detergent-permeabilized samples were observed by total internal reflection fluorescence microscopy. We found that, like antivinculin, fluorescent phalloidin derivatives and antibodies to alpha-actinin, filamin, and talin preferentially labeled the contact domains. This suggests that bundles of microfilaments associate with the membrane at sites of myotube-substrate attachment. In contrast, a 43K protein, closely associated with AChR, was present only at AChR domains. A monoclonal antibody to actin labeled both AChR and contact domains, suggesting that actin is enriched over both regions. Our results suggest that, like the plasma membrane of AChR clusters, the underlying membrane skeleton is organized into at least two distinct domains.