Showing papers by "Paul Matsudaira published in 1994"

The COOH terminus of the c-Abl tyrosine kinase contains distinct F- and G-actin binding domains with bundling activity.

Abstract: The myristoylated form of c-Abl protein, as well as the P210bcr/abl protein, have been shown by indirect immunofluorescence to associate with F-actin stress fibers in fibroblasts. Analysis of deletion mutants of c-Abl stably expressed in fibroblasts maps the domain responsible for this interaction to the extreme COOH-terminus of Abl. This domain mediates the association of a heterologous protein with F-actin filaments after microinjection into NIH 3T3 cells, and directly binds to F-actin in a cosedimentation assay. Microinjection and cosedimentation assays localize the actin-binding domain to a 58 amino acid region, including a charged motif at the extreme COOH-terminus that is important for efficient binding. F-actin binding by Abl is calcium independent, and Abl competes with gelsolin for binding to F-actin. In addition to the F-actin binding domain, the COOH-terminus of Abl contains a proline-rich region that mediates binding and sequestration of G-actin, and the Abl F- and G-actin binding domains cooperate to bundle F-actin filaments in vitro. The COOH terminus of Abl thus confers several novel localizing functions upon the protein, including actin binding, nuclear localization, and DNA binding. Abl may modify and receive signals from the F-actin cytoskeleton in vivo, and is an ideal candidate to mediate signal transduction from the cell surface and cytoskeleton to the nucleus.

Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein.

Abstract: A novel human mRNA whose expression is induced over 200-fold in B lymphocytes by latent Epstein-Barr virus (EBV) infection was reverse transcribed, cloned, and sequenced. The mRNA is predicted to encode a protein containing four peptides which precisely match amino acid sequences from a previously identified 55-kDa actin-bundling protein, p55. In vitro translation of the cDNA results in a 55-kDa protein which binds to actin filaments in the presence of purified p55 from HeLa cells. The p55 mRNA is undetectable in non-EBV-infected B- and T-cell lines or in a myelomonocytic cell line (U937). Newly infected primary human B lymphocytes, EBV-transformed B-cell lines, latently infected Burkitt tumor cells expressing EBNA2 and LMP1, a chronic myelogenous leukemia cell line (K562), and an osteosarcoma cell line (TK143) contain high levels of p55 mRNA or protein. In EBV-transformed B cells, p55 localizes to perinuclear cytoplasm and to cell surface processes that resemble filopodia. The p55 mRNA is detected at high levels in spleen and brain tissues, at moderate levels in lung and placenta tissues, and at low levels in skeletal muscle, liver, and tonsil tissues and is undetectable in heart, kidney, pancreas, and bone marrow tissues. Immunohistochemical staining of human brain tissue demonstrates p55 localization to the perinuclear cytoplasm and dendritic processes of many, but not all, types of cortical or cerebellar neurons, to glial cells, and to capillary endothelial cells. In cultured primary rat neurons, p55 is distributed throughout the perinuclear cytoplasm and in subcortical filamentous structures of dendrites and growth cones. p55 is highly evolutionarily conserved since it shows 40% amino acid sequence identity to the Drosophila singed gene product and 37% identity to fascin, an echinoderm actin-bundling protein. The evolutionary conservation of p55 and its lack of extensive homology to other actin-binding proteins suggest that p55 has specific microfilament-associated functions in cells in which it is differentially expressed, including neural cells and EBV-transformed B lymphocytes.

Identification of I-plastin, a human fimbrin isoform expressed in intestine and kidney.

Abstract: The complete cDNA sequence of human intestine-specific plastin (I-plastin) was determined from a clone derived by PCR. It consists of a 97-bp 5' untranslated region, a 1,887-bp coding region, and a 1,655-bp 3' untranslated region. The coding region predicts a 629-residue polypeptide whose sequence displays 86, 75, and 73% identities with chicken intestine fimbrin, human T-plastin, and human L-plastin, respectively. Recombinant I-plastin cross-linked actin filaments into bundles in the absence but not in the presence of calcium. The I-plastin gene was mapped by PCR to human chromosome 3; the L- and T-plastin genes were previously mapped to chromosomes 13 and X, respectively. I-plastin mRNA was detected in the small intestine, colon, and kidneys; relatively lower levels of expression were detected in the lungs and stomach. In contrast, L-plastin expression was restricted to the spleen and other lymph node-containing organs, while T-plastin was expressed in a variety of organs, including muscle, brain, uterus, and esophagus. In contrast to the situation for the intestine, high levels of L- and T-plastin mRNAs were detected in Caco-2, a human colon-derived cell line. Immunofluorescence microscopy detected I-plastin in the brush border of the small intestine and colon. These results identify I-plastin as the human homolog of chicken intestine fimbrin and as a third plastin isoform in humans.

Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin.

Abstract: Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits.

Solution structure of villin 14T, a domain conserved among actin‐severing proteins

Abstract: The solution structure of the N-terminal domain of the actin-severing protein villin has been determined by multidimensional heteronuclear resonance spectroscopy. Villin is a member of a family of actin-severing proteins that regulate the organization of actin in the eukaryotic cytoskeleton. Members of this family are built from 3 or 6 homologous repeats of a structural domain of approximately 130 amino acids that is unrelated to any previously known structure. The N-terminal domain of villin (14T) contains a central beta-sheet with 4 antiparallel strands and a fifth parallel strand at one edge. This sheet is sandwiched between 2 helices on one side and a 2-stranded parallel beta-sheet with another helix on the other side. The strongly conserved sequence characteristic of the protein family corresponds to internal hydrophobic residues. Calcium titration experiments suggest that there are 2 binding sites for Ca2+, a stronger site near the N-terminal end of the longest helix, with a Kd of 1.8 +/- 0.4 mM, and a weaker site near the C-terminal end of the same helix, with a Kd of 11 +/- 2 mM. Mutational and biochemical studies of this domain in several members of the family suggest that the actin monomer binding site is near the parallel strand at the edge of the central beta-sheet.

The fimbrin and alpha-actinin footprint on actin.

Abstract: satisfying experience in science occurs when information from several areas converge to give a big picture about an important problem. In three papers (Holtzman et al., 1994; Honts et al., 1994; McGough et al., 1994) in this issue of The Journal of Cell Biology, the disciplines of genetics and structural biology have revealed that two actin cross-linking proteins, fimbrin and alpha-actinin, bind to the same region on actin. Because both proteins belong to a superfamily of actin cross-linking proteins, we have some confidence that other members of the superfamily, including filamin, spectrin, dystrophin, and ABP-120, also bind the same region of actin. This information about binding sites is an important step toward understanding how actin illaments are organized by cross-linking proteins into bundles and supramolecular networks. This story is rooted in the classic biochemical studies of muscle proteins. Alpha-actinin was first purified from skeletal muscle (Maruyama and Ebashi, 1965) and characterized as a Z-line component; its rodlike shape was revealed by electron microscopy (see Meyer and Aebi, 1990). Later, spurred by cell biologists who demonstrated that cytoplasmic extracts of noumuscle cells could undergo reversible solgel transformations, a generation of biologists isolated and characterized a number of actin gelation and bundling proteins, including ABP-280 and filamin, speetrin, fascin, and ABP-120, from ameba (reviewed in Pollard and Cooper, 1986; Stossel et al., 1985; Weeds, 1982); alpha-actinin was rediscovered as a nonmuscle actin cross-linking protein (Burridge and Feramisco, 1981). During the late 1980's, eDNA sequencing showed that many of these gelation factors belonged to a superfamily that shares a common 27-kD NH2-terminal domain (Baron et al., 1987) that binds actin filaments. Fimbrin bundles actin filaments in intestinal brush border microvilli, and it is also located where actin bundles terminate at membrane adhesion plaques (Bretscher and Weber, 1980). The sequence databases (de Arruda et al., 1990) showed that fimbrin was also involved in cell transformation (Lin et al., 1994), and that it was a target of phosphorylation when leukocytes are activated by growth factors and mitogens (Zu et al., 1990). Hence, fimbrin is also called plastin and pp70. An important structural theme emerged from the sequences: cross-linking proteins in this superfamily are

1H, 15N, 13C and 13CO resonance assignments and secondary structure of villin 14T, a domain conserved among actin-severing proteins

Abstract: Sequence-specific assignments have been made for the 1H, 15N, 13C and 13CO resonances of 14T, the 126-residue amino-terminal domain of the actin-severing protein villin. Villin is a member of a family of proteins that regulate cytoskeletal actin by severing, capping and nucleating actin filaments. Actin binding is dependent on calcium and disrupted by phosphatidyl inositol 4,5-bisphosphate. Actin-severing proteins are built from three or six repeats of a conserved domain, represented by 14T. Expression in Escherichia coli facilitated incorporation of 15N and 13C isotopes and application of triple-resonance, backbone-directed strategies for the sequential assignments. Elements of regular secondary structure have been identified by characteristic patterns of NOE cross peaks and values of vicinal 3JH n Hα coupling constants. Amide protons that exchange slowly (rates less than 1.0×10-4 per min) are concentrated in the central β-sheet and the second and third α-helices, suggesting that these elements of secondary structure form very stable hydrogen bonds. Assignments for the amide nitrogens and protons have been examined as a function of pH and calcium concentration. Based on the conservation of chemical shifts in the core of the domain, villin 14T maintains the same overall fold in the pH range from 4.15 to 6.91 and the calcium range from 0 to 50 mM. The calcium data indicate the presence of two calcium-binding sites and suggest their locations.