Phosphorylation of the cytoskeletal protein talin by protein kinase C

Talin, a component of the focal contact of cultured cells, is an in vitro substrate for protein kinase C. Immunoprecipitation confirms that talin is the phosphorylated protein. Phosphorylation is dependent on both phosphatidylserine and calcium and reaches a level of incorporation of 0.8 mol phosphate/mol protein. Phosphoamino acid analysis demonstrates the presence of phosphoserine and phosphothreonine, but no phosphotyrosine. Two dimensional mapping of tryptic peptides, and V8 peptides reveals the existence of multiple phosphorylation sites. The identification of talin as a substrate for protein kinase C implicates talin as a potential regulator of focal contact organization and perhaps cell morphology.     

The cytoskeletal protein talin is O-glycosylated.

Talin is a 215-kDa cytoskeletal protein implicated in linking actin filaments to the plasma membrane. We show here that chicken gizzard talin is galactosylated by incubation with UDP-[3H]galactose and galactosyl-transferase. The labeled carbohydrate moiety is removed by beta-elimination and comigrates with Gal beta 1-4GlcNAcitol, indicating that talin belongs to a recently discovered class of cytosolic proteins carrying N-acetylglucosamine (GlcNAc) O-linked to serine or threonine (Holt, G. D., and Hart, G. W. (1986) J. Biol. Chem. 261, 8049-8057). Two glycosylated sequences were identified in the tail domain of talin: ANQAIQMAXQNLVDPAXTQ and GILANQLTNDYGQLAQQ, corresponding to amino acids 1470-1488 and 1883-1899, respectively, of the mouse talin amino acid sequence (Rees, D. J. G., Ades, S. E., Singer, S. J., and Hynes, R. O. (1990) Nature 347, 685-689). The putative glycosylation sites are PAXTQ and QLTND. At most 6% of chicken gizzard talin and 3% of porcine stomach talin are galactosylated by galactosyltransferase. Furthermore, human platelet talin is not labeled at all by the procedure, indicating that it may not be glycosylated.     

Structural and biophysical properties of the integrin-associated cytoskeletal protein talin

Talin is a large cytoskeletal protein (2541 amino acid residues) which plays a key role in integrin-mediated events that are crucial for cell adhesion, migration, proliferation and survival. This review summarises recent work on the structure of talin and on some of the structurally better defined interactions with other proteins. The N-terminal talin head (approx. 50 kDa) consists of an atypical FERM domain linked to a long flexible rod (approx. 220 kDa) made up of a series of amphipathic helical bundle domains. The F3 FERM subdomain in the head binds the cytoplasmic tail of integrins, but this interaction can be inhibited by an interaction of F3 with a helical bundle in the talin rod, the so-called “autoinhibited form” of the molecule. The talin rod contains a second integrin-binding site, at least two actin-binding sites and a large number of binding sites for vinculin, which is important in reinforcing the initial integrin–actin link mediated by talin. The vinculin binding sites are defined by hydrophobic residues buried within helical bundles, and these must unfold to allow vinculin binding. Recent experiments suggest that this unfolding may be mediated by mechanical force exerted on the talin molecule by actomyosin contraction.     

Membrane fusion induced by the major lipid-binding domain of the cytoskeletal protein talin

Secondary structure predictions have led to the identification of a major membrane-anchoring domain of the cytoskeletal protein talin spanning from amino acid 385 to 406. Using a synthetically derived peptide of this region, researchers have shown that it inserts into POPC/POPG phospholipid membranes with a partition coefficient of K(app)=1.1+/-0.2 x 10(5) M(-1) and has an average molar reaction enthalpy of DeltaH=-2.5 kcal/mol, as determined by monolayer expansion technique and isothermic titration calorimetry [J. Biol. Chem. 275, 17954]. We applied resonance energy transfer (RET) assays to analyze the fusogenic properties of this peptide by lipid mixing and used liposomes containing carboxyfluorescein to measure the contents leakage. We directly visualized talin peptide-induced vesicle membrane fusion using cryo-electron microscopy. This is the first example of a cytoskeletal protein domain that can trigger membrane fusion that might be of importance for understanding membrane targeting and motile events at the leading edge of the cell.     

cDNAs encoding members of a family of proteins related to human sterol carrier protein 2 and assignment of the gene to human chromosome 1 p21----pter.

Sterol carrier protein 2 (SCP2) is believed to play a key role in intracellular lipid movement. Here we report the cloning and nucleotide sequences of cDNAs encoding SCP2-related proteins of 58.85 kD and 30.8 kD and the assignment of the SCP2 gene to human chromosome 1 p21-pter. The SCP2-related proteins share common deduced carboxyl amino acid sequences with SCP2 and the cDNAs have a common 3' untranslated nucleotide sequence. The mRNAs encoding these proteins increased in a coordinate fashion as human placental cytotrophoblasts differentiated into syncytiotrophoblasts in culture. Our observations document the existence of a family of related proteins encoded by the human SCP2 gene.     

Localisation of merosin-positive congenital muscular dystrophy to chromosome 4p16.3

The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders, which present within the first 6 months of life with hypotonia, muscle weakness and contractures, associated with dystrophic changes on skeletal muscle biopsy. We have previously reported a large consanguineous family segregating merosin-positive congenital muscular dystrophy, in which involvement of known CMD loci was excluded. A genome-wide linkage search of the family conducted using microsatellite markers spaced at 10-Mb intervals failed to identify a disease locus. A second scan using a high-density SNP array, however, permitted a novel CMD locus on 4p16.3 to be identified (multipoint LOD score 3.4). Four additional consanguineous CMD families with a similar phenotype were evaluated for linkage to a 4.14-Mb interval on 4p16.3; however, none showed any evidence of linkage to the region. Our findings further illustrate the utility of highly informative SNP arrays compared with standard panels of microsatellite markers for the mapping of recessive disease loci.     

Localisation of the human blue cone pigment gene to chromosome band 7q31.3-32

Blue cone pigment (BCP) is one of three types of cone photoreceptors responsible for normal colour vision. In this study, the BCP gene has been localised to chromosome 7q31.3-32 by fluorescent in situ hybridisation of cosmid clones containing the gene. This is consistent with previous mapping of the BCP gene to chromosome 7q31-35.     

Localization of the human gene encoding heterogeneous nuclear RNA ribonucleoprotein G (hnRNP-G) to chromosome 6p12

Summary The gene encoding nuclear RNA ribonucleoprotein G (hnRNP-G), a p43 glycoprotein, has been mapped to human chromosome 6, band p12, by radioactive in situ hybridization.     

The gene encoding human protective protein (PPGB) is on chromosome 20

Normal lymphocyte prometaphase chromosome spreads were hybridized in situ using single- and double-color fluorescence techniques. The results obtained with either the 1.8-kb protective protein cDNA or a 12-kb genomic fragment of the human protective protein gene as probe demonstrate that the PPGB gene is localized on the long arm of chromosome 20. This assignment was confirmed by hybridization with whole chromosome DNA libraries.     

Localization of the human HuR gene to chromosome 19p13.2

The HuR gene encodes a specific RNA binding protein that is a member of the human Elav-like gene family. This family of proteins, which includes HuD, HuC and Hel-N1, is involved in cellular differentiation. Alterations of HuD and Hel-N1 structure are associated with small cell lung tumors and medulloblastomas. To investigate a possible linkage of the HuR gene to malignancy, the locus of the gene was mapped on human metaphase chromosomes. Analysis of the fluorescence signals on banded chromosomes showed that the HuR gene is localized to human chromosome 19p13.2. Received: 6 June 1996 / Revised: 8 August 1996     

Localization of the expressed human p58 protein kinase chromosomal gene to chromosome 1p36 and a highly related sequence to chromosome 15.

The gene for the human p58 protein kinase, a cell division control-related gene, has been mapped by somatic cell hybrid analyses, in situ localization with the chromosomal gene, and nested polymerase chain reaction amplification of microdissected chromosomes. These studies indicate that the expressed p58 chromosomal gene maps to 1p36, while a highly related p58 sequence of unknown nature maps to chromosome 15. Assignment of a p34cdc2-related gene to 1p36 may have implications for numerous tumors that involve deletion of this region, including neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma, and endocrine neoplasia.     

Localisation of the human N-ras oncogene to chromosome 1cen - p21 by in situ hybridisation.

The N-ras gene is a transforming gene isolated from a variety of human tumour cell lines and is a member of a family of related ras genes. Somatic cell hybrids have previously shown that the N-ras gene is located on chromosome 1. We have confirmed this localisation by in situ hybridisation to metaphase preparations of lymphocytes and localised the gene to the region 1cen - p21. A survey has found 47 reported cases of malignancy involving deletions in the short arm of chromosome 1. Fifteen of the 47 involved a deletion in this region.     

Localization of the gene for human heart fatty acid binding protein to chromosome 1p32-1p33

Among 639 spontaneous abortions between the 8th and 14th week of gestation 342 (53.5%) revealed an abnormal karyotype. While the rate of trisomies distinctly increased with advancing maternal age, a decrease in the rate of 45,X conceptuses and polyploidies was observed among abortions from older women. The overall relation of XXXXXXYY among the tetraploidies was 1411 and that of XXXXXYXYY among the triploidies was 26 361. However, when the latter was related to maternal age, a reversal of the XXXXXY ratio of 12 in the younger to 21 in the older age groups became evident. Furthermore a decrease in the rate of paternally derived partial hydatidiform moles was found among the triploid abortion specimens from older women. From these observations we conclude that digyny plays a major role in the origin of triploidy in the increased maternal age groups, while diandry related to immaturity of oocytes and impairment of oocyte cortical function is more frequent in triploid abortions from younger women.     

Identification of a processed pseudogene related to the functional gene encoding the GM2 activator protein: localization of the pseudogene to human chromosome 3 and the functional gene to human chromosome 5.

The GM2 activator protein is an essential substrate cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A (EC 3.2.1.52). There have been conflicting reports as to the chromosomal localization of the gene encoding the activator. We demonstrate here that these conflicts were caused by the presence of a previously unidentified processed activator-pseudogene on chromosome 3, and we confirm a previous ELISA-based localization of the functional activator gene to chromosome 5. Our data indicate that the functional activator locus can still be considered a candidate site for defects causing some forms of spinal muscular atrophy.     

Localization of the intronless gene coding for calmodulin-like protein CLP to human chromosome 10p13-ter

The functional intronless gene coding for a calmodulin-like protein (CLP) has been localized to human chromosome 10p13-ter. Chromosomal assignment was performed by Southern blot analysis of DNA from human-rodent somatic cell hybrids and amplification of a CLP gene-specific 1090-bp DNA fragment by the polymerase chain reaction (PCR) on DNA from human-hamster cell hybrids. Chromosomal sublocalization was carried out by in situ hybridization of human chromosome metaphase spreads. The CLP gene is the first member of the human calmodulin/calmodulin-like gene family to be chromosomally sublocalized. Its presence near the telomeric end of the short arm of chromosome 10 may be of significance with respect to its highly (epithelial) cell-type restricted expression in vivo and strong downregulation upon malignant transformation. The generation of a human CLP gene-specific sequence tag site specified by the two primers used for PCR should prove useful for future linkage studies.     

The cytoskeletal protein talin contains at least two distinct vinculin binding domains

We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2- 6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin.