Eric H. Ball

Quantitation of proteins by elution of Coomassie brilliant blue R from stained bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

A simple method for the extraction of Coomassie brilliant blue R from stained protein bands excised from polyacrylamide gels is described. Spectrophotometric measurement of the eluted dye forms the basis of a sensitive assay to quantitate proteins in gels in the range 0.5-10 micrograms. The method requires no unusual equipment and is suitable for measurement of multiple samples. The polypeptide is not extracted and remains available for further analysis. The technique has been applied to three proteins and gels of various acrylamide percentages.

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.

Stomatin-Like Protein 2 Binds Cardiolipin and Regulates Mitochondrial Biogenesis and Function

ABSTRACT Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched microdomains in which electron transport complexes are optimally assembled. Likely through the prohibitin functional interactome, SLP-2 then regulates mitochondrial biogenesis and function.

Regulation of T-Cell Activation by Phosphodiesterase 4B2 Requires Its Dynamic Redistribution during Immunological Synapse Formation

ABSTRACT Stimulation of T cells through their antigen receptors (TCRs) causes a transient increase in the intracellular concentration of cyclic AMP (cAMP). However, sustained high levels of cAMP inhibit T-cell responses, suggesting that TCR signaling is coordinated with the activation of cyclic nucleotide phosphodiesterases (PDEs). The molecular basis of such a pathway is unknown. Here we show that TCR-dependent signaling activates PDE4B2 and that this enhances interleukin-2 production. Such an effect requires the regulatory N terminus of PDE4B2 and correlates with partitioning within lipid rafts, early targeting of this PDE to the immunological synapse, and subsequent accumulation in the antipodal pole of the T cell as activation proceeds.

Inhibition of protein kinase C by sphingosine correlates with the presence of positive charge

The role of the 2-amino group of sphingosine on the in vitro inhibition of protein kinase C was investigated by comparing protein kinase C activity in the presence and absence of sphingosine at various pHs. Inhibition by sphingosine was found to be pH dependent. Above pH 7.75, sphingosine has little or no inhibitory effect. In fact, at pH 8.5, sphingosine slightly enhances enzyme activity above that which occurs when the enzyme is stimulated by diacylglycerol and phosphatidylserine. After correcting for electrostatic repulsion, we find that the intrinsic pK for sphingosine in Triton micelles is 8.5. Inhibition of protein kinase C by sphingosine at physiological pHs therefore correlates with the presence of a positive charge.

The Relationship Between the Bilayer to Hexagonal Phase Transition Temperature in Membranes and Protein Kinase C Activity

A number of substances affect the activity of protein kinase C. Among uncharged and zwitterionic compounds, those which activate protein kinase C also lower the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine while substances which inhibit protein kinase C raise this transition temperature. Using this criteria, we have identified 3β-chloro-5-cholestene, 5β-cholan-24-ol and eicosane as new protein kinase C activators and have shown that Z-Ser-Leu-NH2, Z-Gly-Leu-NH2, Z-Tyr-Leu-NH2, cyclosporin A and cholestan-3β, 5α, 6β-triol are protein kinase C inhibitors.

A collagen-binding protein involved in the differentiation of myoblasts recognizes the Arg―Gly―Asp sequence

We had earlier demonstrated that a 46-kDa glycoprotein is involved in the differentiation of rat skeletal myoblasts. We now show that the binding of this glycoprotein to collagen and gelatin is disrupted by Arg-Gly-Asp (RGD) containing peptide but not by Arg-Gly-Glu (RGE). The former peptide also selectively elutes the 46-kDa glycoprotein bound to gelatin-Sepharose. Since all other proteins which bind RGD sequences have been found at the cell surface, we attempted to localize the 46-kDa glycoprotein by means of immuno fluorescent staining and radioiodine labeling. Surprisingly, the majority of the protein was found to be localized in the endoplasmic reticulum. Protease treatment of a microsomal fraction revealed that the protein is in the interior of the reticulum. Immunoprecipitation experiments, using a polyclonal antibody against the 46-kDa protein, demonstrated that no closely related proteins exist in myoblasts and also confirmed that the protein was not a fragment of a cell-surface localized protein. These findings suggest that the RGD sequence is also used in protein recognition within the cell.

Interaction of the N- and C-terminal Domains of Vinculin CHARACTERIZATION AND MAPPING STUDIES

The vinculin head to tail intramolecular self-association controls its binding sites for other components of focal adhesions. To study this interaction, the head and tail domains were expressed, purified, and assayed for various characteristics of complex formation. Analytical centrifugation demonstrated a strong interaction in solution and formation of a complex more asymmetric than either of the individual domains. A survey of binding conditions using a solid-phase binding assay revealed characteristics of both electrostatic and hydrophobic forces involved in the binding. In addition, circular dichroism of the individual domains and the complex demonstrated that conformational changes likely occur in both domains during association. The interaction sites were more closely mapped on the protein sequence by deletion mutagenesis. Amino acids 181–226, a basic region within the acidic head domain, were identified as a binding site for the vinculin tail, and residues 1009–1066 were identified as sufficient for binding the head. Moreover, mutation of an acidic patch in the tail (residues 1013–1015) almost completely eliminated its ability to interact with the head domain further supporting the significance of ionic interactions in the binding. Our data indicate that the interaction between the head and tail domains of vinculin occurs through oppositely charged contact sites and results in conformational changes in both domains.

Partial characterization of a collagen-binding, differentiation-related glycoprotein from skeletal myoblasts

A 46-kDa glycoprotein, gp46, which binds collagen has been purified to homogeneity from L6 rat skeletal myoblasts. The procedure involves extraction of crude myoblast membranes with 1% sodium dodecyl sulfate followed by concanavalin A affinity chromatography and preparative gel electrophoresis. The sequence of 15 N-terminal amino acids had some resemblance to a sequence in myosin light chains. The oligosaccharide chains of the glycoprotein can be released by treatment with endoglycosidase H, suggesting that gp46 has high-mannose type of glycans. Galactose and sialic acid are not detected in the purified protein. gp46 is widely distributed and conserved in different cell lines as determined by immunoblotting using a monoclonal anti-gp46 antibody. High levels of gp46 were found in several fibroblastic and myogenic cell lines, but not in a hematopoietic cell line. Undifferentiated F9 embryonal carcinoma cells lacked gp46 but the glycoprotein was induced when the cells were made to differentiate in the presence of retinoic acid. Broad survey of gp46 in different cell lines also suggests that it is present mainly in those cell lines which attach to the substratum and produce collagens. Although the function of gp46 is not yet known, the evidence suggests that it is developmentally regulated and is probably involved in the synthesis or assembly of collagen in the endoplasmic reticulum.

Apparent dominance of serine auxotrophy and the absence of expression of muscle-specific proteins in rat myoblast × mouse L-cell hybrids

Hybrids obtained from crosses of rat myoblasts and mouse L-cells are unable to differentiate into myotubes. The synthesis of muscle-specific proteins, creatine phosphokinase (CPK), myosin, and adenyl cyclase is also suppressed in the hybrids. Adenyl cyclase produced in the hybrids is not activated by isoproterenol. The glycine auxotrophy of myoblasts is phenotypically expressed as a dominant trait in hybrids. Genotypically, however, all the enzymes of the phosphorylated pathway of serine biosynthesis and serine hydroxymethyltransferase, the enzyme inter converting serine and glycine, are present in normal concentrations in the hybrids, just as they are in the mouse L-cells. Unlike either of the parental lines, the hybrid cells are unable to grow on serine-supplemented medium, but do so when glycine is also added. Glycine by itself supports growth of the hybrids in the absence of added serine. All the curious effects of glycine/serine on growth of hybrids are rationalized and accounted for by the hypothesis that in these cells the enzyme serine hydroxy-methyltransferase, for unknown reasons, does not function in vivo.