Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific antibodies: relation to cytoskeletal structures.

The intracellular precipitation of nonerythrocyte spectrin has been achieved by the microinjection into cells of either a monoclonal antibody (IgM) directed against the alpha chain of nonerythrocyte spectrin or an affinity-purified polyclonal antibody raised against bovine brain spectrin (fodrin). This antibody-induced precipitation of spectrin was observed in fibroblastic and epithelial cell types, including embryonic bovine tracheal fibroblasts, a bovine kidney epithelial cell line (MDBK), Hela cells, gerbil fibroma cells, and fibroblast lines of human and mouse origins. The precipitation of the spectrin was specific and two proteins with a similar distribution to the nonerythrocyte spectrin were not induced to co-precipitate in the spectrin aggregates. Comparing the two types of antibody microinjected, the affinity-purified polyclonal antibody resulted in more compact aggregates of spectrin and these were frequently aligned with microfilament bundles. The rate at which the spectrin aggregates were cleared into presumptive lysosomes varied with different cell types: in some such as the bovine kidney epithelial cells, this appeared complete within 3 h after microinjection, whereas in some of the fibroblasts the spectrin aggregates were prominent in the cytoplasm at 24 and even 48 h after microinjection. Microfilament bundles appeared unaffected by the aggregation of spectrin. We conclude that the integrity of the actin microfilament bundles does not require nonerythrocyte spectrin and that most probably these structures are linked at their termini to the membrane through proteins other than nonerythrocyte spectrin. No effect of the intracellular spectrin precipitation was observed on cell shape, or on the distribution of coated vesicles or microtubules. The aggregation of the nonerythrocyte spectrin, however, did affect the distribution of the vimentin type of intermediate filaments in most of the cell types studied. These filaments became more distorted and condensed, but generally did not collapse around the nucleus as occurs following microtubule disruption induced by colchicine treatment. The clumped intermediate filaments were frequently seen to coincide with regions of aggregated spectrin. This aggregation of intermediate filaments was not induced by microinjection of irrelevant antibodies, nor was it induced by the monoclonal antibody against spectrin in cells with which it did not cross-react.(ABSTRACT TRUNCATED AT 400 WORDS)

Cathepsin D in breast cancer cells can digest extracellular matrix in large acidic vesicles.

In breast cancer cell lines, pro-cathepsin D is synthesized in excess and abnormally processed, resulting in its slower maturation and increased secretion into the culture medium. Since this lysosomal protease is only active at acidic pH, we have searched for acidic compartments other than lysosomes where cathepsin D might be active when MCF7 cells are plated on corneal extracellular matrix. We found large acidic intracellular vesicles (1.5 to 20 microns in diameter) by acridine orange and 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine staining, two fluorescent probes which reveal acidic compartments. These vesicles were actively acidified. They were 2- to 20-fold more abundant in MCF7 breast cancer cells and primary cultures of human breast cancers cells than in primary cultures of normal mammary epithelial cells. In living MCF7 cells, high resolution video-enhanced microscopy showed that these vesicles were mobile and intracellular. Double immunolocalization indicated that they contained mature cathepsin D (but no detectable pro-cathepsin D) and endocytosed extracellular material. This material (dextran, transferrin, and extracellular matrix) and the association with other lysosomal enzymes varied according to the vesicles, suggesting their heterogeneity (large endosomes or phagosomes). We conclude that, in breast cancer cells, cathepsin D may digest intracellularly phagocytosed and/or endocytosed extracellular matrix in large acidic vesicles. We propose that the higher expression of cathepsin D associated with the increased number of large acidic vesicles in breast cancer cells may facilitate digestion of basement membrane and consequently metastasis.

Characterization of the cyclic AMP-dependent protein kinase from rat pancreas, further purification of the catalytic subunit, substrate specificity, effect of the pancreatic heat stable inhibitor.

In order to investigate the sequence of events triggered by cyclic AMP and cyclic GMP in exocrine pancreatic cells, the identification of the various protein kinases possibly present in this tissue is of major interest. Further analysis of the two cyclic AMP-dependent protein kinases previously reported [11] suggests that KI is a degraded form of KII. It is therefore likely that a single holoenzyme is present in exocrine cells. In addition no protein kinase, specifically stimulated by cyclic GMP, has been detected in any fraction obtained in the course of purification of the cyclic AMP-dependent protein kinase. A faster and more efficient method than the one previously described [11] allows the purification (5000 times) of the protein kinase catalytic subunit. Analysis of the subunit by sodium dodecyl sulphate polyacrylamide gel electrophoresis indicates a molecular weight of 40 000 +/- 1 000. The enzyme phosphorylates specifically histone H2B (Vm = 236 min(-1), Km = 1.15 10(-5) M) and to a lesser extent H2A, H5 and H1 (Vm = 55--77 min(-1), Km 5--25 10(-5) M). Histones H3 and H4 are not phosphorylated. The effect of the heat stable inhibitor, extracted from rat pancreas, on the phosphorylation of H2B has been investigated. The inhibition is of the non competitive type with respect to ATP. The inhibition at various histone concentrations cannot be described by the Michaelis-Menten equation.

Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell-cell adhesion.

The spleen tyrosine kinase Syk has predominantly been studied in hematopoietic cells in which it is involved in immunoreceptor-mediated signaling. Recently, Syk expression was evidenced in numerous nonhematopoietic cells and shown to be involved in tumor formation and progression. The Syk downstream signaling effectors in nonhematopoietic cells remain, however, to be uncovered, and were investigated using MS-based quantitative phosphoproteomics. Two strategies, based on the inhibition of the Syk catalytic activity and on the loss of Syk expression were employed to identify phosphotyrosine-dependent complexes. Quantitative measurements were obtained on 350 proteins purified with phosphotyrosine affinity columns using the SILAC method. Forty-one proteins are dependent on both Syk expression and catalytic activity and were selected as signaling effectors. They are involved in a variety of biological processes such as signal transduction, cell-cell adhesion and cell polarization. We investigated the functional involvement of Syk in cell-cell adhesion and demonstrated the phosphorylation of E-cadherin and alpha-catenin. In addition, Syk is localized at cell-cell contacts, and Syk-mediated phosphorylation of E-cadherin seems to be important for the proper localization of p120-catenin at adherens junctions. Identification of the biochemical pathways regulated by Syk in human cancer cells will help to uncover its role in tumor formation and progression.

Interaction of biological membranes with the cytoskeletal framework of living cells.

The review is focused on the molecular structure and function of the proteins composing the actin-based cytoskeletal cortex, located at the cytoplasmic face of plasma membranes of eucaryotic cells, which stabilizes integral membrane proteins in separate domains of cell membranes. It includes a survey of the molecular properties of the proteins of the erythrocyte membrane skeleton such as spectrin, ankyrin, protein 4.1, and adducin. The properties of the immunological counterparts of erythroid cortical proteins found in nonerythroid tissues and cells are compared. The structural organization and function of the newly discovered class of calcium-binding proteins, nonerythroid peripheral membrane proteins, calpactins, are also described. Finally, the discussion of some experimental models illustrates that the membrane skeleton of living cells is actively involved in a wide variety of essential biological functions ranging from differentiation, to maintenance of cell polarity and cell shape, and regulation of exocytotic processes.

Binding of HeLa spectrin to a specific HeLa membrane fraction.

From 30-40 g of Hela-S3 cells grown in suspension, 0.25-0.50 mg of spectrin has been purified by conventional biochemical procedures starting from a low ionic strength extraction at alkaline pH of crude Hela membranes. Hela spectrin consists in its native form of a tetramer alpha 2 beta 2 of two high molecular weight polypeptides (240,000 and 230,000 daltons). Three different populations of Hela membranes depleted of both spectrin and actin have been prepared on discontinuous sucrose gradients. Surprisingly, spectrin will reassociate with only the heavier membrane fraction. This reassociation is specific for Hela spectrin, since three other purified Hela proteins as well as human erythrocyte spectrin do not reassociate under the same conditions. This binding is not due to the presence of traces of actin still present in the membrane fraction since two Hela actin-binding proteins (filamin I and II) do not show any significant binding to this fraction. The nature of the membrane-binding site for Hela spectrin is discussed.

Characterization of very acidic phagosomes in breast cancer cells and their association with invasion.

Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5-10 microns) in which endocytosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcourrier et al. (1990). Cancer Res. 50, 6045-6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epifluorescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 microns diameter latex beads and fluorescence Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are heterophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H(+)-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (< 4), lower than the lysosomal value (pH approximately 5) in the same cells and in specialized phagocytotic cells such as macrophages. We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.

Ezrin is a cyclic AMP-dependent protein kinase anchoring protein.

cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.

The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells.

Syk is a protein tyrosine kinase that is widely expressed in haematopoietic cells. It is involved in coupling activated immunoreceptors to downstream signalling events that mediate diverse cellular responses including proliferation, differentiation and phagocytosis. Syk expression has been reported in cell lines of epithelial origin, but its function in these cells remains unknown. Here we show that Syk is commonly expressed in normal human breast tissue, benign breast lesions and low-tumorigenic breast cancer cell lines. Syk messenger RNA and protein, however, are low or undetectable in invasive breast carcinoma tissue and cell lines. Transfection of wild-type Syk into a Syk-negative breast cancer cell line markedly inhibited its tumour growth and metastasis formation in athymic mice. Conversely, overexpression of a kinase-deficient Syk in a Syk-positive breast cancer cell line significantly increased its tumour incidence and growth. Suppression of tumour growth by the reintroduction of Syk appeared to be the result of aberrant mitosis and cytokinesis. We propose that Syk is a potent modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas.