Isolation of a major human placental substrate for the epidermal growth factor (urogastrone) receptor kinase: immunological cross-reactivity with transducin and sequence homology with lipocortin

Using as a starting material either a detergent extract or a protein fraction eluted from membranes with ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, we have isolated from human placental membranes a major substrate for the epidermal growth factor (urogastrone) receptor kinase (EGF kinase). The substrate was isolated both in an intact form, having a molecular mass of approximately 38-kDa (p38), and in a 35-kDa form (p35) representing a proteolytic cleavage product of p38. Both p38 and p35 cross-reacted with antibodies directed against bovine retinal transducin, but did not cross-react with antibodies directed against the 35-kDa beta subunit of human placental G-protein. Antisera directed against the placental EGF kinase substrate failed to react with either bovine or human placental src kinase substrate, p36. Conversely, antisera directed against p36 reacted only poorly with placental p38 or p35. Although p38 had a blocked amino terminus that precluded sequence analysis, p35 yielded an N-terminal sequence that was identical with residues 13-36 of human lipocortin. Our data clearly distinguish p38 from the previously described intestinal calcium binding protein calpactin I or p36 that is also a tyrosine kinase substrate, and our work points to a close relationship (if not identity) between p35 and a 35-kDa EGF receptor kinase substrate previously characterized in A431 cells. We conclude that p38 and p35, which very likely represent human placental lipocortin, may share only limited epitope homology with transducin alpha subunit; however, the possibility that p38, along with intestinal p36 and with a family of related calcium binding proteins, may, like transducin, play a role in receptor-mediated transmembrane signaling is discussed.     

Calcium-dependent conformational changes in the 36-kDa subunit of intestinal protein I related to the cellular 36-kDa target of Rous sarcoma virus tyrosine kinase

Protein I from intestinal epithelium is biochemically and immunologically related to the fibroblast 36-kDa substrate of the Rous sarcoma virus-encoded tyrosine protein kinase (Gerke and Weber (1984) EMBO J. 3, 227-233). Protein I is a Ca2+-binding protein containing two copies each of a 36- and 10-kDa subunit. Denaturation/renaturation experiments show that the 36-kDa subunit is a monomer, whereas the 10-kDa subunit forms a dimer. Mixing of the subunits leads to reconstituted protein I. Physicochemical properties of protein I and its isolated subunits reveal a Ca2+-dependent conformational change in the 36-kDa subunit which involves the exposure of 1 or more tyrosine residues to a more aqueous environment. This change points to a Ca2+ binding constant of about 10(4) M-1 in the presence of 2 mM Mg2+ and induces the ability of protein I and the 36-kDa subunit to bind in vitro to F-actin and nonerythroid spectrin. The same high Ca2+ requirement has been reported for the in vitro tyrosine phosphorylation of a 35-kDa protein from A-431 carcinoma cells by the epidermal growth factor receptor kinase (Fava and Cohen (1984) J. Biol. Chem. 259, 2636-2645). Here we show that this 35-kDa substrate is biochemically and immunologically related to the 36-kDa subunit of protein I, which in turn corresponds to the substrate of the Rous sarcoma virus kinase. The protein of A-431 cells exists not only as a monomer but also as a dimer. The latter fraction contains a 10-kDa polypeptide immunologically related to the corresponding subunit of protein I. Given past results on the A-431 system, we speculate that the monomer rather than the dimer is the preferred in vitro substrate for the epidermal growth factor receptor kinase. Thus, the 10-kDa subunit, which induces dimerization of the phosphorylatable large subunit, may act as an inhibitor.     

Functional Analysis of Putative Adhesion Factors in Lactobacillus acidophilus NCFM

Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.     

Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM

Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.     

Disruption of arabinogalactan proteins disorganizes cortical microtubules in the root of Arabidopsis thaliana

The cortical array of microtubules inside the cell and arabinogalactan proteins on the external surface of the cell are each implicated in plant morphogenesis. To determine whether the cortical array is influenced by arabinogalactan proteins, we first treated Arabidopsis roots with a Yariv reagent that binds arabinogalactan proteins. Cortical microtubules were markedly disorganized by 1 microM beta-D-glucosyl (active) Yariv but not by up to 10 microM beta-D-mannosyl (inactive) Yariv. This was observed for 24-h treatments in wild-type roots, fixed and stained with anti-tubulin antibodies, as well as in living roots expressing a green fluorescent protein (GFP) reporter for microtubules. Using the reporter line, microtubule disorganization was evident within 10 min of treatment with 5 microM active Yariv and extensive by 30 min. Active Yariv (5 microM) disorganized cortical microtubules after gadolinium pre-treatment, suggesting that this effect is independent of calcium influx across the plasma membrane. Similar effects on cortical microtubules, over a similar time scale, were induced by two anti-arabinogalactan-protein antibodies (JIM13 and JIM14) but not by antibodies recognizing pectin or xyloglucan epitopes. Active Yariv, JIM13, and JIM14 caused arabinogalactan proteins to aggregate rapidly, as assessed either in fixed wild-type roots or in the living cells of a line expressing a plasma membrane-anchored arabinogalactan protein from tomato fused to GFP. Finally, electron microscopy of roots prepared by high-pressure freezing showed that treatment with 5 microM active Yariv for 2 h significantly increased the distance between cortical microtubules and the plasma membrane. These findings demonstrate that cell surface arabinogalactan proteins influence the organization of cortical microtubules.     

Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme

Many terrestrial plant species are able to form symbiotic associations with arbuscular mycorrhizal fungi. Here we have identified three cDNA clones representing genes whose expression is induced during the arbuscular mycorrhizal symbiosis formed between Medicago truncatula and an arbuscular mycorrhizal fungus, Glomus versiforme. The three clones represent M. truncatula genes and encode novel proteins: a xyloglucan endotransglycosylase-related protein, a putative arabinogalactan protein (AGP), and a putative homologue of the mammalian p110 subunit of initiation factor 3 (eIF3). These genes show little or no expression in M. truncatula roots prior to formation of the symbiosis and are significantly induced following colonization by G. versiforme. The genes are not induced in roots in response to increases in phosphate. This suggests that induction of expression during the symbiosis is due to the interaction with the fungus and is not a secondary effect of improved phosphate nutrition. In situ hybridization revealed that the putative AGP is expressed specifically in cortical cells containing arbuscules. The identification of two mycorrhiza-induced genes encoding proteins predicted to be involved in cell wall structure is consistent with previous electron microscopy data that indicated major alterations in the extracellular matrix of the cortical cells following colonization by mycorrhizal fungi.     

Sonic hedgehog is associated with H+-K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation

Sonic hedgehog (Shh) is found within gastric parietal cells and processed from a 45-kDa to a 19-kDa bioactive protein by an acid- and protease-dependent mechanism. To investigate whether Shh is associated with the parietal cell membrane compartment that becomes exposed to both acid and proteolytic enzymes during acid secretion, the cellular location of Shh within resting and stimulated gastric parietal cells was examined. Immunofluorescence microscopy of rabbit stomach sections showed that Shh colocalized predominantly with parietal and pit, not chief/zymogen or neck, cell markers. In resting and histamine-stimulated rabbit gastric glands Shh was expressed only in parietal cells close to H+-K+-ATPase-containing tubulovesicular and secretory membranes with some colocalizing with gamma-actin at the basolateral membrane. Gastric gland microsomal membranes were prepared by differential and sucrose gradient centrifugation and immunoisolation with an anti-H+-K+-ATPase-alpha subunit antibody. The 45- and 19-kDa Shh proteins were detected by immunoblot in immunopurified H+-K+-ATPase-containing membranes from resting and stimulated gastric glands, respectively. Incubating glands with a high KCl concentration removed Shh from the membranes. Histamine stimulated 19-kDa Shh secretion from gastric glands into the medium. In human gastric cancer 23132/87 cells cultured on permeable membranes, histamine increased 19-kDa Shh secretion into both apical and basolateral media. These findings show that Shh is a peripheral protein associated with resting and stimulated H+-K+-ATPase-expressing membranes. In addition, Shh appears to be expressed at or close to the basolateral membrane of parietal cells.     

Phosphorylation of p36 in vitro by protein kinase C

The 36kDa subunit of protein I (p36) is a major substrate of several tyrosine protein kinases. Here we demonstrate that protein kinase C catalyzes the incorporation of 1.7 moles of phosphate per mole of protein I. Phosphorylation is absolutely dependent on the presence of both calcium and phospholipid, and is specific for serine and threonine residues. Phosphorylation of protein I by the c-AMP dependent protein kinase, phosphorylase kinase, casein kinase I, and casein kinase II was not observed. The in vivo significance of protein kinase C dependent phosphorylation of p36 is discussed.     

Comparison of Ca++-regulated events in the intestinal brush border

The intestinal epithelial cell and specifically the cytoskeleton of the brush border are thought to be controlled by micromolar levels of free calcium. Calcium-binding proteins of this system include intestinal calcium binding protein (CaBP), calmodulin (CaM), villin, and a 36,000-mol-wt protein substrate of tyrosine kinases. To assess the sequence of events as the intracellular Ca++ level rises, we determined the amount of CaM and CaBP in the intestinal epithelium by western blotting and tested the Ca++ binding of CaM and CaBP by equilibrium dialysis. The Ca++-dependent actin severing activity of villin was analyzed in the presence of physiological CaM levels and increasing calcium concentrations. In addition, we analyzed the Ca++ levels required for interaction between CaM and the microvillus 110,000-mol-wt protein as well as fodrin and the interaction between a polypeptide of 36,000 mol wt (P-36) and actin. The results suggest that CaBP serves as the predominant Ca++ buffer in the cell, but CaM can effectively buffer ionic calcium in the microvillus and thus protect actin from the severing activity of villin. CaM binds to its cytoskeletal receptors, 110,000-mol-wt protein and fodrin differently, governed by the free Ca++ and pH. The interaction between P-36 and actin, however, appears to require an unphysiologically high calcium concentration (10(-4) to 10(-3) M) to be meaningful. The results provide a coherent picture of the different Ca++ regulated events occurring when the free calcium rises into the micromolar level in this unique system. This study would suggest that as the Ca++ rises in the intestinal epithelial cell an ordered sequence of Ca++ saturation of intracellular receptors occurs with the order from the lowest to highest Ca++ requirements being CaBP less than CaM less than villin less than P-36.