Phosphorylation of caldesmon by p34cdc2 kinase. Identification of phosphorylation sites

It has recently been shown that caldesmon from non-muscle (Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172) and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding to F-actin and Ca(2+)-calmodulin. In this study, we have identified five phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667, Thr673, Thr696, and Ser702. All the sites bear some resemblance to the S(T)-P-X-X motif recognized by p34cdc2. The preferred site of phosphorylation at Thr673 accounts for about 40% of the total phosphorylation. Four of the sites occur in two pairs of closely spaced sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in each pair inhibits strongly the phosphorylation of the second site in the same pair, presumably due to the close proximity of the two sites. Similar negative cooperativity in phosphorylation of Ser667 and Thr673 was observed using a 22-residue synthetic peptide containing the two sites. Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90% of the total level of phosphorylation and these sites are located within the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to bind actin and Ca(2+)-calmodulin.     

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.     

Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis

We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.     

Base-catalyzed degradation of permethylated 3-O-glycosyl- glycopyranosid-2-uloses

In a modification of the Svensson degradation, otherwise permethylated glycopyranosid-2-uloses bearing 4-O-glycosyl substituents are formed by the Swern oxidation. Base-catalyzed elimination on treatment with triethylamine then gives 4-deoxy-3-O-methylglyc-3-enopyranosid-2-ulose-terminat ed oligosaccharides with liberation of glycosyl substituents as reducing sugars but without further degradation. Mild acid hydrolysis results in removal of the unsaturated sugar residues so that the overall depolymerization occurs with net loss only of the initially oxidized sugar residue.     

A novel L-fuco-4-O-methyl-D-glucurono-D-xylan from Hyptis suaveolens

The acidic polysaccharide from the seed-coat mucilage of Hyptis suaveolens is a highly branched L-fuco-4-O-methyl-D-glucurono-D-xylan for which a structure is proposed having a 4-linked beta-D-xylan backbone carrying side chains of single 4-O-methyl-alpha-D-glucuronic acid residues at O-2 and 2-O-L-fucopyranosyl-D-xylopyranose units at O-3. The structural analysis involves base-catalyzed beta-elimination of uronic acid residues from the methylated glycan followed by degradation using a modified Svensson oxidation-elimination sequence.     

Sequence and characterization of Bacillus subtilis CheW.

A Bacillus subtilis open reading frame (ORF) encoding a predicted polypeptide of 156 amino acids was subcloned and sequenced. The polypeptide was found to be homologous to CheW of Escherichia coli, sharing 28.6% amino acid identity. The ORF was verified by using a bacteriophage T7 expression system in E. coli. The gene was inactivated by insertion of a nonpolor chloramphenicol acetyltransferase cassette in its N-terminal region. In the absence of chemoeffectors, the mutant displayed a smooth swimming bias, with some tumbling. The CheW- mutant was defective on swarm plates but was complemented by a plasmid that expressed wild type CheW. Addition of attractant or repellent to the CheW- mutant resulted in transient smooth swimming or tumbling, respectively. However, capillary assays revealed that chemotaxis was substantially impaired in the mutant strain.     

Epidermal growth factor receptor: Elements of intracellular communication

While EGF has an important function in cell growth regulation, the molecular mechanisms by which intracellular signal connect the EGF: receptor complex on the plasma membrane with the initiation of DNA synthesis and mitogenesis is not well understood. The discovery that rasGAP, PI-3 kinase and PLC-gamma 1 are substrates for the EGF receptor tyrosine kinase has provided a beginning in understanding the biochemistry underlying growth factor receptor transduction.     

Inhibition of AIDS virus replication by acemannan in vitro.

Acemannan (ACE-M), a beta-(1,4)-linked acetylated mannan, was evaluated for in vitro activity against human immunodeficiency virus type 1 (HIV-1). Castanospermine (CAS), deoxymannojirimycin (DMN), swainsonine (SWS), azidothymidine (AZT), and dideoxythymidine (DDC) were tested in parallel as control compounds. In vitro antiviral efficacy of ACE-M was evaluated in a variety of cell lines including human peripheral mononuclear, CEM-SS1 and MT-2(2) cells. The virus strain, number of infectious units per cell, and target cell line were important factors in determining the degree of inhibition of viral cytopathic effect in the presence of ACE-M and other control compounds tested. Maximum inhibitory effect was observed in CEM-SS cells infected with the RFII strain of HIV-1. This inhibitory effect was determined to be concentration-dependent. Assay design included primary screening to measure cell viabilities of infected target cells in the presence and absence of test compounds. When tested on HIV-1/RFII-infected CEM-SS cells, the 50% inhibitory effect of CAS (IC50 = 28), an inhibitor of alpha-glucosidase I, was determined to be similar to that observed for ACE-M (IC50 = 45). However, DMN and SWS, inhibitors of mannosidase I and II, tested in parallel to CAS and ACE-M, exhibited no IC50 values. Antiviral potential of ACE-M as an inhibitor of syncytia formation was also explored using CEM-SS cells. Suppression of syncytia formation was observed at an ACE-M concentration of 31.25 micrograms/ml, and complete inhibition was observed at 62.5 micrograms/ml. In addition, HIV-1 RNA levels were studied to establish the antiviral potential of ACE-M in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of protein perturbants on phospholipid bilayers

Series of alcohols, amides, ureas, and sulfoxides with increasingly longer hydrocarbon chains have been shown to lower progressively the thermal denaturation temperature of proteins. This effect is presumably due to a hydrophobic interaction between the solute and nonpolar domains of the protein. Theoretically, these interactions should occur between the solute and any macromolecular structure having a nonpolar region to which the solute has access. A recent review by Arakawa et al. has summarized evidence for such an interaction between organic solutes and proteins and suggested that these interactions are favored at higher temperatures. The present study investigates the effects of several classes of compounds on the stability of phospholipid vesicles. The results show that many compounds that are known to perturb protein function also destabilize phospholipid bilayers as reflected by solute-induced loss of vesicle contents.     

Co-purification of proteases with basic fibroblast growth factor (FGF)

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.