Emergence and disappearance of an immune molecule,an antimicrobial lectin,in basal metazoa. A tachylectin-related protein in the sponge Suberites domuncula

Sponges (phylum Porifera) represent the evolutionarily oldest metazoans that comprise already a complex immune system and are related to the crown taxa of the protostomians and the deuterostomians. Here, we demonstrate the existence of a tachylectin-related protein in the demosponge Suberites domuncula, termed Suberites lectin. The MAPK pathway was activated in response to lipopolysaccharide treatment of the three-dimensional cell aggregates, the primmorphs; this process was abolished by the monosaccharide D-GlcNAc. The cDNA encoding the S. domuncula lectin was identified and cloned; it comprises 238 amino acids (26 kDa) in the open reading frame. The deduced protein has one potential transmembrane region, three characteristic Cys residues, and six internal tandem repeats; it shares the highest sequence similarity with lectins from the horseshoe crab Tachypleus trunculus. The steady-state level of expression of the Suberites lectin rises in primmorphs in response to lipopolysaccharide, an effect that was prevented by co-incubation with D-GlcNAc. The natural sponge lectin was purified by affinity chromatography; it has a size of 27 kDa and displays antibacterial activity against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus. The putative protein, deduced from the cloned gene, is identical/similar to the purified natural protein, as demonstrated by immunological cross-reactivity with specific antibodies. We conclude that the S. domuncula lectin acts as an antibacterial molecule involved in immune defense against bacterial invaders.     

Dephosphorylation of the hepatic insulin receptor: absence of intrinsic phosphatase activity in purified receptors

We have compared here the reversibility of phosphorylation of insulin receptors either partially purified by lectin chromatography, or highly purified by specific immunoprecipitation with anti-receptor antibodies. We found that the beta subunit of partially purified insulin receptors was rapidly dephosphorylated (t 1/2 = 15 min). In contrast, the level of phosphorylation of immunoprecipitated receptors remained unchanged for up to 4 hours at 37 degrees C. However, cytosolic phosphatases, which are inhibited by vanadate, were able to induce a complete dephosphorylation of immunoprecipitated receptors. These results show that 1. phosphorylation of insulin receptors is reversible; and 2. no phosphatase activity is contained in the insulin receptor structure itself.     

Sucrose induces rapid activation of CfSAPK, a mitogen-activated protein kinase, in Cephalostachyum fuchsianum Gamble cells

Sucrose was recently demonstrated to function as a molecular signal. However, sucrose-specific sensing and signalling pathways remain largely undefined. Here, we show that Cephalostachyum fuchsianum sucrose-activated protein kinase (CfSAPK) is transiently and specifically activated by sucrose in C. fuchsianum Gamble suspension cells. The result suggested that CfSAPK participates in a sucrose-signalling pathway. CfSAPK was partially purified from sucrose-treated cells and further analysed. Kinase activity assays revealed that CfSAPK preferentially used myelin basic protein (MBP) as substrate in vitro and strongly phosphorylate MBP threonine residue(s) and weakly phosphorylated MBP serine residue(s). Of the divalent cations tested, Mg(2+) was required for CfSAPK activation. Phosphatase treatment of CfSAPK abolished its kinase activity, indicating that phosphorylation is required for CfSAPK activation. Seven internal tryptic peptides identified from CfSAPK matched mitogen-activated protein kinases (MAPKs) in plants. CfSAPK cDNA was cloned using RT-PCR and rapid amplification of cDNA ends (RACE). CfSAPK cDNA encodes a 382-amino acid protein with a calculated molecular mass of 43,466.9 Da. The CfSAPK protein contains all 11 conserved kinase subdomains found in other Ser/Thr kinases. The amino acids sequence of CfSAPK is highly homologous to group A MAPKs in monocotyledon plants.     

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.     

Insulin-like effect of trypsin on the phosphorylation of rat adipocyte insulin receptor

Trypsin treatment of a partially purified insulin receptor preparation from rat adipocytes stimulated the phosphorylation of 90,000- and 72,000-Da polypeptides immunoprecipitated by anti-insulin receptor antibody. The phosphorylation of tyrosine residues alone was observed in both polypeptides. Trypsin concentrations which stimulated insulin receptor phosphorylation were the same as those previously shown to activate rat adipocyte glycogen synthase. Trypsin treatment of the insulin receptor fraction also stimulated the phosphorylation of an exogenous substrate of tyrosine kinase similarly to insulin treatment. Trypsin treatment of a highly purified insulin receptor from human placenta also activated the phosphorylation of the receptor-derived peptides. These results suggest that the insulin-stimulated protein kinase, a component of the insulin receptor, was activated by tryptic digestion to phosphorylate polypeptides derived from the insulin receptor itself. Thus, it is suggested that stimulation by trypsin of phosphorylation of the insulin receptor may be related to the insulin-like metabolic actions of trypsin observed in rat adipocytes.     

Insulin stimulates the phosphorylation level of v-Ha-ras protein in membrane fraction

Insulin was found to stimulate the phosphorylation of the 21,000-dalton protein encoded by the ras oncogene of Harvey murine sarcoma virus in membrane fraction both in vivo and in vitro. When the human ras proteins expressed in E. coli were reconstituted with purified human insulin receptor, GTPase activity of normal or its mutated oncogenic ras protein was not stimulated by the addition of insulin. Likewise, tyrosine kinase activity or insulin binding capacity of the receptor was not influenced when assayed in the presence of the ras proteins. These results suggest that ras proteins may be coupled with the insulin receptor system through some unidentified membrane factors.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

Protein kinase C phosphorylates the sponge aggregation receptor after its binding to the homologous aggregation factor

The aggregation factor from the sponge Geodia cydonium functions also as a growth factor after binding to the aggregation receptor (= growth factor receptor) on the plasma membrane of homologous cells. We have recently shown that protein kinase C is involved in the pathway transducing the growth factor signal. Here we report that the aggregation receptor (a polypeptide with an Mr of 43,500) is phosphorylated by protein kinase C. Using a plasma membrane fraction only this phosphoprotein (pp) 43.5 became phosphorylated by kinase C. The phosphorylation of pp43.5 in intact cells in response to the binding of the aggregation factor to this polypeptide was a late event and occurred 10 to 15 h after addition of the aggregation factor. Based on studies with phorbol esters it appears to be very likely that protein kinase C also phosphorylates pp43.5 in vitro. The degree of phosphorylation of pp43.5 paralleled with both the extent of DNA synthesis and ras oncogene expression. The latter process resulted in a switch of the responsiveness of the cells to growth factors signals: 10 to 15 h after addition of the aggregation factor to dissociated cells, this factor lost its growth factor function while the homologous lectin gained the ability to stimulate cell proliferation (to be published). These results support the idea that phosphorylation of pp43.5 (= aggregation receptor) results in an inhibition of its function, i.e., the transduction of the growth factor (= aggregation factor) signal.     

Inhibition of protein kinase C zeta subspecies blocks the activation of an NF-kappa B-like activity in Xenopus laevis oocytes.

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a large variety of cellular genes. However, the mechanism whereby this nuclear factor is activated remains to be determined. In this report, we present evidence that in oocytes from Xenopus laevis, (i) ras p21- and phospholipase C (PLC)-mediated phosphatidylcholine (PC) hydrolysis activates NF-kappa B and (ii) protein kinase C zeta subspecies is involved in the activation of NF-kappa B in response to insulin/ras p21/PC-PLC. Thus, the microinjection of either ras p21 or PC-PLC, or the exposure of oocytes to insulin, promotes a significant translocation to the nucleus of an NF-kappa B-like activity. This effect is not observed when oocytes are incubated with phorbol myristate acetate or progesterone, both of which utilize a ras p21-independent pathway for oocyte activation. These data strongly suggest a critical role of the insulin/ras p21/PC-PLC/protein kinase C zeta pathway in the control of NF-kappa B activation.     

Molecular cloning, characterization, and expression of a human 14-kDa lectin

Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta. Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library. The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin. This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library. The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence. Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated. Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene. The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography.     

Studies on ras proteins. Catalytic properties of normal and activated ras proteins purified in the absence of protein denaturants

Normal (Gly12) and activated (Val12) Ha-ras proteins were produced in Escherichia coli, and purified to an apparent homogeneity without using any protein denaturants. The purified proteins contained an equimolar amount of GDP. They were stable in the presence of 5 mM Mg2+ and 25% (v/v) glycerol when incubated at 60 degrees C for 5 min. The binding of GDP to the protein was greatly stabilized by Mg2+. In the presence of 10 mM Mg2+, the bound GDP hardly exchanged with external guanine nucleotides, even at 30 degrees C. The exchange reaction was markedly enhanced in the presence of 10 mM EDTA or 120 mM ammonium sulfate. The rate-limiting step of the exchange reaction was the dissociation of the bound GDP from the ras protein, and this step was facilitated 40- to 100-fold by the addition of EDTA or ammonium sulfate. The dissociation rate of the normal (Gly12) ras protein was 2- to 3-fold faster than that of the activated (Val12) protein. The dissociation constants (Kd) for GDP of the normal and activated ras proteins were 1.2 X 10(-8) and 3.1 X 10(-9) M, respectively. The overall turnover rate of GTPase activity of the normal ras protein (10.8 mmol.mol-1.min-1) was about 10-fold higher than that of the activated protein (1.1 mmol.mol-1.min-1) in the absence of Mg2+ (less than 10(-8) M).     

A Lectin-like Molecule is Discharged from Mucocysts of Tetrahymena pyriformis in the Presence of Insulin

ABSTRACT. By use of a monoclonal antibody directed against purified lectin from the sponge Geodia cydonium it was demonstrated that the mucocysts of Tetrahymena pyriformis contain a substance immunologically similar to that found in G. cydonium . In extracts of T. pyriformis the monoclonal antibody recognizes a 36 kDa protein; binding could be abolished by adsorption of the antibody with (i) crude extract, (ii) purified lectin from G. cydonium and (iii) a 29 aa long peptide. In addition the data show that 10^-6 M of insulin causes first the release of mucocyst material, which reacts with the lectin antibody, and second its subsequent redistribution on the surface of the somatic cilia and the oral field.     

Effect of Cryphonectria hypovirus 1 (CHV1) infection on Cpkk1, a mitogen-activated protein kinase kinase of the filamentous fungus Cryphonectria parasitica

We screened Cryphonectria parasitica genomic and cDNA libraries with a probe obtained from the amplification of a conserved region among the sequence of known mitogen activated protein kinase kinases (MAPKK) and obtained genomic and cDNA clones. Sequence comparisons of the clones obtained confirmed the identification of a C. parasitica homologue to other fungal MAPKK, which we named Cpkk1. Polyclonal antibodies raised against a purified Cpkk1 fusion protein expressed in Escherichia coli were used to detect Cpkk1 protein in extracts of CHV1-infected and uninfected C. parasitica grown in liquid culture. Differences in the dynamics of phosphorylation and dephosphorylation were noticed. Under the conditions investigated, Cpkk1 protein expression is associated with active mycelial growth, before the onset of a senescent developmental stage. We hypothesize that differences in Cpkk1 phosphorylation state between CHV1 infected and virus free strains are due to a delay of the onset of the developmental stage caused by the presence of the virus.     

Protein phosphorylation in isolated human adipocytes-adrenergic control of the phosphorylation of hormone-sensitive lipase

The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32PO4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the beta-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (Mr 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the alpha-2 agonist clonidine. Epinephrine, a combined alpha and beta agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the alpha-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.     

The ral gene: a new ras related gene isolated by the use of a synthetic probe.

We synthesized a set of 20-mer oligonucleotides corresponding to a sequence of seven amino acids strictly conserved in all the different ras proteins, from yeast to man, as well as in rho and YPT, two proteins distantly related to p21 ras (approximately 30% amino acid homology). This oligonucleotide probe was used to search for new members of the ras family. We describe here a new ras related gene named ral, isolated from a cDNA library of immortalized simian B-lymphocytes. The ral gene codes for a 206 amino acid protein of expected mol. wt 23.5 kd that shares greater than 50% homology with H-ras, K-ras or N-ras. The GTP binding regions of p21 ras and a C-terminal cysteine involved in membrane anchoring are also present in ral; this strongly suggests that ral is a GTP binding protein with membrane localization. Furthermore, several external regions of p21 ras presumably involved in the interaction with effector, receptor and/or regulatory proteins are highly homologous to the corresponding regions in ral. Therefore some of the proteins that interact with ral might be identical or closely related to those interacting with p21 ras.     

Diacylglycerols modulate phosphorylation of the insulin receptor from human mononuclear cells

It has been found that 1,2- but not 1,3-diacylglycerols stimulated phosphorylation of the insulin receptor of cultured human monocyte-like (U-937) and lymphoblastoid (IM-9) cells both in the intact- and broken-cell systems. The stimulation of the receptor's beta-subunit phosphorylation was dose-dependent, with optimal effect at 100 micrograms/ml of diacylglycerol. The effects of insulin and 1,2-diacylglycerols on the phosphorylation of partially purified insulin receptors were additive. Phosphoamino acid analysis showed a major effect of diacylglycerols on phosphorylation of tyrosine residues. The diacylglycerols also stimulated tyrosine kinase activity of the partially purified U-937 and IM-9 insulin receptors 2.5-3.5-fold when measured by phosphorylation of an exogenous substrate, poly(Glu80Tyr20) in the absence of any added insulin, calcium or phospholipid. Since this diacylglycerol effect could not be reproduced under conditions optimal for protein kinase C activation and the purified protein kinase C did not stimulate phosphorylation of the beta-subunit of the insulin receptor in this system, it is unlikely that the diacylglycerol effect was mediated by protein kinase C. Since these exogenous 1,2-diacylglycerols at the same high concentration also inhibited 125I-insulin binding to the insulin receptor of the intact U-937 and IM-9 cells, diacylglycerols could modulate the function of the insulin receptor and insulin action in human mononuclear cells.     

香蕉MuMADS1基因的生物信息学分析

通过筛选用采后0h和48h的香蕉果实构建的果实成熟的SSH(抑制差减杂交)文库,得到一条命名为MuMADS1长度为888bp的片段。通过互联网数据库及生物信息学分析工具对香蕉MuMADS1基因及其编码蛋白进行理化性质预测、序列与结构分析和功能预测。结果表明:MuMADS1基因编码蛋白分子式为C1171H1879N351O367S7,属于亲水的不稳定蛋白;保守结构域分析含有保守的MADS盒和半保守的K-box盒;二级结构主要是以α螺旋为主;具有多种磷酸化位点和核定位信号;同源性比较发现与许多植物的花器官决定基因具有较高的相似性,推测它们可能为同源基因,具有相似的生物学功能。     

Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1

The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines.