小麦翻译控制肿瘤蛋白TCTP与蔗糖非酵解型蛋白激酶SnRK1的相互作用

翻译控制肿瘤蛋白(Translationally controlled tumor protein, TCTP)广泛存在于真核细胞中,参与调节细胞分裂、植物生长发育,并介导植物抵御病原物侵染。蔗糖非酵解型蛋白激酶(SNF1-related protein kinase, SnRK1)在酵母、动物和植物中非常保守,并参与包括糖代谢和抵抗非生物和生物胁迫在内的一系列生理过程。本实验室前期工作证明TaTCTP响应叶锈菌侵染并参与诱发寄主产生防卫反应。为了深入探讨TaTCTP在叶锈菌侵染小麦诱发的防卫反应中发挥的作用,采用串联亲和纯化(TAP)与质谱(MS)联用技术,鉴定出SnRK1可能为TaTCTP潜在互作蛋白。文中对TCTP和SnRK1的相互作用进行了研究。酵母双杂交结果表明,同时携带TCTP和SnRK1的酵母可以在SD/-Leu/-Trp/-His/-Ade(SD/-LWHA,四缺)培养基上生长,说明TCTP与SnRK1在酵母双杂交系统中可以发生相互作用;通过双分子荧光互补实验,发现TCTP与SnRK1发生相互作用的荧光信号分布在细胞质中;进一步用Co-IP实验证明TCTP和SnRK1可以发生相互作用。本研究为深入研究TaTCTP在小麦与叶锈菌互作过程中的作用机制奠定了重要基础,对进一步完善小麦抵御叶锈菌侵染的分子机理具有重要意义。     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Metabolic signalling and carbon partitioning: role of Snf1-related (SnRK1) protein kinase

A protein kinase that plays a key role in the global control of plant carbon metabolism is SnRK1 (sucrose non-fermenting-1-related protein kinase 1), so-called because of its homology and functional similarity with sucrose non-fermenting 1 (SNF1) of yeast. This article reviews studies on the characterization of SnRK1 gene families, SnRK1 regulation and function, interacting proteins, and the effects of manipulating SnRK1 activity on carbon metabolism and development.     

Differential expression of two barley SNF1-related protein kinase genes

We have amplified and cloned DNA sequences derived from a gene encoding a SNF1 (sucrose-non-fermenting 1)-related protein kinase which differs from that previously reported from barley. Northern blot and polymerase chain reaction (PCR) analysis of RNA populations, using specific probes and oligonucleotide primers, indicated that the two SNF1-related genes are differentially regulated. One is expressed in all tissues, whereas the other is expressed at high levels in the seed endosperm and aleurone, but at levels undetectable by northern blot analysis in other tissues. Comparisons with other plant SNF1-related protein kinase genes suggest that the form which is expressed at greatly enhanced levels in the seed is less similar to the other plant homologues which have been reported and may be unique to cereals.     

Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate

One of the major protein kinases (PK(III)) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1-related family of protein kinases. In the present study, we have developed a fluorescence-based continuous assay for measurement of PK(III) activity. Using the continuous assay, along with the fixed-time-point (32)P-incorporation assay, we demonstrate that PK(III) activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8. 5 to 5.5 and by reducing the concentration of Mg(2+) in the assay from 10 to 2 mM. Under likely physiological conditions (pH 7.0 and 2 mM Mg(2+)), 10 mM Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PK(III) in the following order: Glc-6-P > mannose-6-P, fructose-1,6P(2) > ribose-5-P, 3-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PK(III) activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.     

A cell cycle role for a plant sucrose nonfermenting-1-related protein kinase (SnRK1) is indicated by expression in yeast

Plant sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRK1s) have been shown to restore carbon catabolite derepression of gene expression in the yeast Saccharomyces cerevisiae when expressed in snf1 mutants. SNF1 has been implicated in the mediation of cell cycle control in response to nutrient levels and, in the present study, we show that expression of the rye (Secale cereale) SnRK1, RKIN1, in a yeast snf1 mutant has a dramatic effect on the size of cells growing on a minimal medium where SNF1 function is essential. The mean volume of the yeast cells which were expressing RKIN1 was two-thirds that of the whi1 mutant, the smallest viable cells known in S. cerevisiae, and the cells died after 3 days unless rescued onto complex medium. This is the first experimental evidence of a role for SnRK1s in plant cell cycle control.     

Cloning and characterization of Hunk, a novel mammalian SNF1-related protein kinase

We previously identified a novel protein kinase, Hunk, by means of a degenerate PCR screen designed to isolate kinases expressed in the murine mammary gland. We now describe the molecular cloning, chromosomal localization, and activity of this kinase and characterize its spatial and temporal pattern of expression in the mouse. We have isolated a 5.0-kb full-length cDNA clone that contains the 714-amino-acid open reading frame encoding Hunk. Analysis of this cDNA reveals that Hunk is most closely related to the SNF1 family of serine/threonine kinases and contains a newly described SNF1 homology domain. Accordingly, antisera specific for Hunk detect an 80-kDa polypeptide with associated phosphotransferase activity. Hunk is located on distal mouse chromosome 16 in a region of conserved synteny with human chromosome 21q22. During fetal development and in the adult mouse, Hunk mRNA expression is developmentally regulated and tissue-specific. Moreover, in situ hybridization analysis reveals that Hunk expression is restricted to subsets of cells within a variety of organs in the adult mouse. These findings suggest a role for Hunk in murine development.     

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.     

A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain

A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.     

Rapid analysis of an osmotic stress responsive promoter by transient expression in intact wheat embryos

Expression of the wheat ‘Em’ genes in embryos isstrongly induced both by abscisic acid and by subjection toosmotic stress. We have examined the basis of this inductionin an homologous system by con structing fusions between thepromoter sequences of a cloned ‘Em’ gene and theGUS reporter gene. The ABA-and stress-mediated expression ofthese constructs has been assayed following delivery to intactwheat embryos by particle bombardment. Staining of bombardedembryos with the chromogenic substrate X-gluc enabled a simpleand rapid visual identification of promoter activity by scoringthe numbers of stained spots. Although not rigorously quantitative,a general correspondence between number of expression events(spots) and promoter activity could be inferred when the resultsobtained with bombarded embryos were compared with those obtainedby the fluorlmetnc measurement of GUS activity in cereal aleuroneprotoplasts transfected with the same constructs. Analysis ofa series of 5'-deletions of the ‘Em’ promoters indicatedthat the stress-responsive cis-acting regulatory elements comprisethe same sequences as those responsible for ABA-mediated geneexpression. Key words: Abscisic acid, osmotic stress, Em genes, wheat embryos, transient expression     

A Complex Containing SNF1-Related Kinase (SnRK1) and Adenosine Kinase in Arabidopsis

SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5′-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5′-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK in vitro by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for in vivo linkage but also revealing that in vivo regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.     

Regulation of spinach SNF1-related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 5'-AMP

Members of the SNF1-related protein kinase-1 (SnRK1) subfamily of protein kinases are higher plant homologues of mammalian AMP-activated and yeast SNF1 protein kinases. Based on analogies with the mammalian system, we surmised that the SnRK1 kinases would be regulated by phosphorylation on a threonine [equivalent to Thr175 in Arabidopsis thaliana SnRK1 (AKIN10)] within the 'T loop' between the conserved DFG and APE motifs. We have raised an antibody against a phosphopeptide based on this sequence, and used it to show that inactivation of two spinach SnRK1 kinases by protein phosphatases, and reactivation by a mammalian upstream protein kinase, is associated with changes in the phosphorylation state of this threonine. We also show that dephosphorylation of this threonine by protein phosphatases, and consequent inactivation, is inhibited by low concentrations of 5'-AMP, via binding to the substrate (i.e. the kinase). This is the first report showing that the plant SnRK1 kinases are regulated by AMP in a manner similar to their mammalian counterparts. The possible physiological significance of these findings is discussed.     

Serum protein depletion by cultured rat embryos (1)

Cultures of 10-day rat embryos depleted a protein band with a molecular weight of approximately 125,000 from the rat serum medium. Delayed centrifuged serum differed from immediately centrifuged serum by a reduction in the staining intensity of the 125,000 molecular weight protein band and by the absence of two high molecular weight (greater than 200,000) protein bands.