Original articleSNF1-related protein kinase (SnRK1) phosphorylates class I heat shock protein
Introduction
Small heat shock proteins (HSPs) are found in all eukaryotes and appear to be of particular importance in plants, where there are at least six different gene families, the products of which target to different cell compartments. Two forms (class I and II) are cytosolic whilst others target the chloroplast, mitochondrion and endoplasmic reticulum. Small HSPs can accumulate to high levels in most parts of the plant following heat shock and as such represent the major plant heat shock response [34]. Small HSPs, along with other HSPs, are also induced by exposure of plants to heavy metals such as cadmium [20].
Small HSPs act as molecular chaperones in vitro. For example, two conserved cytosolic plant forms from pea have been shown to enhance refolding of chemically-denatured enzymes and also to prevent thermal aggregation and thermal inactivation [18]. Aggregation of HSPs into large, insoluble cytoplasmic “heat shock granules” during heat treatment has been observed commonly in vivo and has also been seen in chloroplasts [21]. These granules are often associated with mRNA.
In the majority of plant tissues, small HSPs remain undetectable under normal conditions. Nevertheless, expression under developmental control without heat shock has been observed in the embryo, endosperm, pollen and fruit [34]. In the case of seeds, such expression has been shown to be regulated transcriptionally [22]. It is possible that peripheral tissues such as these are more likely to undergo heat stress and are primed to respond to it. Alternatively, it has been suggested that HSPs in some of these organs are contributing to desiccation tolerance [36]. There is evidence for induction of small HSPs by water-stress in sunflower, although the response is confined to stems and roots [2], and over-expression of a class II small HSP in arabidopsis increased osmotolerance [30]. In the resurrection plant (Craterostigma plantagineum), desiccation tolerance is maintained in all vegetative tissues and small HSPs are expressed highly in the roots and some desiccation-induced expression is observed in leaves [1].
Sucrose nonfermenting-1 (SNF1) is a yeast protein kinase [6] that is activated in response to low cellular glucose levels. It is required for the derepression of many genes [10], the arrest of the cell cycle [32] and the inactivation of acetyl-CoA carboxylase, glycogen synthase and other metabolic enzymes [16], [37].
The mammalian homologue of SNF1 is AMP-activated protein kinase (AMPK) which, as its name suggests, is activated by the metabolite, 5′-adenosine monophosphate (AMP), high levels of which are symptomatic of low cellular energy levels [14]. Like SNF1, AMPK phosphorylates and inactivates enzymes of anabolic pathways, such as acetyl-CoA carboxylase (fatty acid synthesis) and HMG-CoA reductase (sterol/isoprenoid synthesis). AMPK is also implicated in the regulation of insulin action and secretion and, thereby, type 2 diabetes mellitus and obesity in humans, while mutations in AMPK are associated with a severe heart defect (hypertrophy and arrhythmia).
The plant homologue of SNF1 and AMPK is SnRK1 (SNF1-related protein kinase-1) [12], [13]. SnRK1 is regulated at the transcriptional level and through phosphorylation by an upstream protein kinase. It is not activated directly by AMP but AMP does affect its phosphorylation state [28]. There is also evidence that SnRK1 is inhibited by glucose-6-phosphate [33] and it may be activated in response to high sucrose as well as low glucose levels [12], [13].
SnRK1 phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoA reductase), sucrose phosphate synthase (SPS) and nitrate reductase (NR) in vitro (inactivation of NR also requires the binding of a 14-3-3 protein to the phosphorylation site) [12], [13]. However, like its yeast counterpart, it is also involved in regulating the expression of genes involved in carbohydrate metabolism. For example, expression of an antisense potato SnRK1 sequence in transgenic potato has been shown to cause a reduction in sucrose synthase gene expression in tubers and loss of sucrose-inducibility of sucrose synthase gene expression in leaves [24]. Similarly, co-bombardment with an antisense SnRK1 gene was found to repress transient activity of an α-amylase (α-Amy2) gene promoter in cultured wheat embryos [17].
Genetic manipulation of SnRK1 activity in plants has profound effects on development. For example, antisense SnRK1 potato tubers do not sprout at all if kept at 5 °C [13], possibly because mobilisation of stored starch to support sprouting is impaired. Furthermore, expression of an antisense SnRK1 sequence causes abnormal pollen development and male sterility in barley [39].
In order to elucidate further the signalling pathways in which SnRK1 is operating, particularly in cereal seeds, we have undertaken two-hybrid screening of a barley endosperm cDNA library using barley SnRK1 as “bait”. We reported previously the cloning of BSnIP1, a cDNA encoding a protein related to the yeast regulatory subunit, SNF4, by this method [27]. Here we describe the cloning of a class I HSP, its expression analysis, and the demonstration that it will act as a substrate for SnRK1.
Section snippets
Cloning of class I small HSP from a barley seed cDNA library
We have described previously [27] the isolation of five cDNAs, BSnIP1-5, through the two-hybrid screening of a mid-stage barley endosperm cDNA library using barley SnRK1 as “bait”. BSnIP1 was found to encode a member of a class of proteins that are unique to plants but have some similarity with the yeast regulatory protein, SNF4 [27]. In the present study, we undertook the analysis of the second cDNA isolated in this screen, BSnIP2. It was isolated three times, independently, from one library
Discussion
The two-hybrid system has been exploited previously to identify a number of protein factors that interact with yeast SNF1 [38]. We chose to follow this approach to identify components of signalling pathways associated with barley endosperm SnRK1. We have described in a previous report the isolation by this method of cBSnIP1, a cDNA encoding a member of a family of proteins that are unique to plants but have some similarity with the yeast regulatory protein, SNF4 [27]. In the present paper, we
Cloning of class I, 17 kDa small HSP
The production of a barley endosperm (16–22 days post-anthesis) cDNA library in plasmid vector pGAD10 (Clontech, USA), the two-hybrid screening of this library using barley SnRK1 as bait and the isolation of five cDNAs, BSnIP1-5, which encode proteins that interact with SnRK1, has been described previously [27]. Nucleotide sequencing of BSnIP2 was performed using Sequenase (Amersham, UK), with Clontech and custom primers (Sigma-Genosys, UK). Database searches were carried out with the BLAST
Acknowledgements
Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. PGD thanks the BBSRC for a CASE studentship.
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Present address: School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom.