Filip Rolland | KU Leuven (original) (raw)
Papers by Filip Rolland
Journal of Experimental Botany, 2011
Over the past decades, considerable advances have been made in understanding the crucial role and... more Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/ N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.
Journal of Experimental Botany, 2012
Over the past decades, considerable advances have been made in understanding the crucial role and... more Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/ N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.
Fems Yeast Research - FEMS YEAST RES, 2001
Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detect... more Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may ...
Yeast, 1998
In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (= GGS1 = FDP1 = BYP1 = CIF... more In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (= GGS1 = FDP1 = BYP1 = CIF1 = GLC6 = TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1 delta mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total D-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, +/- 10 mM in a hxk1 delta hxk2 delta glk1 delta and 2-3 mM in a tps1 delta strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity.
Trends in Biochemical Sciences, 2001
Glucose is a ubiquitous nutrient for eukaryotic cells, serving as a source of carbon and energy. ... more Glucose is a ubiquitous nutrient for eukaryotic cells, serving as a source of carbon and energy. In the presence of glucose, a variety of metabolic pathways are affected, resulting in innumerable changes in metabolic intermediates, cofactors and end-products. In addition, different eukaryotic cells use specific mechanisms to sense the presence of glucose ). The physiological role of these mechanisms depends on the particular cell type.
Plant Molecular Biology, 2006
The disaccharide trehalose has dramatic effects on plant metabolism, growth and development. Arab... more The disaccharide trehalose has dramatic effects on plant metabolism, growth and development. Arabidopsis seedlings grown on trehalose-containing medium without sucrose display increased expression of the starch synthesis gene ApL3, hyper-accumulation of starch in the cotyledons and inhibition of root growth. Here we show that the ABI4 transcription factor mediates the effects of trehalose on starch metabolism and growth, independently of abscisic acid (ABA) synthesis and hexokinase (HXK1) signaling. Surprisingly, although the abi4 mutation partially rescued trehalose inhibition of root elongation, ApL3 expression levels were still enhanced. Gene expression analysis suggests that trehalose affects both starch synthesis and starch breakdown. The expression of genes involved in starch breakdown, such as SEX1 and the b-amylase gene BMY8/BAM3, was strongly down-regulated in WT plants grown on trehalose but not in abi4 mutants. Addition of trehalose to liquid-grown WT seedlings also Electronic Supplementary Material Supplementary material is available in the online version of this article at http:// dx.
Molecular Microbiology, 2000
FEMS Yeast Research, 2001
Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detect... more Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low. ß
FEMS Yeast Research, 2002
Glucose has dramatic effects on the regulation of carbon metabolism and on many other properties ... more Glucose has dramatic effects on the regulation of carbon metabolism and on many other properties of yeast cells. Several sensing and signalling pathways are involved. For many years attention has focussed on the main glucose-repression pathway which is responsible for the downregulation of respiration, gluconeogenesis and the transport and catabolic capacity of alternative sugars during growth on glucose. The hexokinase 2-dependent glucose-sensing mechanism of this pathway is not well understood but the downstream part of the pathway has been elucidated in great detail. Two putative glucose sensors, the Snf3 and Rgt2 non-transporting glucose carrier homologs, control the expression of many functional glucose carriers. Recently, several new components of this glucose-induction pathway have been identified. The Ras-cAMP pathway controls a wide variety of cellular properties in correlation with cellular proliferation. Glucose is a potent activator of cAMP synthesis. In this case glucose sensing is carried out by two systems, a G-protein-coupled receptor system and a still elusive glucose-phosphorylation-dependent system. The understanding of glucose sensing and signalling in yeast has made dramatic advances in recent years and has become a strong paradigm for the elucidation of nutrient-sensing mechanisms in other eukaryotic organisms. ß
Enzyme and Microbial Technology, 2000
Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strong... more Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strongly in a variety of physiological properties compared to cells growing in the absence of glucose. Part of these differences appear to be caused by the protein kinase A (PKA) and related signal transduction pathways. Addition of glucose to cells previously deprived of glucose triggers cAMP accumulation, which is apparently mediated by the Gpr1-Gpa2 G-protein coupled receptor system. However, the resulting effect on PKA-controlled properties is only transient when there is no complete growth medium present. When an essential nutrient is lacking, the cells arrest in the stationary phase G0. At the same time they acquire all characteristics of cells with low PKA activity, even if there is ample glucose present. When the essential nutrient is added again, a similar PKA-dependent protein phosphorylation cascade is triggered as observed after addition of glucose to glucosedeprived cells, but which is not cAMP-mediated. Because the pathway involved requires a fermentable carbon source and a complete growth medium, at least for its sustained activation, it has been called "fermentable growth medium (FGM)-induced pathway."
Annual Review of Plant Biology, 2006
Article, publication date, and citation information can be found at www.plantcell.org/cgi/
FEBS Journal, 2011
All life forms on earth require a continuous input and monitoring of carbon and energy supplies. ... more All life forms on earth require a continuous input and monitoring of carbon and energy supplies. The AMP-activated kinase (AMPK) ⁄ sucrose non-fermenting1 (SNF1) ⁄ Snf1-related kinase1 (SnRK1) protein kinases are evolutionarily conserved metabolic sensors found in all eukaryotic organisms from simple unicellular fungi (yeast SNF1) to animals (AMPK) and plants (SnRK1). Activated by starvation and energy-depleting stress conditions, they enable energy homeostasis and survival by up-regulating energyconserving and energy-producing catabolic processes, and by limiting energy-consuming anabolic metabolism. In addition, they control normal growth and development as well as metabolic homeostasis at the organismal level. As such, the AMPK ⁄ SNF1 ⁄ SnRK1 kinases act in concert with other central signaling components to control carbohydrate uptake and metabolism, fatty acid and lipid biosynthesis and the storage of carbon energy reserves. Moreover, they have a tremendous impact on developmental processes that are triggered by environmental changes such as nutrient depletion or stress. Although intensive research by many groups has partly unveiled the factors that regulate AMPK ⁄ SNF1 ⁄ SnRK1 kinase activity as well as the pathways and substrates they control, several fundamental issues still await to be clarified. In this review, we will highlight these issues and focus on the structure, function and regulation of the AMPK ⁄ SNF1 ⁄ SnRK1 kinases.
Journal of Experimental Botany, 2011
Over the past decades, considerable advances have been made in understanding the crucial role and... more Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/ N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.
Journal of Experimental Botany, 2012
Over the past decades, considerable advances have been made in understanding the crucial role and... more Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/ N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.
Fems Yeast Research - FEMS YEAST RES, 2001
Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detect... more Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may ...
Yeast, 1998
In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (= GGS1 = FDP1 = BYP1 = CIF... more In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (= GGS1 = FDP1 = BYP1 = CIF1 = GLC6 = TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1 delta mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total D-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, +/- 10 mM in a hxk1 delta hxk2 delta glk1 delta and 2-3 mM in a tps1 delta strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity.
Trends in Biochemical Sciences, 2001
Glucose is a ubiquitous nutrient for eukaryotic cells, serving as a source of carbon and energy. ... more Glucose is a ubiquitous nutrient for eukaryotic cells, serving as a source of carbon and energy. In the presence of glucose, a variety of metabolic pathways are affected, resulting in innumerable changes in metabolic intermediates, cofactors and end-products. In addition, different eukaryotic cells use specific mechanisms to sense the presence of glucose ). The physiological role of these mechanisms depends on the particular cell type.
Plant Molecular Biology, 2006
The disaccharide trehalose has dramatic effects on plant metabolism, growth and development. Arab... more The disaccharide trehalose has dramatic effects on plant metabolism, growth and development. Arabidopsis seedlings grown on trehalose-containing medium without sucrose display increased expression of the starch synthesis gene ApL3, hyper-accumulation of starch in the cotyledons and inhibition of root growth. Here we show that the ABI4 transcription factor mediates the effects of trehalose on starch metabolism and growth, independently of abscisic acid (ABA) synthesis and hexokinase (HXK1) signaling. Surprisingly, although the abi4 mutation partially rescued trehalose inhibition of root elongation, ApL3 expression levels were still enhanced. Gene expression analysis suggests that trehalose affects both starch synthesis and starch breakdown. The expression of genes involved in starch breakdown, such as SEX1 and the b-amylase gene BMY8/BAM3, was strongly down-regulated in WT plants grown on trehalose but not in abi4 mutants. Addition of trehalose to liquid-grown WT seedlings also Electronic Supplementary Material Supplementary material is available in the online version of this article at http:// dx.
Molecular Microbiology, 2000
FEMS Yeast Research, 2001
Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detect... more Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low. ß
FEMS Yeast Research, 2002
Glucose has dramatic effects on the regulation of carbon metabolism and on many other properties ... more Glucose has dramatic effects on the regulation of carbon metabolism and on many other properties of yeast cells. Several sensing and signalling pathways are involved. For many years attention has focussed on the main glucose-repression pathway which is responsible for the downregulation of respiration, gluconeogenesis and the transport and catabolic capacity of alternative sugars during growth on glucose. The hexokinase 2-dependent glucose-sensing mechanism of this pathway is not well understood but the downstream part of the pathway has been elucidated in great detail. Two putative glucose sensors, the Snf3 and Rgt2 non-transporting glucose carrier homologs, control the expression of many functional glucose carriers. Recently, several new components of this glucose-induction pathway have been identified. The Ras-cAMP pathway controls a wide variety of cellular properties in correlation with cellular proliferation. Glucose is a potent activator of cAMP synthesis. In this case glucose sensing is carried out by two systems, a G-protein-coupled receptor system and a still elusive glucose-phosphorylation-dependent system. The understanding of glucose sensing and signalling in yeast has made dramatic advances in recent years and has become a strong paradigm for the elucidation of nutrient-sensing mechanisms in other eukaryotic organisms. ß
Enzyme and Microbial Technology, 2000
Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strong... more Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strongly in a variety of physiological properties compared to cells growing in the absence of glucose. Part of these differences appear to be caused by the protein kinase A (PKA) and related signal transduction pathways. Addition of glucose to cells previously deprived of glucose triggers cAMP accumulation, which is apparently mediated by the Gpr1-Gpa2 G-protein coupled receptor system. However, the resulting effect on PKA-controlled properties is only transient when there is no complete growth medium present. When an essential nutrient is lacking, the cells arrest in the stationary phase G0. At the same time they acquire all characteristics of cells with low PKA activity, even if there is ample glucose present. When the essential nutrient is added again, a similar PKA-dependent protein phosphorylation cascade is triggered as observed after addition of glucose to glucosedeprived cells, but which is not cAMP-mediated. Because the pathway involved requires a fermentable carbon source and a complete growth medium, at least for its sustained activation, it has been called "fermentable growth medium (FGM)-induced pathway."
Annual Review of Plant Biology, 2006
Article, publication date, and citation information can be found at www.plantcell.org/cgi/
FEBS Journal, 2011
All life forms on earth require a continuous input and monitoring of carbon and energy supplies. ... more All life forms on earth require a continuous input and monitoring of carbon and energy supplies. The AMP-activated kinase (AMPK) ⁄ sucrose non-fermenting1 (SNF1) ⁄ Snf1-related kinase1 (SnRK1) protein kinases are evolutionarily conserved metabolic sensors found in all eukaryotic organisms from simple unicellular fungi (yeast SNF1) to animals (AMPK) and plants (SnRK1). Activated by starvation and energy-depleting stress conditions, they enable energy homeostasis and survival by up-regulating energyconserving and energy-producing catabolic processes, and by limiting energy-consuming anabolic metabolism. In addition, they control normal growth and development as well as metabolic homeostasis at the organismal level. As such, the AMPK ⁄ SNF1 ⁄ SnRK1 kinases act in concert with other central signaling components to control carbohydrate uptake and metabolism, fatty acid and lipid biosynthesis and the storage of carbon energy reserves. Moreover, they have a tremendous impact on developmental processes that are triggered by environmental changes such as nutrient depletion or stress. Although intensive research by many groups has partly unveiled the factors that regulate AMPK ⁄ SNF1 ⁄ SnRK1 kinase activity as well as the pathways and substrates they control, several fundamental issues still await to be clarified. In this review, we will highlight these issues and focus on the structure, function and regulation of the AMPK ⁄ SNF1 ⁄ SnRK1 kinases.