CDPK1, a calcium-dependent protein kinase, regulates transcriptional activator RSG in response to gibberellins

Abstract

The homeostasis of gibberellins (GAs) is maintained by negative-feedback regulation in plant cells. REPRESSION OF SHOOT GROWTH (RSG) is a transcriptional activator with a basic Leu zipper domain suggested to contribute GA feedback regulation by the transcriptional regulation of genes encoding GA biosynthetic enzymes. The 14-3-3 signaling proteins negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The phosphorylation on Ser-114 of RSG is essential for 14-3-3 binding of RSG; however, the kinase that catalyzes the reaction is unknown. Recently a Ca²⁺-dependent protein kinase (CDPK) was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of the Ser-114 of RSG. Our results suggest that CDPK decodes the Ca²⁺ signal produced by GAs and regulates the intracellular localization of RSG in plant cells.

To communicate between cells, multicellular organisms, whether plants or animals, coordinate their growth and development at a tissue and organ level via small signaling molecules named hormones. Compared with animals, plants have acquired marvelous developmental plasticity during their evolution by integrating their endogenous program for morphogenesis with the ever-fluctuating environments throughout their lives because of their sessility. Plant hormones crucially contribute to orchestrate innate processes of gene expression in a spatiotemporally specialized manner and also to transduce exterior environmental stimuli to nuclei. Gibberellins (GAs), which are tetracyclic diterpenoid growth factors, are the essential regulators of many aspects of plant growth and development, including seed germination, stem elongation, flower induction and anther development, and their endogenous level is delicately tuned by feedback control at several steps in the metabolic pathway.¹ Although GA feedback regulation is shown to depend on GA signaling components,² its molecular mechanisms have not yet been explained.

RSG is a transcriptional activator, which is involved in the regulation of endogenous amounts of GAs.³ Expression of the dominant negative form of RSG repressed the transcription of the ent-kaurene oxidase gene of GA biosynthesis in transformed plants, resulting in a severe dwarf phenotype. The function of RSG is negatively regulated by 14-3-3 proteins⁴ that bind to RSG depending on the RSG phosphorylation of Ser-114, and thereby sequester RSG in the cytoplasm so that it is unable to regulate its targets in the nucleus.⁵ One of the signals that dominate the intracellular localization of RSG is the endogenous level of GAs, that is, RSG is translocated into the nucleus in response to a reduction in GA levels, and GA treatment could reverse this nuclear accumulation.⁵ The GA-dependent nuclear export of RSG requires 14-3-3 binding and Ser/Thr kinase activity.⁵ However, there is little information on the molecular mechanisms by which GA regulates the intracellular localization of RSG. Of particular importance is the identification and characterization of the protein kinase that promotes the association of RSG with 14-3-3 proteins through phosphorylation of the Ser-114 of RSG in response to GAs.

Signaling pathways are complex networks of biochemical reactions, which culminate in the alteration of a gene expression pattern mediated by transcriptional mechanisms. Sequence-specific transcription factors collectively function as the key interface between genetic information encoded in DNA sequence and signaling systems in response to internal and external stimuli. Intensive studies have revealed the post-translational regulation of transcription factors, including covalent modifications and interactions with coactivators and general transcription factors.⁶ Phosphorylation is one of the most frequent and significant post-translational modifications of transcription factors. However, information on the protein kinases that directly phosphorylate transcription factors is limited in plants.

One of the fastest known responses to GA is an increase in the concentration of cytosolic Ca²⁺.⁷ In response to diverse internal and external stimuli, cells generate transient increases in the concentration of intracellular free Ca²⁺, as a second messenger, with various spatiotemporal profiles varying in amplitude, frequency, duration, intracellular location, and timing.⁸ Important information regarding the nature of the stimulus may be encoded in the different spatiotemporal profiles of increases in the concentration of Ca²⁺.⁹ Different Ca²⁺ sensors recognize specific Ca²⁺ signatures and bring about changes in metabolism and gene expression.¹⁰ Among Ca²⁺-binding sensory proteins in plants, Ca²⁺-dependent Ser/Thr protein kinases (CDPKs), only found in plants and some protozoans, are considered to be a major molecular decoder of Ca²⁺ signals because the protein kinase C and the conventional calmodulin-dependent protein kinase (CaMK), which represent the two major types of Ca²⁺-regulated kinases in animal systems, are missing from plants such as Arabidopsis.¹¹ There are 34 genes encoding CDPKs in Arabidopsis and 29 genes in rice.^12,13 There is evidence that CDPKs are targeted to multiple cellular locations,¹⁴ suggesting that CDPKs participate in various physiological processes including the accumulation of storage starch and protein in immature seeds of rice,¹⁵ tolerance to cold, salt and drought stress in rice,¹⁶ a defense response in tobacco,¹⁷ development and regulation of nodule number in Medicago truncatula,¹⁸ abscisic acid response in Arabidopsis,¹⁹ and pollen tube growth in petunia.²⁰ To understand how CDPKs affect plant physiology, their specific target proteins must be clarified. However, very little is known about the physiological targets of CDPKs.

Recently, we identified CDPK1 that is an RSG kinase and promotes 14-3-3 binding of RSG by phosphorylation of the Ser-114 of RSG (Fig. 1).²¹ Inhibition of CDPK1 repressed the GA-induced phosphorylation of Ser-114 of RSG and GA-induced nuclear export of RSG. GA induced phosphorylation of CDPK1 and the interaction between CDPK1 and RSG. Furthermore, overexpression of CDPK1 inhibited the transcriptional activation of a GA 20-oxidase gene in a process of feedback control and resulted in the sensitization to a GA biosynthetic inhibitor. Our results suggest that a CDPK decodes the Ca²⁺ signal produced by GAs and regulates the intracellular localization of RSG.

Figure 1.

A schematic model of the signal transduction cascade in GA feedback regulation.

Phylogenetic analyses have suggested that the CDPK family evolutionally arose through the fusion of a CaMK and a calmodulin.²² Mammalian CaMKII phosphorylates histone deacetylases (HDACs), one of corepressors of the transcriptional regulation in eukaryotes, and promotes nuclear exclusion of HDACs through 14-3-3 binding in skeletal muscle differentiation and cardiac growth.^23,24 Forkhead transcription factors FOXOs of animals are exported from the nucleus to the cytoplasm through phosphorylation by Akt kinase and 14-3-3 binding in response to growth factors.²⁵ Accordingly, the nuclear-cytoplasmic partitioning of transcriptional regulators by 14-3-3 proteins depending on Ca²⁺ signaling appears to be an evolutionarily ancient mechanism or to have evolved in parallel in both kingdoms.

Molecular genetics with mutants of Arabidopsis and rice definitely demonstrated that a soluble GA receptor, GID1-mediated degradation of nuclear DELLA proteins that function as repressors of GA-dependent processes is the central regulatory switch in GA signaling in plant cells.^26,27 However, correlation of DELLA proteins to Ca²⁺ signaling is completely unknown. Several physiological studies suggested that there are alternative GA signaling pathways, that is, the existence of a membrane-localized GA receptor,^28,29 involvement of heterotrimeric G protein in GA signaling and a plasma membrane GA receptor.³⁰ To elucidate the molecular relationship between the DELLA pathway and the CDPK1-RSG/14-3-3 pathway would provide a significant clue for the understanding of a new aspect of the GA signaling machinery beginning to emerge as a complicated network composed from various branches and intersections beyond our expectations.

Abbreviations

GA

gibberellin

RSG

repression of shoot growth

CDPK

Ca²⁺-dependent Ser/Thr protein kinase

CaMK

calmodulin-dependent protein kinase

HDAC

histone deacetylase

GID

GA-insensitive dwarf