Phosphorylation-independent binding of 14–3–3 to NtCDPK1 by a new mode

Abstract

14–3–3 pproteins play essential roles in diverse cellular processes through the direct binding to target proteins. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in gibberellin (GA) feedback regulation. The 14–3–3 proteins bind to RSG depending on the RSG phosphorylation of Ser-114 and negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The Ca²⁺-dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14–3–3 binding of RSG by phosphorylation of RSG. 14–3–3 weakly binds to NtCDPK1 by a new mode. The autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14–3–3 but not for its maintenance. In this study, we showed that 14–3–3 binding to NtCDPK1 does not require the autophosphorylation when RSG was bound to NtCDPK1. These data suggest that 14–3–3 binds to an unphosphorylated motif in NtCDPK1 exposed by a conformational change in NtCDPK1 but not to a phosphate group generated by autophosphorylation of NtCDPK1.

Abbreviations

CDPK

Ca²⁺-dependent protein kinase

RSG

repression of shoot growth

GA

gibberellin

14–3–3 signaling proteins form a highly conserved family of homo- and heterodimeric proteins in eukaryotes.^1,2 Previous studies uncovered that binding to a target in the amphipathic groove of a 14–3–3 dimer often requires a phosphorylated consensus motif and can have a range of variation.³ Through these binding reactions, the 14–3–3 proteins appear to act as molecular scaffolds or chaperones. The biological roles of 14–3–3 proteins have been demonstrated in signal transduction, development, and cell cycle control by promotion of protein-protein interaction, subcellular targeting, and protection of phosphorylation sites from phosphatases.⁴ Recent studies including the 14–3–3 interactome have been reported that 14–3–3 proteins can also interact with unphosphorylated targets.^5-10

A tobacco transcription factor RSG, which is involved in the gibberellin homeostasis, is negatively regulated by 14–3–3 signaling proteins.^11-14 The 14–3–3 proteins 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 target genes in the nucleus.¹⁵ NtCDPK1 was identified as an RSG kinase that promotes 14–3–3 binding to RSG by phosphorylation of Ser-114 of RSG.¹⁶ NtCDPK1 interacts with RSG in vivo and in vitro in a Ca²⁺-dependent manner and specifically phosphorylates Ser-114 of RSG. NtCDPK1 decodes the Ca²⁺ signal produced by GAs and regulates the intracellular localization of RSG.

CDPKs are unique Ca²⁺ decoders that are only found in plants and some protozoans.¹⁷ CDPKs have been shown to play important roles in various physiological processes, including plant growth and development and abiotic and biotic stress responses in plants.^18-20 CDPKs are Ser/Thr protein kinases that are composed of a variable N-terminal domain, a catalytic domain, a junction domain containing an autoinhibitory segment, and a calmodulin-like domain. The variable N-terminal domain of CDPKs, which is diversified not only in amino acid sequence but also in length, is involved in the substrate recognition.^21,22 The autoinhibitory segment blocks the substrate binding site of the catalytic domain in the absence of Ca²⁺. Ca²⁺ binding triggers the translocation of all the regions downstream of the catalytic domain (i.e., the junction domain and calmodulin-like domain). This movement enables CDPK to phosphorylate substrates.^23,24

In a recent publication, we showed that 14–3–3 interacts with autophosphorylated NtCDPK1 in the presence of Ca²⁺.²⁵ 14–3–3 and RSG bound to the catalytic domain and the variable N-terminal domain of NtCDPK1, respectively, and NtCDPK1 formed a heterotrimer with RSG and 14–3–3. 14–3–3 was transferred from NtCDPK1 to the immediate product, phosphorylated RSG, suggesting that NtCDPK1 not only phosphorylates the substrate RSG but may also play a role as a scaffold that promotes the binding between phosphorylated RSG and 14–3–3. The binding between NtCDPK1 and 14–3–3 was evidently weaker than that between RSG and 14–3–3. This weak binding of 14–3–3 to NtCDPK1 could allow NtCDPK1 to transfer 14–3–3 to RSG.

14–3–3 binds to NtCDPK1 by a new mode, which results in the weak binding between 14–3–3 and NtCDPK1 and enables 14–3–3 to be transferred from NtCDPK1 to RSG.²⁵ When 14–3–3-bound NtCDPK1 was treated with λ-phosphatase, NtCDPK1 was completely dephosphorylated, although 14–3–3 maintained the binding to NtCDPK1. Namely, autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14–3–3 but not for its maintenance. This result indicated that 14–3–3 does not directly bind to a phosphate group on NtCDPK1 generated by autophosphorylation. The autophosphorylation may induce a conformational change in NtCDPK1, which appears to expose an unphosphorylated motif in NtCDPK1. Therefore, 14–3–3 could recognize the unphosphorylated motif but not the phosphate group. Alternatively, the conformational change necessary for 14–3–3 binding could be induced by Ca²⁺ and ATP.

In this study, we examined whether RSG-binding to NtCDPK1 affects autophosphorylation-dependent binding of 14–3–3 to NtCDPK1. In vitro pull-down assay showed that 14–3–3 binds to NtCDPK1-RSG complex independently of autophosphorylation of NtCDPK1 (Fig. 1). However, we could not distinguish whether 14–3–3 binds to NtCDPK1 or RSG. Autophosphorylation of NtCDPK1 is not necessary for the maintenance of the binding between NtCDPK1 and 14–3–3, while phosphorylation of RSG is required for that between RSG and 14–3–3.²⁵ Therefore, this result suggests that 14–3–3 binds to unphosphorylated NtCDPK1 rather than unphosphorylated RSG. RSG binds to the variable N-terminal domain of NtCDPK1.^21,22 Therefore, the variable N-terminal domain may inhibit the 14–3–3 binding to NtCDPK1 by masking the 14–3–3 binding site in NtCDPK1 and the binding of RSG to the variable N-terminal domain may prevent the inhibition by the variable N-terminal domain. The autophosphorylation reaction of NtCDPK1 also triggers the binding of 14–3–3 to NtCDPK1, suggesting that the autophosphorylation reaction may also suppress the inhibition by the variable N-terminal domain. Although the autophosphorylation of NtCDPK1 in response to Ca²⁺ precedes the RSG binding to NtCDPK1 in vivo, this data supports that 14–3–3 binds to NtCDPK1 by recognizing the unphosphorylated motif in NtCDPK1 exposed by the conformational change but not the phosphate group on NtCDPK1. Our findings suggests that not only the strong binding through a phosphorylated consensus motif but also the unconventional weak binding through an unphosphorylated motif is required for the physiological function of 14–3–3. Although decades of research have focused on the strong binding between proteins, the investigation of the weak interactions in many proteins will also become increasingly important in future.

Figure 1.

14–3–3 does not need the autophosphorylation of NtCDPK1 for the binding to NtCDPK1 when RSG was bound to NtCDPK1. Purified recombinant glutathione S-transferase (GST)–NtCDPK1 was autophosphorylated in a reaction mixture with ATP. Autophosphorylated or unphosphorylated GST–NtCDPK1 was absorbed into glutathione beads. The immobilized GST–NtCDPK1s were incubated with or without maltose binding protein (MBP)–RSG. Glutathione beads into which GST–NtCDPK1 and MBP–RSG had been absorbed were washed with a pull-down buffer and incubated with His-tagged (His)–14–3–3. NtCDPK1-bound proteins were subjected to SDS-PAGE, followed by immunoblot analysis with anti-14–3–3 and anti-MBP for the detection of His–14–3–3 and MBP–RSG, respectively. GST–NtCDPK1 was visualized by Coomassie brilliant blue (CBB) staining.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.