An autophosphorylation site database for leucine‐rich repeat receptor‐like kinases in Arabidopsis thaliana

Leucine‐rich repeat receptor‐like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single‐pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high‐throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His‐tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho‐Ser autophosphorylation.     

Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor‐like kinases

The Arabidopsis thaliana somatic embryogenesis receptor‐like kinase (SERK) family consists of five leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)‐mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC‐MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C‐terminally located residue Ser‐562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr‐462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP‐tagged SERK1 from plant extracts followed by MS/MS identified Ser‐303, Thr‐337, Thr‐459, Thr‐462, Thr‐463, Thr‐468, and Ser‐612 or Thr‐613 or Tyr‐614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser‐299 and Thr‐462. This suggests both intra‐ and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser‐887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.     

BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis

Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor‐like cytoplasmic kinase sub‐family (RLCK‐XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T‐DNA insertion lines for eight BSK genes (BSK1–BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24‐epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR‐responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3‐like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3‐like kinases.     

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.     

Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis

During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 (⁶²⁵RRSRRL⁶³⁰) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.     

OST1‐mediated BTF3L phosphorylation positively regulates CBFs during plant cold responses

Cold stress is a major environmental factor that negatively affects plant growth and survival. OST1 has been identified as a key protein kinase in plant response to cold stress; however, little is known about the underlying molecular mechanism. In this study, we identified BTF3 and BTF3L (BTF3‐like), β‐subunits of a nascent polypeptide‐associated complex (NAC), as OST1 substrates that positively regulate freezing tolerance. OST1 phosphorylates BTF3 and BTF3L in vitro and in vivo, and facilitates their interaction with C‐repeat‐binding factors (CBFs) to promote CBF stability under cold stress. The phosphorylation of BTF3L at the Ser50 residue by OST1 is required for its function in regulating freezing tolerance. In addition, BTF3 and BTF3L proteins positively regulate the expression of CBF genes. These findings unravel a molecular mechanism by which OST1‐BTF3‐CBF module regulates plant response to cold stress.     

Auxiliary Photosynthetic Functions of Arabidopsis thaliana—Studies in vitro and in vivo

An improved cultivation system for Arabidopsis thaliana was developed, allowing advanced biochemical studies in vitro and in vivo of this important model plant. Highly functional Arabidopsis thylakoids were isolated and used to study both basic and regulatory photosynthetic functions with the aim to create a platform for the characterization of mutants deficient in auxiliary proteins. Light-induced proteolytic degradation of the D1 protein could be followed and shown to be a subsequent event to photoinactivation of electron transport. The phosphorylation and dephosphorylation of thylakoid proteins resembled that seen in spinach leaves although phospho-CP43 revealed an unusual regulatory behavior.     

Arabidopsis Raf‐like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling

Given their sessile nature, land plants must use various mechanisms to manage dehydration under water‐deficit conditions. Osmostress‐induced activation of the SNF1‐related protein kinase 2 (SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors [PYR (pyrabactin resistance)/PYL (pyrabactin resistance‐like)/RCAR (regulatory component of ABA receptors) proteins] and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) of the B3 clade of the mitogen‐activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2‐mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress‐induced subclass III SnRK2 activity. Our findings identify a specific type of B3‐MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.