A. Gururaj Rao

Identification of in Vitro Autophosphorylation Sites and Effects of Phosphorylation on the Arabidopsis CRINKLY4 (ACR4) Receptor-like Kinase Intracellular Domain: Insights into Conformation, Oligomerization, and Activity

Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase (RLK) that consists of an extracellular domain and an intracellular domain (ICD) with serine/threonine kinase activity. While genetic and cell biology experiments have demonstrated that ACR4 is important in cell fate specification and overall development of the plant, little is known about the biochemical properties of the kinase domain and the mechanisms that underlie the overall function of the receptor. To complement in planta studies of the function of ACR4, we have expressed the ICD in Escherichia coli as a soluble C-terminal fusion to the N-utilization substance A (NusA) protein, purified the recombinant protein, and characterized the enzymatic and conformational properties. The protein autophosphorylates via an intramolecular mechanism, prefers Mn(2+) over Mg(2+) as the divalent cation, and displays typical Michaelis-Menten kinetics with respect to ATP with an apparent K(m) of 6.67 ± 2.07 μM and a V(max) of 1.83 ± 0.18 nmol min(-1) mg(-1). Autophosphorylation is accompanied by a conformational change as demonstrated by circular dichroism, fluorescence spectroscopy, and limited proteolysis with trypsin. Analysis by nanoliquid chromatography and mass spectrometry revealed 16 confirmed sites of phosphorylation at Ser and Thr residues. Sedimentation velocity and gel filtration experiments indicate that the ICD has a propensity to oligomerize and that this property is lost upon autophosphorylation.

Polypyrimidine tract-binding proteins of potato mediate tuberization through an interaction with StBEL5 RNA

Highlight PTB proteins of potato bind to the mobile RNA, StBEL5, and enhance stability and trafficking of the RNA to select organs. This protein–RNA interaction leads to enhanced tuber production.

PP2A-3 interacts with ACR4 and regulates formative cell division in the Arabidopsis root

Significance Plant growth and development are mediated through a wide range of proteins, including receptor kinases and phosphatases. The receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4) is part of a mechanism controlling formative cell divisions in the Arabidopsis root. However, the regulation of ACR4 signaling and how it affects cell divisions remains completely unknown. We discovered that ACR4 phosphorylates the PROTEIN PHOSPHATASE 2A-3 (PP2A-3) catalytic subunit of the PP2A phosphatase holoenzyme and that PP2A dephosphorylates ACR4. These data exposed a tightly regulated point in the associated biochemical network regulating formative cell divisions in plant roots. In plants, the generation of new cell types and tissues depends on coordinated and oriented formative cell divisions. The plasma membrane-localized receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4) is part of a mechanism controlling formative cell divisions in the Arabidopsis root. Despite its important role in plant development, very little is known about the molecular mechanism with which ACR4 is affiliated and its network of interactions. Here, we used various complementary proteomic approaches to identify ACR4-interacting protein candidates that are likely regulators of formative cell divisions and that could pave the way to unraveling the molecular basis behind ACR4-mediated signaling. We identified PROTEIN PHOSPHATASE 2A-3 (PP2A-3), a catalytic subunit of PP2A holoenzymes, as a previously unidentified regulator of formative cell divisions and as one of the first described substrates of ACR4. Our in vitro data argue for the existence of a tight posttranslational regulation in the associated biochemical network through reciprocal regulation between ACR4 and PP2A-3 at the phosphorylation level.

Evidence for Intermolecular Interactions between the Intracellular Domains of the Arabidopsis Receptor-Like Kinase ACR4, Its Homologs and the Wox5 Transcription Factor

Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase (RLK) involved in the global development of the plant. The Arabidopsis genome encodes four homologs of ACR4 that contain sequence similarity and analogous architectural elements to ACR4, termed Arabidopsis CRINKLY4 Related (AtCRRs) proteins. Additionally, a signaling module has been previously proposed including a postulated peptide ligand, CLE40, the ACR4 RLK, and the WOX5 transcription factor that engage in a possible feedback mechanism controlling stem cell differentiation. However, little biochemical evidence is available to ascertain the molecular aspects of receptor heterodimerization and the role of phosphorylation in these interactions. Therefore, we have undertaken an investigation of the in vitro interactions between the intracellular domains (ICD) of ACR4, the CRRs and WOX5. We demonstrate that interaction can occur between ACR4 and all four CRRs in the unphosphorylated state. However, phosphorylation dependency is observed for the interaction between ACR4 and CRR3. Furthermore, sequence analysis of the ACR4 gene family has revealed a conserved ‘KDSAF’ motif that may be involved in protein-protein interactions among the receptor family. We demonstrate that peptides harboring this conserved motif in CRR3 and CRK1are able to bind to the ACR4 kinase domain. Our investigations also indicate that the ACR4 ICD can interact with and phosphorylate the transcription factor WOX5.

Biochemical and biophysical characterization of maize-derived HBsAg for the development of an oral vaccine.

Although a vaccine against hepatitis B virus (HBV) has been available since 1982, it is estimated that 600,000 people die every year due to HBV. An affordable oral vaccine could help alleviate the disease burden and to this end the hepatitis B surface antigen (HBsAg) was expressed in maize. Orally delivered maize material induced the strongest immune response in mice when lipid was extracted by CO2 supercritical fluid extraction (SFE), compared to full fat and hexane-extracted material. The present study provides a biochemical and biophysical basis for these immunological differences by comparing the active ingredient in the differently treated maize material. Purified maize-derived HBsAg underwent biophysical characterization by gel filtration, transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-CD, and fluorescence. Gel filtration showed that HBsAg forms higher-order oligomers and TEM demonstrated virus-like particle (VLP) formation. The VLPs obtained from SFE were more regular in shape and size compared to hexane or full fat material. In addition, SFE-derived HBsAg showed the greatest extent of α-helical structure by far UV-CD spectrum. Fluorescence experiments also revealed differences in protein conformation. This work establishes SFE-treated maize material as a viable oral vaccine candidate and advances the development of the first oral subunit vaccine.

Insights into molecular interactions between the juxtamembrane and kinase subdomains of the Arabidopsis Crinkly-4 receptor-like kinase

Arabidopsis CRINKLY4 (ACR4), a receptor-like kinase required for plant growth and development, possesses an extracellular ligand binding domain, a transmembrane helix, and an intracellular domain (ICD). The ICD contains the juxtamembrane (JMD) and the C-terminal (CTD) subdomains, which flank the core kinase domain (KD), with at least 16 autophosphorylation sites. Phosphorylation sites are often docking sites for the modification-dependent recruitment of interacting proteins that orchestrate many downstream signaling events. In this context, we have specifically probed the role of the two phosphorylation sites Ser(475) and Thr(478) in the JMD using mutagenesis and phage-peptide screening techniques. Thus, naïve and phosphorylated 15-mer peptides derived from the JMD were panned against a 21-amino acid random phage peptide library. The phosphorylated peptide preferentially recognized the consensus sequence LxSLL. This sequence harbors the LxxLL motif, a known protein-protein interaction motif that is also present in the N-terminal lobe of the KD. We demonstrate the binding of JMD peptides to the KD and also show through kinetic analyses of mutants that phosphorylation of Ser(475) and Thr(478) in the JMD is necessary for optimal substrate phosphorylation in vitro. Our experiments suggest that an intramolecular interaction can occur between the JM and the N-terminal lobe of the KD.

The growing story of (ARABIDOPSIS) CRINKLY 4

Receptor kinases play important roles in plant growth and development, but only few of them have been functionally characterized in depth. Over the past decade CRINKLY 4 (CR4)-related research has peaked as a result of a newly discovered role of ARABIDOPSIS CR4 (ACR4) in the root. Here, we comprehensively review the available (A)CR4 literature and describe its role in embryo, seed, shoot, and root development, but we also flag an unexpected role in plant defence. In addition, we discuss ACR4 domains and protein structure, describe known ACR4-interacting proteins and substrates, and elaborate on the transcriptional regulation of ACR4 Finally, we address the missing knowledge in our understanding of ACR4 signalling.

Mapping and Characterization of the Interaction Interface between Two Polypyrimidine-Tract Binding Proteins and a Nova-Type Protein of Solanum tuberosum

Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that generally contain four RNA recognition motifs (RRMs). In potato, six cDNAs encoding full-length PTB proteins have been identified. In the present study Nova1-like protein, designated StNova1, was identified as a potential interacting partner of the StPTB proteins via yeast two-hybrid screening. Nova protein is a RNA-binding protein that contains three K-homology (KH) domains. In humans, these proteins are involved in regulation of neuronal RNA metabolism but the role of Nova-like proteins in plants is poorly understood. We have validated this interaction and mapped the protein binding region on StNova1 and StPTB1 and −6 using a novel domain interaction phage display (DIPP) technique. The interaction between the two RNA-binding proteins StPTB1/6 and StNova1 is mediated through linker regions that are distinctly separated from the RRMs. Furthermore, using a random 21-mer phage-peptide library, we have identified a number of peptides with the consensus sequence motif [S/G][V/I][L/V]G that recognize the StPTB proteins. One over-represented peptide that recognizes StPTB6 contains the GVLGPWP sequence that is similar to the GIGGRYP sequence in the glycine-rich linker region between the KH2 and KH3 domains of StNova1. We show, through site-specific mutations, the importance of glycine and proline residues in StNova1-StPTB interactions.

Investigation of Autophosphorylation Sites of Plant Receptor Kinases and Phosphorylation of Interacting Partners

The optimal kinase activity of plant receptor-like kinases (RLKs) is often regulated by autophosphorylation of specific sites. Many of these phosphorylated residues then serve as recruiting sites for downstream interacting proteins. Therefore, identification of the phosphosites can be an important first step in delineating the signaling network. This chapter describes a protocol for identification of phosphorylated residues by mass spectrometry as well as a protocol to determine if an interacting partner can be phosphorylated in vitro.