Michael B. Goshe

Sequential Transphosphorylation of the BRI1/BAK1 Receptor Kinase Complex Impacts Early Events in Brassinosteroid Signaling

Brassinosteroids (BRs) regulate plant development through a signal transduction pathway involving the BRI1 and BAK1 transmembrane receptor kinases. The detailed molecular mechanisms of phosphorylation, kinase activation, and oligomerization of the BRI1/BAK1 complex in response to BRs are uncertain. We demonstrate that BR-dependent activation of BRI1 precedes association with BAK1 in planta, and that BRI1 positively regulates BAK1 phosphorylation levels in vivo. BRI1 transphosphorylates BAK1 in vitro on specific kinase-domain residues critical for BAK1 function. BAK1 also transphosphorylates BRI1, thereby quantitatively increasing BRI1 kinase activity toward a specific substrate. We propose a sequential transphosphorylation model in which BRI1 controls signaling specificity by direct BR binding followed by substrate phosphorylation. The coreceptor BAK1 is then activated by BRI1-dependent transphosphorylation and subsequently enhances signaling output through reciprocal BRI1 transphosphorylation. This model suggests both conservation and distinct differences between the molecular mechanisms regulating phosphorylation-dependent kinase activation in plant and animal receptor kinases.

Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase.

Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography–tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.

Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis

Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: brassinosteroid insensitive 1 (BRI1), which is the BR receptor, and its coreceptor BRI1-associated kinase 1 (BAK1). Both proteins are classified as serine/threonine protein kinases, but here we report that recombinant cytoplasmic domains of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases. With BRI1, Tyr-831 and Tyr-956 are identified as autophosphorylation sites in vitro and in vivo. Interestingly, Tyr-956 in kinase subdomain V is essential for activity, because the Y956F mutant is catalytically inactive and thus this site cannot be simply manipulated by mutagenesis. In contrast, Tyr-831 in the juxtamembrane domain is not essential for kinase activity but plays an important role in BR signaling in vivo, because expression of BRI1(Y831F)-Flag in transgenic bri1–5 plants results in plants with larger leaves (but altered leaf shape) and early flowering relative to plants expressing wild-type BRI1-Flag. Acidic substitutions of Tyr-831 restored normal leaf size (but not shape) and normal flowering time. This is an example where a specific tyrosine residue has been shown to play an important role in vivo in plant receptor kinase function. Interestingly, 6 additional LRR-RLKs (of the 23 tested) were also found to autophosphorylate on tyrosine in addition to serine and threonine, suggesting that tyrosine signaling should be considered with other plant receptor kinases as well.

Stable isotope-coded proteomic mass spectrometry

Developing the ability to quantify changes in protein abundance between cells subjected to a variety of physiological and environmental conditions is an extremely active area of proteome research. Although advances in chromatography, mass spectrometry instrumentation, and bioinformatics have contributed to producing a viable method for comparative proteome-wide analyses, the highest precision of quantitation is based, in part, upon improved methods for chemical and metabolic stable isotope labeling of proteins and peptides. The ability to quantify differences in protein expression and post-translational modifications using stable isotope labeling has been achieved, but insights into the biochemical mechanisms that will contribute to the development of new biotechnologies have yet to be realized.

Absolute Protein Quantification by LC/MSE for Global Analysis of Salicylic Acid-Induced Plant Protein Secretion Responses

The plant cell wall is a dynamic cellular compartment consisting of a complex matrix of components that can change dramatically in response to environmental stresses. During pathogen attack, for instance, a wide spectrum of proteins that participate in various sequential processes involved in plant defense is secreted into the cell wall. In this study, a mass spectrometry, data-independent acquisition approach known as LC/MS (E) was used to assess temporal changes in the cell wall proteome in response to different levels of an endogenous inducer of plant disease defense responses, salicylic acid (SA). LC/MS (E) was used as a label-free method that enabled simultaneous protein identification and absolute femtomole quantification of each protein secreted into the extracellular matrix. A total of 74 secreted proteins were identified, 63 of which showed increased specific secretion in response to SA. A majority of this induced secretion occurred within 2 h of treatment, indicating that many proteins are involved in the early stages of plant defenses. We also identified a number of apparently nonclassically secreted proteins, suggesting that, as in many nonplant systems, Golgi/ER-independent mechanisms exist for plant protein secretion. These results provide new insight into plant apoplastic defense mechanisms and demonstrate that LC/MS (E) is a powerful tool for obtaining both relative and absolute proteome-scale quantification that can be applied to complex, time- and dose-dependent experimental designs.

SILIP: a novel stable isotope labeling method for in planta quantitative proteomic analysis

Due to ease of manipulation, metabolic isotope coding of samples for proteomic analysis is typically performed in cell culture, thus preventing an accurate in vivo quantitative analysis, which is only achievable in intact organisms. To address this issue in plant biology, we developed SILIP (stable isotope labeling in planta) using tomato plants (Solanum lycopersicum cv. Rutgers) as a method that allows soil-grown plants to be efficiently labeled using a 14N/15N isotope coding strategy. After 2 months of growth on 14N- and 15N-enriched nitrogen sources, proteins were extracted from four distinct tomato tissues (roots, stems, leaves and flowers), digested, and analyzed by LC/MS/MS (data-dependent acquisition, DDA) and alternating low- and elevated-energy MS scans (data-independent acquisition, MS(E)). Using a derived relationship to generate a theoretical standard curve, the measured ratio of the M (monoisotopic) and M-1 isotopologues of 70 identified 15N-labeled peptides from 16 different proteins indicated that 15N incorporation was almost 99%, which is in excellent agreement with the 99.3% 15N-enriched nitrate used in the soil-based medium. Values for the various tissues ranged from 98.2 +/- 0.3% 15N incorporation in leaves to 98.8 2 +/- 0.2% in stems, demonstrating uniform labeling throughout the plant. In addition, SILIP is compatible with root-knot nematode (Meloidogyne spp.) development, and thus provides a new quantitative proteomics tool to study both plant and plant-microorganism systems.

An efficient organic solvent based extraction method for the proteomic analysis of Arabidopsis plasma membranes.

Membrane proteins are involved in diverse cellular processes and are an integral component of many signaling cascades, but due to their highly hydrophobic nature and the complexities associated with studying these proteins in planta, alternative methods are being developed to better characterize these proteins on a proteome-wide scale. In our previous work ( Mitra , S. K. et al. J. Proteome Res. 2007 , 6 , ( 5 ), 1933 - 50 ), methanol-assisted solubilization was determined to facilitate the identification of both hydrophobic and hydrophilic membrane proteins compared to Brij-58 solubilization and was particularly effective for leucine-rich repeat receptor-like kinases (LRR RLKs). To improve peptide identification and to overcome sample losses after tryptic digestion, we have developed an effective chloroform extraction method to promote plasma membrane protein identification. The use of chloroform extraction over traditional solid-phase extraction (SPE) prior to off-line strong cation exchange liquid chromatography (SCXC) and reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis facilitated the removal of chlorophylls, major contaminants of plant tissue preparations that can affect downstream analysis, in addition to the effective removal of trypsin used in the digestion. On the basis of a statistically derived 5% false discovery rate, the chloroform extraction procedure increased the identification of unique peptides for plasma membrane proteins over SPE by 70% which produced nearly a 2-fold increase in detection of membrane transporters and LRR RLKs without increased identification of contaminating Rubisco and ribosomal peptides. Overall, the combined use of methanol and chloroform provides an effective method to study membrane proteins and can be readily applied to other tissues and cells types for proteomic analysis.

The BRASSINOSTEROID INSENSITIVE1–LIKE3 Signalosome Complex Regulates Arabidopsis Root Development

Brassinosteroids are essential regulators of plant growth and development. While BRI1 signaling regulates cellular growth in the majority of plant tissues, our study uncovers the composition of the BRL3 receptor complex in Arabidopsis and addresses cell type–specific roles for BRL3 signaling in root development. Brassinosteroid (BR) hormones are primarily perceived at the cell surface by the leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1 (BRI1). In Arabidopsis thaliana, BRI1 has two close homologs, BRI1-LIKE1 (BRL1) and BRL3, respectively, which are expressed in the vascular tissues and regulate shoot vascular development. Here, we identify novel components of the BRL3 receptor complex in planta by immunoprecipitation and mass spectrometry analysis. Whereas BRI1 ASSOCIATED KINASE1 (BAK1) and several other known BRI1 interactors coimmunoprecipitated with BRL3, no evidence was found of a direct interaction between BRI1 and BRL3. In addition, we confirmed that BAK1 interacts with the BRL1 receptor by coimmunoprecipitation and fluorescence microscopy analysis. Importantly, genetic analysis of brl1 brl3 bak1-3 triple mutants revealed that BAK1, BRL1, and BRL3 signaling modulate root growth and development by contributing to the cellular activities of provascular and quiescent center cells. This provides functional relevance to the observed protein–protein interactions of the BRL3 signalosome. Overall, our study demonstrates that cell-specific BR receptor complexes can be assembled to perform different cellular activities during plant root growth, while highlighting that immunoprecipitation of leucine-rich repeat receptor kinases in plants is a powerful approach for unveiling signaling mechanisms with cellular resolution in plant development.

Data‐driven modeling reconciles kinetics of ERK phosphorylation, localization, and activity states

The extracellular signal‐regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live‐cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERKs modification of and release from nuclear substrate anchors. Thus, adaptation of whole‐cell ERK phosphorylation is a by‐product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose‐dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed.

Development and Application of a Phosphoproteomic Method Using Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC), IMAC, and LC–MS/MS Analysis to Study Marek’s Disease Virus Infection

Mareks Disease (MD) is an avian neoplastic disease caused by Mareks Disease Virus (MDV). The mechanism of virus transition between the lytic and latent cycle is still being investigated; however, post-translational modifications, especially phosphorylation, have been thought to play an important role. Previously, our group has used strong cation exchange chromatography in conjunction with reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the changes in global proteomic expression upon MDV infection (Ramaroson , M. F.; Ruby, J.; Goshe, M. B.; Liu , H.-C. S. J. Proteome Res. 2008, 7, 4346-4358). Here, we extend our study by developing an effective separation and enrichment approach to investigate the changes occurring in the phosphoproteome using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to fractionate peptides from chicken embryo fibroblast (CEF) digests and incorporating a subsequent IMAC enrichment step to selectively target phosphorylated peptides for LC-MS/MS analysis. To monitor the multidimensional separation between mock- and MDV-infected CEF samples, a casein phosphopeptide mixture was used as an internal standard. With LC-MS/MS analysis alone, no CEF phosphopeptides were detected, while with ERLIC fractionation only 1.2% of all identified peptides were phosphorylated. However, the incorporation of IMAC enrichment with ERLIC fractionation provided a 50-fold increase in the percentage of identified phosphopeptides. Overall, a total of 581 unique phosphopeptides were identified (p < 0.05) with those of the MDV-infected CEF sample containing nearly twice as many as the mock-infected control of which 11% were unique to MDV proteins. The changes in the phosphoproteome are discussed including the role that microtubule-associated proteins may play in MDV infection mechanisms.