Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by ³²P_i revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.The regulation of protein function by phosphorylation and dephosphorylation is a key mechanism of intracellular signaling pathways in eukaryotic organisms. Protein phosphorylation is catalyzed by protein kinases, which are themselves often regulated by phosphorylation (1). The specificity of protein kinases is essential for their cellular functions. In some groups of protein kinases, the specificity is achieved by means of “docking interactions.” Protein kinase docking interactions involve a recognition site on the kinase or a flanking domain that is different from the active site. The most notable example, MAP kinases, uses a docking interaction to specifically recognize substrates, upstream kinases, and phosphatases. Despite the large amount of data on protein kinase docking interactions, e.g. in the MAP kinase field, there is very little information on how these essential interactions are regulated (2–4).3-Phosphoinositide-dependent protein kinase 1 (PDK1)³ belongs to the AGC family of protein kinases and is the activation loop kinase for several other AGC kinases (5). A key feature of the AGC kinase family members except PDK1 is the presence of a C-terminal extension (CT) to the catalytic core that contains a conserved hydrophobic motif (HM) harboring a phosphorylation site. In many AGC kinases, the HM mediates a docking interaction with PDK1. For example, p90 ribosomal S6 kinase (RSK), p70 S6 kinase (S6K) and serum- and glucocorticoid-induced protein kinase (SGK) interact with PDK1 upon phosphorylation of the HM site (6–9). The phosphorylated HM binds to a HM-binding pocket in the catalytic core of PDK1 that was originally termed the PIF-binding pocket (6, 10).Besides its role in the docking of substrates to PDK1, the HM/PIF-binding pocket was also identified as a ubiquitous and key regulatory site in likely all AGC kinases (7, 11). Thus, in AGC kinases studied up to now, the HM/PIF-binding pocket serves as an intramolecular docking site for the phosphorylated HM. In summary, the HM has a dual function in AGC kinase activation, (i) mediating the intermolecular interaction with PDK1 and (ii) acting as an intramolecular allosteric activator that stabilizes the active conformation of the kinase domain via binding to the HM/PIF-binding pocket.The CT of AGC kinases additionally contains a second regulatory phosphorylation site traditionally termed the “turn motif” (TM), and more recently the zipper (Z) site. The Z/TM phosphate interacts with a binding site within the kinase domain, acting like a zipper which serves to support the intramolecular binding of the phosphorylated HM to the HM/PIF-binding pocket (12). Hence, AGC kinases are synergistically activated by phosphorylation at the activation loop, the HM, and the Z/TM sites.Protein kinase C-related protein kinases (PRKs) (13) (also named PAK for protease-activated kinase (14–16) and PKN for protein kinase N (17)) represent a subfamily of AGC kinases. So far, three PRK isoforms were identified, PRK1, PRK2, and PKN3, which are effectors of the small GTP-binding protein Rho. PRKs, as well as the Rho-associated kinases (ROCKs), are considered to be the protein kinases that mediate the phosphorylation events downstream of Rho activation and both can be inhibited by the highly specific protein kinase inhibitor Y27632 (18). The most notable role described for PRK2 is the control of entry into mitosis and exit from cytokinesis (19). In addition, PRK2 phosphorylates the hepatitis C virus (HCV) RNA polymerase (20). In support of a function in HCV RNA replication, PRK2 inhibitors like Y27632 suppress HCV replication (21).The N-terminal region of PRK2 possesses three Rho effector (HR1) domains (13), a pseudosubstrate region that is thought to have an autoinhibitory function (22) and a C2-like domain, which is a potential binding site for lipid activators. The C-terminal region of PRK2 harbors the HM that mediates the docking interaction with the HM/PIF-binding pocket in its upstream kinase PDK1 (10, 23). Interestingly, PRKs and also atypical protein kinase Cs (PKCs, PKCζ, and PKCι/λ), contain an acidic residue instead of a phosphorylatable amino acid at the site equivalent to the HM phosphorylation site in other AGC kinases. Therefore, the molecular events that regulate the interaction of PRK2 and PKCζ with PDK1 must be different from the mechanism characterized for S6K, SGK, and RSK.In the present work we extended and refined the model of docking interaction between PRK2 and PDK1 and characterized C-terminal regions of PRK2 that participate in the regulation of this interaction. The work sheds light on the common as well as specific mechanisms that operate in the regulation of PDK1 docking interaction with its different substrates.     

The AGC Kinase MtIRE: A Link to Phospholipid Signaling During Nodulation?

The development of nitrogen fixing root nodules is complex and involves an interplay of signaling processes. During maturation of plant host cells and their endocytosed rhizobia in symbiosomes, host cells and symbiosomes expand. This expansion is accompanied by a large quantity of membrane biogenesis. We recently characterized an AGC kinase gene, MtIRE, that could play a role in this expansion. MtIRE''s expression coincides with host cell and symbiosome expansion in the proximal side of the invasion zone in developing Medicago truncatula nodules. MtIRE''s closest homolog is the Arabidopsis AGC kinase family IRE gene, which regulates root hair elongation. AGC kinases are regulated by phospholipid signaling in animals and fungi as well as in the several instances where they have been studied in plants. Here we suggest that a phospholipid signaling pathway may also activate MtIRE activity and propose possible upstream activators of MtIRE protein''s presumed AGC kinase activity.Key Words: AGC kinase, nitrogen fixation, nodulation, Medicago truncatula, Sinorhizobium meliloti, infection zone, 3-phosphoinositide-dependent kinase, root hair elongationDuring symbiotic nitrogen-fixing nodule development, both plant cells and rhizobia undergo cell division and expansion.^1–3 In legume roots, nodule organogenesis is triggered by rhizobial Nod factor at the emerging root hair zone. In the indeterminate Medicago-Sinorhizobium symbiosis, inner cortical cell divisions form nodule primordia which emerge from the root and differentiate into complex nodule structures. Rhizobia enter the nodules through plant derived conduits, the infection threads (ITs). ITs begin in curled root hairs, grow through several cell layers and end at nodule primordia where rhizobia are deposited into host cell symbiosomes.² In mature nodules, the meristematic zone I at the nodule apex contains dividing cells. Rhizobia from ITs infect these cells as they exit zone I and enter the infection zone, zone II. The newly released rhizobia, now termed bacteroids, are rod-shaped. In the distal part of zone II, bacteroids divide along with the symbiosome membrane (also called the peribacteroid membrane) that contains them.⁴ As the plant cells with their internalized bacteroids progress toward the proximal end of zone II, bacteroid division ceases. Bacteroid elongation and expansion of the surrounding symbiosome space and membrane is a feature of the proximal side of zone II.⁴ Enormous membrane biogenesis accompanies progression through zone II. As the cells exit zone II, both host cells and bacteroids stop expanding. Interzone II-III is characterized by starch accumulation and zone III is where nitrogen fixation takes place.Members of the protein kinase AGC (for cAMP dependent, cGMP dependent, and protein kinase C) family have been shown to be important in yeast and mammalian signal transduction. The interaction of growth factors with their receptors leads to the activation of phosphatidylinositol (PtdIns) 3-kinase and the phosphorylation of PtdIns species.⁵ These then activate PDK1 enzymes, 3-phosphoinositide-dependent kinases, also AGC kinases,⁵ which then phosphorylate and activate downstream AGC kinases. Several plant AGC kinases have important roles in development and defense,^6–8 although most plant AGC kinases'' functions are still to be discovered.⁹ Two Arabidopsis AGC kinases, IRE and AGC2-1 have been shown to have roles regulating root hair elongation.^10,11We recently cloned and characterized a Medicago IRE-like AGC kinase gene MtIRE,¹² possibly orthologous to the Arabidopsis IRE gene, AtIRE.¹⁰ Because of MtIRE''s homology to AtIRE we thought it might function during infection, because infection threads can be viewed as inward root hair growth. However, MtIRE''s expression is novel. It is expressed only in nodules and flowers and not in roots or root hairs. During nodule development, its initial expression correlates with the onset of host cell and symbiosome expansion. Expression studies with nodulation mutants demonstrate that MtIRE expression correlates with mutant nodules'' abilities to support host cell and symbiosome expansion.¹² An MtIRE promoter-gusA reporter construct (Fig. 1A) shows expression in the proximal part of zone II, the site of continued host cell expansion and bacteroid and symbiosome elongation. RNA interference experiments were unfortunately inconclusive,¹² probably because of closely related more ubiquitously expressed IRE homologs.Open in a separate windowFigure 1(A) Localization of pMtIRE-gusA expression in wild-type nodulated roots. Composite M. truncatula plants with transgenic roots were grown in the presence of S. meliloti and stained with X-Gluc (blue) for the localization of MtIRE promoter activity. The arrow points to the X-Gluc staining in the proximal side of zone II in a 15 dpi nodule. The arrowhead points to root hairs in which no staining was observed. Bar = 100 µM. (B) Phospholipid signaling pathway that may activate MtIRE protein''s presumed kinase activity.We predict that MtIRE is part of a signal pathway regulating an aspect of host cell expansion or symbiosome elongation, or both. The CCS52A gene has a demonstrated role in host cell expansion, mediating endoreduplication.¹³ In contrast to MtIRE, its expression is found throughout zone II, as well as zone I, where it acts in cell division. One might expect other genes that regulate host cell expansion to also be expressed throughout zone II, which MtIRE is not. A unique feature of the region expressing MtIRE is symbiosome elongation.⁴ Because of MtIRE''s temporal and spatial expression patterns, we favor it having a role in symbiosome expansion, although we cannot rule out a role in the latter stages of host cell expansion.Signaling pathway for MtIRE activation is speculative (Fig. 1B) and based on AGC kinase signaling in other systems. AGC kinases are activated by phosphorylation by phosphoinositide-dependent kinase (PDK1) enzymes, also AGC kinases.⁹ We found 4 tentative consensus sequences (TCs) in the DFCI index (compbio.dfci.harvard.edu) that correspond to PDK1 genes of which 3, TC107355, TC94724 and TC94899, were isolated from expression libraries from roots with developing or mature nodules. PDKs are activated by interaction with lipids. The Arabidopsis PDK1 binds to several signaling lipids, including phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidic acid (PA).¹⁴ Phosphatidylinositol 3-kinase (PI3K) activity produces PtdIns3P and PI3K genes have been observed to be induced during nodule organogenesis in soybean¹⁵ and in M. truncatula.¹⁶ In soybean, two PI3K genes were identified with one specifically expressed during the early stages of nodulation when membrane biogenesis takes place. This gene''s predicted protein has potential phosphorylation sites for cAMP dependent kinases and Ca/calmodulin-dependent kinases.¹⁵ In soybean, PI3K enzymatic activity correlated with membrane proliferation during nodulation.¹⁵ More generally, PI3Ks are implicated in vesicular trafficking and cytoskeletal organization;¹⁷ both are required for host cell and symbiosome elongation. We suggest a model where MtIRE kinase activity is activated by PDK1, which is itself regulated by PI3K through the production of PtdIns3P. More speculatively, PI3K could be under the control of the Nod factor signaling pathway Ca/calmodulin-dependent kinase DMI3.^18,19 DMI3 is induced during nodulation, with highest expression levels found in the distal side of the infection zone,²⁰ before expression of MtIRE. Expression could persist to the proximal side of this zone, similar to the expression of another Nod factor signaling component, DMI2.²¹ Alternatively, MtIRE could be activated by PA in a PDK1-dependent manner similar to Arabidopsis AGC2-1.¹¹ PA can be produced by phospholipase C (PLC) or phospholipase D (PLD) pathways, both of which have been implicated in transducing Nod factor signals.^22–26 Either of these models includes Nod factor signaling in proximal zone II, which has not been well-studied. Expression of rhizobial nod genes has been observed in zone II,²⁷ making Nod factor signaling in this zone plausible. Further examination of zone II and predicted upstream regulators of MtIRE will address this model.     

Linking protein kinase CK2 and auxin transport

Studies performed in different organisms have highlighted the importance of protein kinase CK2 in cell growth and cell viability. However, the plant signaling pathways in which CK2 is involved are largely unknown. We have reported that a dominant-negative mutant of CK2 in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. We demonstrated that auxin transport is, indeed, impaired in these mutant plants, and that this correlates with misexpression and mislocalization of PIN efflux transporters and of PINOID. Our data establishes a link between CK2 activity and the regulation of auxin homeostasis in plants, strongly suggesting that CK2 might be required at multiple points of the pathways regulating auxin fluxes.Key words: protein kinase CK2, root development, auxin, PIN, PINOIDThe plant hormone auxin plays critical roles in plant growth and development.¹ The most abundant natural auxin is the indol-3-acetic acid (IAA), which is synthesized in young apical tissues and then transported to the growing zones of the stem and root. The major route for long distance IAA movement is via the vascular tissue, but, additionally, a slower transport via cell-to-cell (called polar transport) is critical to generate auxin gradients within tissues. Formation of correct auxin gradients is thought to be essential for many plant developmental processes.² In recent years, the IAA transporters have been identified, establishing the molecular basis to understand how auxin transport is regulated. In particular, the identification of the family of plasma-resident PIN proteins, the members of which function as IAA efflux carriers, and the knowledge of their polar localization in the plasma membrane (PM), contributed to generate models predicting the direction of IAA fluxes.^3,4The factors that govern PIN targeting to a particular membrane domain are still not understood. It is known that PIN proteins constitutively undergo cycles of exocytosis and endocytosis to and from the PM, using distinct sorting and recycling endosome trafficking pathways.^5–7 Phosphorylation/dephosphorylation by the Ser/Thr kinase PINOID (PID) and the protein phosphatase 2A, respectively, controls PIN proteins apical/basal localization at the PM, via the GNOM-mediated vesicle trafficking system.⁸ Interestingly, PID is a member of the plant AGC kinases, and, as it happens with its mammals AGC counterparts, is activated by a membrane-associated 3-phosphoinositide-dependent kinase (PDK1).⁹ Moreover, a functional similarity between PIN polar localization in response to auxin and glucose receptor (GLUT4) asymmetrical distribution in response to insulin, has been pointed out.¹⁰ In both cases, cargo proteins (GLUT4 and PIN, respectively) are transported from endosomal vesicles to PM and the process is mediated by PDK1-activated AGC kinases.Protein kinase CK2 is a Ser/Thr kinase evolutionary conserved in eukaryotes, which plays key roles in cell survival, cell division and other cellular processes. A loss-of-function mutant of CK2 in Arabidopsis, obtained by overexpression of a CK2α-inactive subunit, confirmed the essential role of this protein kinase for plant viability.¹¹ Moreover, CK2mut plants showed a dramatic decrease of lateral root formation, inhibition of root growth and overproliferation of root hairs. We have further demonstrated that auxin transport is impaired in this plants, which is concomitant with missexpression of most of the PM-resident PIN proteins, and of PID.¹² In addition, PIN proteins accumulated in endosomal vesicles and auxin gradients were disturbed, both in roots and shoots of CK2mut plants. In particular, root columella cells were depleted of auxin, although the maximum at the quiescent center was unchanged. Starch granule staining with lugol revealed that columella cells retained their fate, although their organization and/or cell shape were clearly affected (Fig. 1).Open in a separate windowFigure 1Lugol-stained starch granules in uninduced (−Dex) and Dex-induced (+Dex) CK2mut roots. In the central part of the figure, a sketch of the main morphogenetic characteristics of mutant roots (right plantlet) as compared to wild-type roots (left plantlet) is shown. Note the shorter roots, wavy phenotype, absence of lateral roots and overproliferation of root hairs in mutant plants.Our results strongly suggest that CK2 is a regulator of auxin-dependent responses, most likely by participating in the regulation of auxin transport. Strikingly, depletion of CK2 activity inhibits some auxin-dependent physiological responses whereas it enhances others. For instance, whereas shoot phototropism was completely absent, root gravitropism was enhanced.¹² Figure 2 shows a time-course of DR5rev::GFP-derived signal after changing the gravity vector, in mutant and control Arabidopsis roots. The progressive auxin translocation to the lower side of the root after gravistimulation is more rapid and sustained in mutant than in control roots, which is likely responsible for the enhanced response to gravity found in mutant roots. Based on these results, we postulate that CK2 might act at different points of the auxin-induced regulatory pathway. As far as is known, the core module that regulates auxin transport is constituted by the protein kinase PID and a protein of the NPH3-domain family. NPH3-containing proteins play important roles in phototropic and gravitropic responses, and regulate polarity and endocytosis of PIN proteins.¹³ As has been proposed by other authors, the participation of one AGC kinase and one NPH3-like protein upstream of an ARF factor might be a common theme in response to different stimulus that are signaled by auxin.¹⁴ We propose that one of the functions of CK2 is the regulation of the activity of core proteins (Fig. 3). Mammalian AGC kinases are well known substrates of CK2 and CK2-dependent phosphorylation is critical for a full display of their activity. The PID and the NPH3-containing protein sequences contain numerous acidic-based motifs that are predicted CK2 phosphorylation sites. Moreover, according to Arabidopsis phosphoproteome databases, several members of the NPH3-containing protein family are predicted to be phosphorylated.¹⁵ In addition, we do not discard the possibility that other proteins involved in PIN transport might also be regulated by CK2-dependent phosphorylation. Experiments are in progress in our laboratory to assess the regulatory role of CK2 in auxin transport.Open in a separate windowFigure 2Time course of auxin relocation during root gravitropic response, as visualized by DR5rev::GFP fluorescence. Root pictures were taken at the indicated times after changing the direction of the gravity vector. Translocation of auxin to the lower part of the root is more rapid in Dex-induced CK2mut plants. Arrows indicate asymmetrical DR5::GFP fluorescence.Open in a separate windowFigure 3Proposed model for the role of CK2 in regulating auxin transport. The core module that regulates auxin transport (shown here as a black box) is constituted by the protein kinase PID and a protein of the NPH3-domain family. PID regulates apical-basal targeting of PIN proteins, by phosphorylating conserved Ser residues present in PIN hydrophilic loops.¹⁶ On the other hand, the family of NPH3-containing proteins regulates polarity and endocytosis of PIN proteins.¹³ There is also a functional similarity between the intracellular transport of PIN proteins and that of the glucose receptor (GLUT4),¹⁰ two processes that are signaled by AGC kinases. We propose that CK2 might be a regulator of the activity of the core proteins, by phosphorylating either the AGC kinase and/or the NPH3-containing protein. Mammalian CK2 is a known regulator of the activity of AGC kinases and other proteins participating in signaling pathways, such as in the Wnt/β-catenin signaling pathway.¹⁷     

Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants

Recently we reported that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase plays an important role in regulating plant cold tolerance. Calcium/calmodulin binds to CRLK1 and upregulates its activity. Gene knockout and complementation studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we show that MEKK1, a member of MAP kinase kinase kinase family, interacts with CRLK1 both in vitro and in planta. The cold triggered MAP kinase activation in wild-type plants was abolished in crlk1 knockout mutants. Similarly, the cold induced expression levels of genes involved in MAP kinase signaling are also altered in crlk1 mutants. These results suggest that calcium/calmodulinregulated CRLK1 modulates cold acclimation through MAP kinase cascade in plants.Key words: calcium, calmodulin, cold stress, MAPK, Arabidopsis, protein phosphorylationCalcium, a universal second messenger in eukaryotic cells, mediates changes in external and internal signals leading to the physiological responses.^1–4 Calcium/calmodulin (Ca²⁺/CaM)-dependent protein kinases (CaMKs) are very important players in calcium/calmodulin mediated signaling in mammalian cells.⁵ In plants, Ca²⁺/CaM-dependent protein phosphorylation was observed more than 25 years ago.⁶ Several calmodulin-regulated protein kinases have been identified and characterized.^7,8 For example, plants have a unique chimeric Ca²⁺/CaM-dependent protein kinase (CCaMK), which exhibits Ca²⁺-dependent autophosphorylation and Ca²⁺/CaM-dependent substrate phosphorylation.⁹ CCaMK is required for bacterial and fungal symbioses in plants.^10–12 Recently, we characterized a novel plant-specific calcium/CaM-regulated receptor-like kinase, CRLK1.¹³ Ca²⁺/CaM binds to CRLK1 and stimulates its kinase activity. Functional studies with CRLK1 indicate that CRLK1 acts as a positive regulator in plant response to chilling and freezing temperatures. To further define the CRLK1-mediated signal pathway, we isolated CRLK1 interacting proteins by co-immunoprecipitation using an anti-CRLK1 antibody. Since cold increases the amount of CRLK1 protein, wildtype plants (WT) were treated at 4°C for 1 hr before co-immunoprecipitation. The resulting CRLK1 immunocomplex was separated by SDS-PAGE. We observed several bands of different sizes only in the wild-type but not in the crlk1 knockout mutant plants (Fig. 1A). Furthermore, the intensity of these bands increased upon cold treatment, suggesting that they are the putative partners or associated proteins of the CRLK1 immunocomplex.Open in a separate windowFigure 1CRLK1 Interacts with MEKK1. (A) One-dimension SDS-PAGE of anti-CRLK1 immunocomplexes from 3-week-old WT or crlk1 plants with or without cold treatment. One mg of total protein was used for immunoprecipitation. (B) A list of putative CRLK1-interacting proteins determined by MALDI-TOF-MS analysis. (C) CRLK1 interacts with MEKK1 as shown by GST pull-down assay. (D) BiFC analysis show that CR LK associates with MEKK1 in vivo. Upper row shows that CRLK and MEKK1 associate both on cell membrane and in endosomes. The middle and last rows are controls. Bar = 10 µm.To determine the identities of these proteins, mass spectrometric analysis was performed with the total immunocomplex.¹⁴ In addition to CRLK1, there were 12 other proteins which matched the Arabidopsis database. Several of them appeared in the pull-down complex from WT, but not from crlk1 mutants. These putative interacting proteins included MEKK1, another unknown protein kinase, a type 2C phosphatase and CaM (Fig. 1B). MEKK1 is one of the 60 putative MAPKKKs in the Arabidopsis genome, and sits on the top of mitogen-activated protein kinase (MAPK) cascade. The MAPK signaling consists of a cascade of three consecutively acting protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and a MAP kinase (MAPK). Plants possess multiple MAPKKKs, MAPKKs and MAPKs, which respond to different upstream signals and activate distinct downstream pathways.^15–17 The specific MAPK module responding to lower temperature has been determined in Arabidopsis.^18,19 MEKK1, a member of MAPKKKs, specifically interacts and phosphorylates MKK2 and regulates COR genes expression in response to cold stress.¹⁹ MEKK1 has been shown to play a role in mediating reactive oxygen species homeostasis.^20,21 Therefore we selected MEKK1 from the putative CRLK1 partners for further studies.     

A Surface of the Kinase Domain Critical for the Allosteric Activation of G Protein-coupled Receptor Kinases

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs and initiate their desensitization. Many prior studies suggest that activated GPCRs dock to an allosteric site on the GRKs and thereby stimulate kinase activity. The extreme N-terminal region of GRKs is clearly involved in this process, but its role is not understood. Using our recent structure of bovine GRK1 as a guide, we generated mutants of solvent-exposed residues in the GRK1 kinase domain that are conserved among GRKs but not in the extended protein kinase A, G, and C family and evaluated their catalytic activity. Mutation of select residues in strands β1 and β3 of the kinase small lobe, αD of the kinase large lobe, and the protein kinase A, G, and C kinase C-tail greatly impaired receptor phosphorylation. The most dramatic effect was observed for mutation of an invariant arginine on the β1-strand (∼1000-fold decrease in k_cat/K_m). These residues form a continuous surface that is uniquely available in GRKs for protein-protein interactions. Surprisingly, these mutants, as well as a 19-amino acid N-terminal truncation of GRK1, also show decreased catalytic efficiency for peptide substrates, although to a lesser extent than for receptor phosphorylation. Our data suggest that the N-terminal region and the newly identified surface interact and stabilize the closed, active conformation of the kinase domain. Receptor binding is proposed to promote this interaction, thereby enhancing GRK activity.G protein-coupled receptor kinases (GRKs)² are members of the protein kinase A (PKA), G, and C (AGC) family that phosphorylate Ser/Thr residues in the cytoplasmic loops and C termini of activated G protein-coupled receptors (GPCRs) (1). Receptor phosphorylation facilitates the binding of arrestin, which uncouples heterotrimeric G proteins, promotes receptor internalization, and activates arrestin-dependent signaling pathways (2, 3). Although playing a beneficial role in receptor desensitization, GRKs are implicated in a range of human diseases, including retinal degeneration, hypertension, heart failure, rheumatoid arthritis, opiate addiction, and various cancers (2, 4). Seven GRKs have been identified in mammals. They can be divided into the following three subgroups based on their sequence homology: GRK1 (GRK1 and GRK7), GRK2 (GRK2 and GRK3), and GRK4 (GRK4, GRK5, and GRK6). The primary structures of the three GRK subgroups are similar, consisting of tandem regulator of G protein signaling homology (RH) and kinase domains. Less conserved sequences involved in phospholipid membrane attachment are found at their C termini (Fig. 1A).Open in a separate windowFIGURE 1.GRK surface residues potentially important for GPCR phosphorylation. A, domain architecture of bGRK1. B, sequence alignment of regions from GRKs that were targeted in this study with other AGC kinases. Colored boxes map these regions back to the domain structure shown in A. Regions of the core kinase domain that contain residues conserved in the GRK subfamily, but not in the extended AGC kinase family, are highlighted in brown. Conserved residues of the AGC kinase C-tail are highlighted in green. Positions investigated in this study are indicated with asterisks. Only one PDB accession code for each kinase of known structure is shown in parentheses. Residue numbers correspond to those of bGRK1. The PXXP, turn, and hydrophobic (HF) motifs (highlighted in gray) are characteristic features found in most AGC kinase C-tails (22). Yeast, Saccharomyces cerevisiae; M.tb, Mycobacterium tuberculosis. C, ribbon diagram of the bGRK1₅₃₅-H₆·ATP complex. The model is a hybrid that contains all the ordered elements from the two unique chains resolved in the crystal structure (PDB accession number 3C4W), such that the observed N terminus and a nearly complete AST region of the kinase C-tail are displayed in a single model. The RH domain is colored gray, and the β-strands and α-helices of the core kinase domain are dark and light brown, respectively. The hinge region joining the kinase small and large lobes (between β5 and αD) is colored yellow. The N-terminal region and the AGC kinase C-tail are shown in green and the AST loop in cyan. The ATP molecule bound in the active site is shown as a stick model, and the two associated Mg²⁺ ions are colored black. D, conservation scores of GRKs mapped onto the surface of bGRK1. The area boxed in C is shown. The conservation scores were calculated by comparing the sequence conservation of residues from the core kinase domain of GRKs with those of the entire AGC family (see text). Residues are colored using a gradient, from magenta (more conserved in GRKs than the AGC kinases) to white (as conserved in GRKs as in AGC kinases) and to yellow (more variable in GRKs than in AGC kinases). The RH domain, which was not included in this calculation, is colored gray. Highest conservation among GRKs cluster at two sites, “site 1” and “site 2.” Site 1 corresponds to a region of the small lobe left exposed by the shorter AST loop found in GRKs relative to other AGC kinases. The AST region of protein kinase B (PDB accession number 1O6K) is superimposed for comparison (blue ribbon).All eukaryotic protein kinases, including GRKs, contain a core catalytic domain of ∼250 amino acids that can be divided into two subdomains, called the small (or N) and large (or C) lobes. The small lobe consists of a five-stranded β-sheet (β1–5) and a conserved helix, αC, whereas the large lobe is mostly α-helical. The active site is located at their interface, with the nucleotide-binding pocket formed primarily by the small lobe and the phosphoacceptor-binding site primarily by the large lobe. In their active conformation, kinases position the hydroxyl group of the phosphoacceptor substrate in the proper orientation with respect to the γ-phosphate of ATP via a network of interactions formed by conserved structural elements from both lobes. Control of this network often underlies the molecular basis for allosteric regulation of protein kinase activity (5–9).In GRKs, this allosteric regulation appears to be mediated by interactions with activated GPCRs. Steady-state kinetics indicate that the K_m values of receptor substrates are in the micromolar range, whereas those of peptide substrates, even those derived from receptors, are in the millimolar range (10–13). Moreover, the catalytic efficiency for peptide phosphorylation by GRKs is much lower than that for receptor phosphorylation, and it can be enhanced in the presence of activated receptors (11, 12, 14). Thus, in addition to the peptide phosphoacceptor-binding site of the large lobe, an additional allosteric receptor-docking site appears to be required to promote catalytic activity in GRKs.The molecular basis for how GRKs interact with activated GPCRs is poorly understood. In vitro, GRKs show little specificity among GPCRs, requiring only that the receptor be in an activated conformation. For example, although GRK1 is the predominant kinase expressed in rod outer segments, GRK1, GRK2, and GRK5 all phosphorylate bovine rhodopsin in a light-dependent manner with comparable catalytic efficiencies (15–17). Therefore, it seems likely that GRKs have a common molecular mechanism for the recognition of activated GPCRs. The region of GRKs most strongly linked to efficient receptor phosphorylation is the highly conserved N-terminal region, which is unique to the GRK subfamily and predicted to form an α-helix (Fig. 1B). Deletion of this region in GRK1, -2, or -5 abolishes receptor phosphorylation (18–20). Additionally, the binding of antibodies (18) or of recoverin (21) to the GRK1 N-terminal region inhibits receptor phosphorylation. In GRK5, it has also been suggested that the N terminus plays a role in phospholipid interactions (20). Another region that is likely involved in the allosteric regulation of GRKs is the AGC kinase C-terminal tail (C-tail), which is an extension of the kinase core domain and often plays a regulatory role in AGC kinases (22–24) (Fig. 1, B and C). The central segment of the C-tail, termed the active site tether (AST), contributes residues to the active site and is only well ordered in kinase domain structures that are in conformations resembling the active state.To date, crystal structures representing each GRK subgroup have been reported, i.e. bovine GRK1 (bGRK1), bovine GRK2 (bGRK2), and human GRK6 (hGRK6) (25–29). Although most of these structures were co-crystallized in the presence of ATP or nucleotide analogs, none adopted the closed, active conformation exhibited by nucleotide-bound PKA (30), and the AST region of their AGC kinase C-tails were either partially or totally disordered. Similarly, the N-terminal region important for receptor phosphorylation was only observed in one crystal structure, namely that of one chain of the bGRK1·ATP complex. Thus, the regions believed to be most important for receptor interaction were largely disordered in these structures, leaving the molecular basis for how GPCRs interact with GRKs unclear. Because the kinase domains in these structures appear to be otherwise competent for catalysis, it is expected that activated GPCRs bind in a manner that promotes full kinase domain closure. Interactions with negatively charged lipids in the cell membrane are also expected to play a role in this transition (20, 31, 32).In this study, we used recently determined structures of bGRK1 as a template to identify surface residues of the kinase domain that are conserved in GRKs but not in the extended AGC family. Biochemical characterization of site-directed mutants of these residues in bGRK1 identified a continuous surface on the small lobe and the AGC kinase C-tail that is critical for GRK activation by GPCRs. The residue whose mutation showed the largest effect on receptor phosphorylation is nearly as important as the N-terminal region, and the analogous residue is also critical in the other two GRK subgroups, represented by bGRK2 and hGRK6. Comparison with other AGC kinases reveals that this surface is uniquely available for protein-protein interactions in the GRK subfamily. A model for activation that involves cooperative interactions between the N-terminal region and the kinase domain is presented.     

The regulatory network of cell cycle progression is fundamentally different in plants versus yeast or metazoans

Plant growth and proliferation control is coming into a global focus due to recent ecological and economical developments. Plants represent not only the largest food supply for mankind but also may serve as a global source of renewable energies. However, plant breeding has to accomplish a tremendous boost in yield to match the growing demand of a still rapidly increasing human population. Moreover, breeding has to adjust to changing environmental conditions, in particular increased drought. Regulation of cell cycle control is a major determinant of plant growth and therefore an obvious target for plant breeding. Furthermore, cell cycle control is also crucial for the DNA damage response, for instance upon irradiation. Thus, an in-depth understanding of plant cell cycle regulation is of importance beyond a scientific point of view. The mere presence of many conserved core cell cycle regulators, e.g., CDKs, cyclins or CDK inhibitors, has formed the idea that the cell cycle in plants is exactly or at least very similarly controlled as in yeast or human cells. Here together with a recent publication we demonstrate that this dogma is not true and show that the control of entry into mitosis is fundamentally different in plants versus yeast or metazoans. Our findings build an important base for the understanding and ultimate modulation of plant growth not only during unperturbed but also under harsh environmental conditions.Key words: cell cycle, phosphorylation, checkpoint, DNA damage, cyclin-dependent kinase, CDK, WEE1, CDC25, ArabidopsisProgression through the cell cycle is not only a decisive event for a single cell but also of key importance for organ growth in multicellular organisms such as plants.^1,2 Moreover, coupled to and overlapping in space and time with proliferation, cell differentiation takes place and thus, a tight control of the cell cycle is one of the foundations of development.³ Thus, not very surprisingly, an elaborated machinery controlling cell cycle regulation has evolved and overall, many proteins appear to be conserved between humans and plants.^4,5 However, there are also clear differences in the repertoire of cell cycle regulators in plants and functional studies have often not yet been conducted to elucidate the specific role of many regulators.In metazoans, a switch-like activation of the central cyclin-dependent kinase, Cdk1 (or its homologous proteins, e.g., Cdc2⁺ or CDC28p) plays one of the most important roles in cell cycle control.⁶ Wee1-type kinases, e.g., Wee1 or Myt1, phosphorylate Cdk1-type kinases at Thr14 and Tyr15 (or the homologous positions) and inhibit their activity (Fig. 1A).⁷ The function of these kinases is opposed by Cdc25 that acts as dual specificity phosphatase and removes these phosphate groups leading to the rapid activation of Cdk1-type kinases. This inhibition of Cdk1 activity by Wee1 and its release by Cdc25 fulfill a fundamental function during metazoan cell cycle control ensures the unidirectionality of the cell cycle.^8,9 The underlying molecular mechanism for this is a wiring of Cdk1 with Cdc25 or Wee1 by positive and antagonistic (double-negative) feedback loops, i.e., Cdk1 activates its activator Cdc25 and inactivates its inhibitor Wee1 (Fig. 1C). Thus, there are only two stable steady states, inactive or active; this bistability generates a biological switch. The transition from one state to the other is thought to be brought about by rising and falling levels of cyclins as activating subunits of CDKs. Moreover, due to the positive feedback wiring, the two steady states are buffered against small changes in cyclin levels, i.e., it takes a much higher concentration of cyclins to switch from G₂-phase into mitosis than to stay in mitosis. This property of feed-back wiring, called hysteresis, greatly reinforces the unidirectionality of the cell cycle (Fig. 1A and C).^10,11Open in a separate windowFigure 1Computational analysis of the switch-like activation of Cdk1-like kinases. (A and B) show steady-state activity of CDKs as a function of cyclin levels. (A) CDK/cyclin activity regulated via inhibitory Tyr15-phosphorylation of the CDK catalytic subunit of the complex. (B) CDK/cyclin activity control is achieved by stoichiometrically acting CDK inhibitors (CKIs). Both switches allow building up inactivated kinase and once a cyclin level has reached a threshold, high levels of kinase activity are rapidly available that can forcefully promote the entry into the next cell cycle phase. Importantly, a small drop in cyclin levels is not sufficient to change the activity state, thus the system is buffered and once the decision is taken to enter the next cell cycle phase, this cannot easily be reverted. (C) Double-negative and positive feedback loops targeting the status of inhibitory CDK phosphorylation. CDK activity is governed via inhibitory phosphorylation by WEE1/MYT1 kinases and activatory dephosphorylation by CDC25 phosphatases. CDK can phosphorylate WEE1/MYT1 to inactivate its own inactivator and CDK activates its own activator CDC25 by phosphorylation.¹¹ (D) Double-negative feedback loop of the CDK-CKI module.⁴³ CKIs inhibit CDKs and, in turn, CDKs promote CKI degradation.⁴²Cdc25 and the feedback loops sketched above are also major targets of a checkpoint response and interruption of these can effectively arrest the cell cycle. For instance, in animals, DNA damage is sensed by ATM and ATR kinases that in turn activate Chk1 and Chk2 kinases which then will phosphorylate and inactivate Cdc25 allowing the cell to repair its damage.^12,13 In parallel, Chk1/2 activate Wee1 by phosphorylation and reinforce the checkpoint.Previously, candidate genes for Cdc25 and Wee1 homologs have been identified in Arabidopsis as well as in other plants.^14–18 Along with the finding that plants contain Cdk1-like kinases with a PSTAIRE cyclin binding signature, designated CDKAs, which can rescue yeast cdc2/cdc28 mutants,^19–22 this has given rise to the notion that the wiring of the regulatory triangle CDKA-CDC25-WEE1 is conserved in plants.Here and in an accompanying publication by Dissmeyer et al.²³ we have probed this notion by a detailed structure-function analysis. Our data demonstrate that the regulatory connection between these three components is not conserved and that plants must have evolved different mechanisms to stably progress through a mitotic cycle and arrest the cell cycle upon DNA damage.     

Autophosphorylation desensitizes phytochrome signal transduction

Plant red/far-red photoreceptor phytochromes are known as autophosphorylating serine/threonine kinases. However, the functional roles of autophosphorylation and kinase activity of phytochromes are largely unknown. We recently reported that the autophosphorylation of phytochrome A (phyA) plays an important role in regulating plant phytochrome signaling by controlling phyA protein stability. Two serine residues in the N-terminal extension (NTE) region were identified as autophosphorylation sites, and phyA mutant proteins with serine-to-alanine mutations were degraded in plants at a significantly slower rate than the wild-type under light conditions, resulting in transgenic plants with hypersensitive light responses. In addition, the autophosphorylation site phyA mutants had normal protein kinase activities. Collectively, our results suggest that phytochrome autophosphorylation provides a mechanism for signal desensitization in phytochrome-mediated light signaling by accelerating the degradation of phytochrome A.Key words: phytochrome, autophosphorylation, phosphorylation, protein kinase, protein degradation, light signaling, signal desensitizationHigher plants continually adapt to their light environments to promote photosynthesis for optimal growth and development. Natural light conditions are monitored by various plant photoreceptors, including red (R)/far-red (FR) photoreceptor phytochromes.^1,2 Phytochromes are dimeric chromoproteins covalently linked to tetrapyrrole chromophore phytochromobilin, and exist as two photo-interconvertible species, red-light absorbing Pr and far-red-light absorbing Pfr forms. Phytochromes are biosynthesized as the Pr form in the dark, and are transformed to the Pfr form upon exposure to red light. This photoactivation of phytochromes induces a highly regulated signaling network for photomorphogenesis in plants.^3,4 Recently, phosphorylation and dephosphorylation have been suggested to play important roles in phytochrome-mediated light signaling;^5,6 for instance, a few phytochrome-associated protein phosphatases have been shown to act as positive regulators of phytochrome signaling.^7–9 However, the functional roles of phytochrome phosphorylation remain to be explored.     

Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes

Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)¹ probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188.ATP binding and hydrolysis are the driving processes in all living organisms. Hundreds of cellular proteins are able to bind and hydrolyze ATP to unfold proteins, transport molecules over membranes, or phosphorylate small molecules or proteins. Proteins with very different structures are able to bind ATP. A large and important class of ATP binding proteins is that of the kinases, which transfer the gamma phosphate from ATP to substrates. Kinases, and particularly protein kinases, play pivotal roles in signaling and protein regulation.The genome of the model plant Arabidopsis thaliana encodes for over 1099 protein kinases and hundreds of other ATP binding proteins (1, 2). Protein kinases are involved in nearly all signaling cascades and regulate processes ranging from cell cycle to flowering and from immunity to germination. Many protein kinases in plants are receptor-like kinases (RLKs), often carrying extracellular leucine-rich repeats (LRRs). The RLK class contains at least 610 members (3), including famous examples such as receptors involved in development (e.g. BRI1, ER, CLV1) and immunity (e.g. FLS2, EFR). Other important classes are mitogen-activated protein (MAP) kinases (MPKs) (20 different members), MPK kinase kinase kinases (MAP3Ks) (60 different members (4)), and calcium-dependent protein kinases (CPKs) (34 different members (5)). Because of their diverse and important roles, protein kinases have been intensively studied in plant science. The current approach is to study protein kinases individually—a daunting task, considering the remaining hundreds of uncharacterized protein kinases. New approaches are necessary in order to study protein kinases and other ATP binding proteins globally rather than individually.ATP binding activities of protein kinases and other proteins can be detected globally by acyl-ATP (AcATP) probes (6, 7) (Fig. 1A). AcATP binds to the ATP pocket of ATP binding proteins and places the acyl group in close proximity to conserved lysine residues in the ATP binding pocket. The acyl phosphonate moiety serves as an electrophilic warhead that can be nucleophilically attacked by the amino group of the lysine, resulting in a covalent attachment of the acyl reporter of the AcATP probe on the lysine and a concomitant release of ATP. The reporter tag is usually a biotin to capture and identify the labeled proteins. Labeled proteins can be displayed on protein blots using streptavidin-HRP. However, because AcATP labels many ATP binding proteins and protein kinases are of relatively low abundance, mass spectrometry is more often used to identify and quantify labeling with AcATP probes. The analysis is preferably done using Xsite, a procedure that involves trypsination of the entire labeled proteome, followed by analysis of the biotinylated peptides rather than the biotinylated proteins (8). This “KiNativ ” approach provides enough depth and resolving power to monitor ∼160 protein kinases in a crude mammalian proteome (7). Of the 518 human protein kinases (9), 394 (76%) have been detected via AcATP labeling (6).Open in a separate windowFig. 1.Structure and mechanism of labeling with BHAcATP. A, BHAcATP contains ATP, an acyl phosphate reactive group, and a biotin tag. When BHAcATP binds to the ATP binding pocket of a protein, the amino group of the nearby lysine reacts with the carbonyl carbon, which results in the covalent binding of the biotin tag to the protein while ATP is released. B, typical BHAcATP labeling profile of Arabidopsis leaf proteome. Arabidopsis leaf extracts were labeled with BHAcATP and the biotinylated proteins were detected on protein blots using streptavidin-HRP. Coomassie Brilliant Blue staining indicates equal loading. Asterisks indicate endogenously biotinylated proteins MCCA and BCCP. White, black, and gray arrowheads indicate bands containing ATBP+RBCL, PGK1, and a mix of ATP binding proteins, respectively. Abbreviations: MCCA, 3-methylcrotonyl-CoA carboxylase; BCCP, biotin carboxyl carrier protein; ATPB, chloroplastic ATPase; RBCL, ribulose-bisphosphate carboxylase; PGK1, phosphoglycerate kinase-1.KiNativ has mostly been used to validate targets of human drugs that target protein kinases using competitive labeling experiments. This approach has been used to identify selective inhibitors of, for example, Parkinson''s disease protein kinase LRRK2 (10), the BMK1 and JNK MAP kinases (11, 12), and the mTOR kinase (13). Importantly, the correlation of the biological activity of protein-kinase-inhibiting drugs with inhibitor affinity detected using KiNativ is better than that achieved when affinities are determined by assays using heterologously expressed protein kinases (7). This improved correlation illustrates that assays in the native environment provide a more realistic measure of protein kinase function.In addition to characterizing inhibitors selectively, AcATP probes can also display differential ATP binding activities of protein kinases. For example, labeling with AcATP probes during infection with dengue virus displayed a 2- to 8-fold activation of a DNA-dependent protein kinase (14) Similarly, AcATP labeling revealed an unexpected Raf kinase activation in extracts upon protein kinase inhibitor treatment (7). In conclusion, profiling with AcATP probes is a powerful approach for monitoring protein kinases and offers unprecedented opportunities to identify selective protein kinase inhibitors and discover protein kinases with differential ATP binding activities.In this work, we introduce AcATP profiling of plant proteomes. In addition to the analysis of labeled peptides, we characterized labeling using gel-based approaches and discovered that biotin is often oxidized in this procedure. We also performed an in-depth analysis of labeling sites in proteins other than protein kinases, which had not been done before. We discuss labeling outside known nucleotide binding pockets and investigate the correlation of labeling sites with protein abundance. We describe 63 labeling sites of known nucleotide binding pockets, of which 24 represent a remarkable diversity of protein kinases, including several LRR-RLKs. This work launches a new approach to study ATP binding proteins in plant science.     

Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

Background

A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive.

Findings

Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca²⁺ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca²⁺ in a concentration-dependent manner. Conversely, increasing Ca²⁺ levels inhibits kinase activity up to 500-fold at 100 nM Ca²⁺.

Conclusions

Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

     

Tyrosine phosphorylation in brassinosteroid signaling

Brassinosteroids (BRs) regulate plant growth and development through a complex signal transduction pathway involving BRASSINOSTEROID INSENSITIVE 1 (BRI1), which is the BR receptor, and its co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1). Both proteins are classified as Ser/Thr protein kinases. Recently, we reported that recombinant cytoplasmic domains (CD) of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases.¹ Two sites of Tyr autophosphorylation were identified that appear to have different effects on BRI1 function. Tyr-831 in the juxtamembrane domain is not essential for kinase activity but has a regulatory role, with phosphorylation of Tyr-831 causing inhibition of growth and delay of flowering. In contrast, Tyr-956 is located in subdomain IV of the kinase domain and is essential for kinase activity, and we are speculating that the free hydroxyl group at this position is essential and thus phosphorylation of Tyr-956 would inhibit BRI1 kinase activity. Expression of BRI1(Y831F)-Flag in the weak allele bri1-5 rescued the dwarf phenotype but plants had rounder leaves, increased shoot biomass, and flowered earlier than plants expressing the BRI1(wild type)-Flag in the bri1-5 background. To further elaborate on earlier results, we present additional phenotypic analysis of transgenic Arabidopsis plants expressing BRI1(Y831F)-Flag or site-directed mutants of other Tyr residues within the kinase domain. The results highlight the unique role of Tyr-831 in regulation of BR signaling in vivo. Elucidating the molecular basis for increased biomass accumulation in plants expressing BRI1(Y831F)-Flag may have applications for agriculture.Key words: brassinosteroids, LRR-RLK, autophosphorylation, tyrosine phosphorylation, signal transduction     

Rat liver endoplasmic reticulum protein kinases

• 1.1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography.
• 2.2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase and casein kinases.
• 3.3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis.
• 4.4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca²⁺ /calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).

     

Phospholipases in action during plant defense signaling

Eukaryotic organisms rely on intricate signaling networks to connect recognition of microbes with the activation of efficient defense reactions. Accumulating evidence indicates that phospholipids are more than mere structural components of biological membranes. Indeed, phospholipid-based signal transduction is widely used in plant cells to relay perception of extracellular signals. Upon perception of the invading microbe, several phospholipid hydrolyzing enzymes are activated that contribute to the establishment of an appropriate defense response. Activation of phospholipases is at the origin of the production of important defense signaling molecules, such as oxylipins and jasmonates, as well as the potent second messenger phosphatidic acid (PA), which has been shown to modulate the activity of a variety of proteins involved in defense signaling. Here, we provide an overview of recent reports describing the different plant phospholipase pathways that are activated during the establishment of plant defense reactions in response to pathogen attack.Key words: lipid signaling, PA, PLA, PLC, PLD, plant immunityIn plant cells, perception of pathogenic microbes largely relies on transmembrane pattern recognition receptors that specifically recognize highly conserved pathogen-derived molecules called PAMPs/MAMPs (pathogen-/microbial-associated molecular patterns), such as bacterial flagellin.¹ PAMP recognition by the plant leads to basal defense responses. A second layer of defense is based on the recognition of specific pathogen-derived molecules, called effectors, primarily by an additional class of plant cytoplasmic receptor proteins [nucleotide-binding leucine-rich repeat (NB-LRR) proteins] but also by protein receptors predicted to be located at the plasma membrane [receptor-like proteins (RLPs) and receptor-like kinases (RLKs)]. This recognition leads to the activation of plant immune responses that are frequently associated to the development of hypersensitive cell death (HR) at the inoculation site, which has been shown to contribute to plant resistance.²The activation of plant immunity involves a variety of early signaling events, including rapid accumulation of reactive oxygen species (ROS), changes in cellular ion fluxes, activation of protein kinase cascades, changes in gene expression and production of stress-related hormones.^3,4 During recent years, a substantial number of reports have also shown the importance of lipids and lipid-related molecules, including glycerolipids, sphingolipids, fatty acids, oxylipins, jasmonates and sterols, in the regulation of plant defense responses.⁵Phospholipids are more than structural components in biological membranes. Indeed, evidence that phospholipases and phospholipid-derived molecules are involved in plant signaling, and more particularly in plant immunity, is rapidly accumulating.^6,7 In plants, phosphatidic acid (PA) can be produced from phospholipids by phospholipase D (PLD) enzymes or from diacylyglycerol (DAG) by DAG kinases (DGKs) in the phospholipase C (PLC) pathway. PA is a potent secondary signal messenger molecule that modulates the activity of kinases, phosphatases, phospholipases and proteins involved in membrane-trafficking, Ca²⁺ signaling and the oxidative burst.^8,9 In addition, a growing body of evidence indicates that phospholipase A (PLA) [and related molecules such as lysophospholipids (LPLs) and free fatty acids (FFAs)] and phospholipase C (PLC) (and its related molecules DAG and DGK) play important roles in the control of the plant defense response to the attack by invading pathogens.⁷Here, we review the recent advances in understanding phospholipase-mediated signaling and its importance in the control of plant immune responses.