Adi3 is a Pdk1-interacting AGC kinase that negatively regulates plant cell death

Bacterial speck disease in tomato is caused by Pseudomonas syringae pv. tomato. Resistance to this disease is conferred by the host Pto kinase, which recognizes P. s. pv. tomato strains that express the effector AvrPto. We report here that an AvrPto-dependent Pto-interacting protein 3 (Adi3) is a member of the AGC family of protein kinases. In mammals, AGC kinases are regulated by 3-phosphoinositide-dependent protein kinase-1 (Pdk1). We characterized tomato Pdk1 and showed that Pdk1 and Pto phosphorylate Adi3. Gene silencing of Adi3 in tomato causes MAPKKKalpha-dependent formation of necrotic lesions. Use of a chemical inhibitor of Pdk1, OSU-03012, also implicates Pdk1 and Adi3 in plant cell death regulation. Adi3 thus appears to function analogously to the mammalian AGC kinase protein kinase B/Akt by negatively regulating cell death via Pdk1 phosphorylation. We speculate that the negative regulatory function of Adi3 might be subverted by interaction with Pto/AvrPto, leading to host cell death that is associated with pathogen attack.     

Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation

The tomato AGC kinase Adi3 is phosphorylated by Pdk1 for activation of its cell death suppression activity. The Pdk1 phosphorylation site for activation of Adi3 is at Ser539. However, there is at least one additional Pdk1 phosphorylation site on Adi3 that has an unknown function. Here we identify an Arabidopsis thaliana sequence homologue of Adi3 termed AGC1-3. Two Pdk1 phosphorylation sites were identified on AGC1-3, activation site Ser596 and Ser269, and by homology Ser212 on Adi3 was identified as a second Pdk1 phosphorylation site. While Ser212 is not required for Adi3 autophosphorylation, Ser212 was shown to be required for full phosphorylation of the Adi3 substrate Gal83.     

A patch of surface-exposed residues mediates negative regulation of immune signaling by tomato Pto kinase

Tomato (Lycopersicon esculentum) Pto kinase specifically recognizes the Pseudomonas effector proteins AvrPto and AvrPtoB, leading to induction of defense responses and hypersensitive cell death. Structural modeling of Pto combined with site-directed mutagenesis identified a patch of surface-exposed residues required for native regulation of signaling. Mutations in this area resulted in constitutive gain-of-function (CGF) forms of Pto that activated AvrPto-independent cell death via the cognate signaling pathway. The patch overlaps the peptide binding region of the kinase catalytic cleft and is part of a broader region required for interaction with bacterial effectors. We propose that the negative regulatory patch is normally occupied by a peptide that represses Pto signaling. Furthermore, we found that Pto kinase activity was required for Avr-dependent activation but dispensable for signaling by CGF forms of Pto. This suggests that Pto signals by a conformational change rather than phosphorylation of downstream substrates in the defense signaling pathway.     

The N-Terminal Domain of the Tomato Immune Protein Prf Contains Multiple Homotypic and Pto Kinase Interaction Sites

Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).     

Ribosomal Protein S3, a New Substrate of Akt,Serves as a Signal Mediator between Neuronal Apoptosis and DNA Repair

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.     

Crystal Structure of the Complex between Pseudomonas Effector AvrPtoB and the Tomato Pto Kinase Reveals Both a Shared and a Unique Interface Compared with AvrPto-Pto

Resistance to bacterial speck disease in tomato (Solanum lycopersicum) is activated upon recognition by the host Pto kinase of either one of two sequence-unrelated effector proteins, AvrPto or AvrPtoB, from Pseudomonas syringae pv tomato (Pst). Pto induces Pst immunity by acting in concert with the Prf protein. The recently reported structure of the AvrPto-Pto complex revealed that interaction of AvrPto with Pto appears to relieve an inhibitory effect of Pto, allowing Pto to activate Prf. Here, we present the crystal structure of the Pto binding domain of AvrPtoB (residues 121 to 205) at a resolution of 1.9Å and of the AvrPtoB_121-205–Pto complex at a resolution of 3.3 Å. AvrPtoB_121-205 exhibits a tertiary fold that is completely different from that of AvrPto, and its conformation remains largely unchanged upon binding to Pto. In common with AvrPto-Pto, the AvrPtoB-Pto complex relies on two interfaces. One of these interfaces is similar in both complexes, although the primary amino acid sequences from the two effector proteins are very different. Amino acid substitutions in Pto at the other interface disrupt the interaction of AvrPtoB-Pto but not that of AvrPto-Pto. Interestingly, substitutions in Pto affecting this unique interface also cause Pto to induce Prf-dependent host cell death independently of either effector protein.     

The Pto kinase of tomato, which regulates plant immunity, is repressed by its myristoylated N terminus

Specific recognition of the Pseudomonas syringae effector proteins AvrPto and AvrPtoB in tomato is mediated by Pto kinase resulting in induction of defense responses, including hypersensitive cell death via a signaling pathway requiring the nucleotide-binding leucine-rich repeats protein Prf. Pto is a myristoylated protein, and N-myristoylation is required for signaling. Here we demonstrated a role for N-myristoylation in controlling Pto kinase activity. A myristoylated peptide corresponding to Pto residues 2-10 significantly impaired the kinase activity of N-truncated Pto. We show that kinase inhibition was specific to the myristoylated form of the peptide and that free myristate supplied in trans was a potent suppressor of Pto kinase activity. Thus, myristate, but not Pto residues 2-10, contributes to suppression of kinase activity in vitro. Accordingly, elimination of the in vivo myristoylation potential of Pto de-repressed kinase activity. The increased potency of free myristate relative to the myristoylated N-peptide inhibitor suggested that the peptide moiety is antagonistic to repression by myristate. Suppression of related protein kinases by myristate declined with similarity to Pto, and the inhibitory activity could be attributed to hydrophobicity. We present evidence that inhibition of Pto by the myristoylated N-peptide is mediated through a previously identified surface regulatory patch. The data show a role for negative regulation of Pto by N-myristoylation, in addition to the previously demonstrated positive role, and are consistent with a model in which the acylated N terminus is sequestered in the catalytic cleft prior to release by Pto activation.     

GIT1 Phosphorylation on Serine 46 by PKD3 Regulates Paxillin Trafficking and Cellular Protrusive Activity

The continuous assembly and disassembly of focal adhesions is required for efficient cell spreading and migration. The G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein whose dynamic localization to sites of cytoskeletal remodeling is critically involved in the regulation of these processes. Here we provide evidence that the subcellular localization of GIT1 is regulated by protein kinase D3 (PKD3) through direct phosphorylation on serine 46. GIT1 phosphorylation on serine 46 was abrograted by PKD3 depletion, thereby identifying GIT1 as the first specific substrate for this kinase. A GIT1 S46D phosphomimetic mutant localized to motile, paxillin-positive cytoplasmic complexes, whereas the phosphorylation-deficient GIT1 S46A was enriched in focal adhesions. We propose that phosphorylation of GIT1 on serine 46 by PKD3 represents a molecular switch by which GIT1 localization, paxillin trafficking, and cellular protrusive activity are regulated.     

Two distinct Pseudomonas effector proteins interact with the Pto kinase and activate plant immunity

The Pto serine/threonine kinase of tomato confers resistance to speck disease by recognizing strains of Pseudomonas syringae that express the protein AvrPto. Pto and AvrPto physically interact, and this interaction is required for activation of host resistance. We identified a second Pseudomonas protein, AvrPtoB, that interacts specifically with Pto and is widely distributed among plant pathogens. AvrPtoB is delivered into the plant cell by the bacterial type III secretion system, and it elicits Pto-specific defenses. AvrPtoB has little overall sequence similarity with AvrPto. However, AvrPto amino acids, which are required for interaction with Pto, are present in AvrPtoB and required for its interaction with Pto. Thus, two distinct bacterial effectors activate plant immunity by interacting with the same host protein kinase through a similar structural mechanism.     

The β-subunit of the SnRK1 complex is phosphorylated by the plant cell death suppressor Adi3

The protein kinase AvrPto-dependent Pto-interacting protein3 (Adi3) is a known suppressor of cell death, and loss of its function has been correlated with cell death induction during the tomato (Solanum lycopersicum) resistance response to its pathogen Pseudomonas syringae pv tomato. However, Adi3 downstream interactors that may play a role in cell death regulation have not been identified. We used a yeast two-hybrid screen to identify the plant SnRK1 (for Sucrose non-Fermenting-1-Related Protein Kinase1) protein as an Adi3-interacting protein. SnRK1 functions as a regulator of carbon metabolism and responses to biotic and abiotic stresses. SnRK1 exists in a heterotrimeric complex with a catalytic α-subunit (SnRK1), a substrate-interacting β-subunit, and a regulatory γ-subunit. Here, we show that Adi3 interacts with, but does not phosphorylate, the SnRK1 α-subunit. The ability of Adi3 to phosphorylate the four identified tomato β-subunits was also examined, and it was found that only the Galactose Metabolism83 (Gal83) β-subunit was phosphorylated by Adi3. This phosphorylation site on Gal83 was identified as serine-26 using a mutational approach and mass spectrometry. In vivo expression of Gal83 indicates that it contains multiple phosphorylation sites, one of which is serine-26. An active SnRK1 complex containing Gal83 as the β-subunit and sucrose nonfermenting4 as the γ-subunit was constructed to examine functional aspects of the Adi3 interaction with SnRK1 and Gal83. These assays revealed that Adi3 is capable of suppressing the kinase activity of the SnRK1 complex through Gal83 phosphorylation plus the interaction with SnRK1 and suggested that this function may be related to the cell death suppression activity of Adi3.     

Tomato Pto encodes a functional N-myristoylation motif that is required for signal transduction in Nicotiana benthamiana

Pto kinase of tomato (Lycopersicon esculentum) confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato expressing avrPto or avrPtoB. Pto interacts directly with these type-III secreted effectors, leading to induction of defence responses including the hypersensitive response (HR). Signalling by Pto requires the nucleotide-binding site-leucine-rich repeat (NBS-LRR) protein Prf. Little is known of how Pto is controlled prior to or during stimulation, although kinase activity is required for Avr-dependent activation. Here we demonstrate a role for the N-terminus in signalling by Pto. N-terminal residues outside the kinase domain were required for induction of the HR in Nicotiana benthamiana. The N-terminus also contributed to both AvrPto-binding and phosphorylation abilities. Pto residues 1-10 comprise a consensus motif for covalent attachment of myristate, a hydrophobic 14-carbon saturated fatty acid, to the Gly-2 residue. Several lines of evidence indicate that this motif is important for Pto function. A heterologous N-myristoylation motif complemented N-terminal deletion mutants of Pto for Prf-dependent signalling. Signalling by wild-type and mutant forms of Pto was strictly dependent on the Gly-2 residue. The N-myristoylation motif of Pto complemented the cognate motif of AvrPto for avirulence function and membrane association. Furthermore, Pto was myristoylated in vivo dependent on the presence of Gly-2. The subcellular localization of Pto was independent of N-myristoylation, indicating that N-myristoylation is required for some function other than membrane affinity. Consistent with this idea, AvrPtoB was also found to be a soluble protein. The data indicate an important role(s) for the myristoylated N-terminus in Pto signalling.     

Plants expressing the Pto disease resistance gene confer resistance to recombinant PVX containing the avirulence gene AvrPto

Elicitation of hypersensitive cell death and induction of plant disease resistance by Pseudomonas syringae pv. tomato (Pst) is dependent on activity of the Pst Hrp secretion system and the gene-for-gene interaction between the tomato resistance gene Pto and the bacterial avirulence gene avrPto. AvrPto was expressed transiently in resistant or susceptible plant lines via a potato virus X (PVX) vector. We found that while PVX is normally virulent on tomato, a PVX derivative expressing avrPto was only capable of infecting plants lacking a functional Pto resistance pathway. Mutations in either the Pto or Prf genes allowed systemic spread of the recombinant virus. These results indicate that recognition of AvrPto by Pto in resistant plant lines triggers a plant defense response that can confer resistance to a viral as well as a bacterial pathogen.     

Pdk1 activity controls proliferation, survival, and growth of developing pancreatic cells

The formation of adequate masses of endocrine and exocrine pancreatic tissues during embryogenesis is essential to ensure proper nutrition and glucose homeostasis at postnatal stages. We generated mice with pancreas-specific ablation of the 3-phosphoinositide-dependent protein kinase 1 (Pdk1) to investigate how signaling downstream of the phosphatidylinositol-3-OH kinase (PI3K) pathway controls pancreas development. Pdk1-conditional knock-out mice were born with conspicuous pancreas hypoplasia, and within a few weeks, they developed severe hyperglycemia. Our detailed characterization of the mutant embryonic pancreas also revealed distinct temporal, cell type-specific requirements of Pdk1 activity in the control of cell proliferation, cell survival, and cell size during pancreas development. These results thus uncover Pdk1 as a novel, crucial regulator of pancreatic growth during embryogenesis. In addition, we provide evidence that Pdk1 activity is required differently in mature pancreatic cell types, since compensatory proliferation and possible mTORC2 activation occurred in exocrine cells but not in β cells of the Pdk1-deficient postnatal pancreas.     

Proteolysis of a Negative Regulator of Innate Immunity Is Dependent on Resistance Genes in Tomato and Nicotiana benthamiana and Induced by Multiple Bacterial Effectors

RPM1-interacting protein 4 (RIN4), a negative regulator of the basal defense response in plants, is targeted by multiple bacterial virulence effectors. We show that RIN4 degradation is induced by the effector AvrPto from Pseudomonas syringae and that this degradation in Solanaceous plants is dependent on the resistance protein, Pto, a protein kinase, and Prf, a nucleotide binding site–leucine-rich repeat protein. Our data demonstrate overlap between two of the best-characterized pathways for recognition of pathogen virulence effectors in plants. RIN4 interacts with multiple plant signaling components and bacterial effectors in yeast and in planta. AvrPto induces an endogenous proteolytic activity in both tomato (Solanum lycopersicum) and Nicotiana benthamiana that degrades RIN4 and requires the consensus site cleaved by the protease effector AvrRpt2. The interaction between AvrPto and Pto, but not the kinase activity of Pto, is required for proteolysis of RIN4. Analysis of many of the effectors comprising the secretome of P. syringae pv tomato DC3000 led to the identification of two additional sequence-unrelated effectors that can also induce degradation of RIN4. Therefore, multiple bacterial effectors besides AvrRpt2 elicit proteolysis of RIN4 in planta.     

Host-mediated phosphorylation of type III effector AvrPto promotes Pseudomonas virulence and avirulence in tomato

The AvrPto protein from Pseudomonas syringae pv tomato is delivered into plant cells by the bacterial type III secretion system, where it either promotes host susceptibility or, in tomato plants expressing the Pto kinase, elicits disease resistance. Using two-dimensional gel electrophoresis, we obtained evidence that AvrPto is phosphorylated when expressed in plant leaves. In vitro phosphorylation of AvrPto by plant extracts occurs independently of Pto and is due to a kinase activity that is conserved in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), and Arabidopsis thaliana. Three Ser residues clustered in the C-terminal 18 amino acids of AvrPto were identified in vitro as putative phosphorylation sites, and one site at S149 was directly confirmed as an in vivo phosphorylation site by mass spectrometry. Substitution of Ala for S149 significantly decreased the ability of AvrPto to enhance disease symptoms and promote growth of P. s. tomato in susceptible tomato leaves. In addition, S149A significantly decreased the avirulence activity of AvrPto in resistant tomato plants. Our observations support a model in which AvrPto has evolved to mimic a substrate of a highly conserved plant kinase to enhance its virulence activity. Furthermore, residues of AvrPto that promote virulence are also monitored by plant defenses.     

Constitutively active Pto induces a Prf-dependent hypersensitive response in the absence of avrPto.

Resistance in tomato to Pseudomonas syringae pv tomato (avrPto) is conferred by the gene Pto in a gene-for-gene relationship. A hypersensitive disease resistance response (HR) is elicited when Pto and avrPto are expressed experimentally within the same plant cell. The kinase capability of Pto was required for AvrPto-dependent HR induction. Systematic mutagenesis of the activation segment of Pto kinase confirmed the homologous P+1 loop as an AvrPto-binding determinant. Specific amino acid substitutions in this region led to constitutive induction of HR upon expression in the plant cell in the absence of AvrPto. Constitutively active Pto mutants required kinase capability for activity, and were unable to interact with proteins previously shown to bind to wild-type Pto. The constitutive gain-of-function phenotype was dependent on a functional Prf gene, demonstrating activation of the cognate disease resistance pathway and precluding a role for Prf upstream of Pto.     

The plant cell death suppressor Adi3 interacts with the autophagic protein Atg8h

The tomato AGC protein kinase Adi3 is known to function as a suppressor of PCD and silencing of Adi3 leads to spontaneous cell death on leaves and stems. In an effort to isolate Adi3 interacting proteins, a yeast two-hybrid screen was carried out and identified the autophagy protein Atg8h as an Adi3 interactor. This interaction occurred independent of the kinase activity status of Adi3. Silencing of genes involved in autophagy is known to eliminate the restriction of pathogen-induced PCD to a few cells and leads to run away PCD. Cosilencing Adi3 with several autophagy genes lead to the same run away cell death suggesting Adi3 may be involved in autophagic regulation of PCD.     

Evidence toward a Dual Phosphatase Mechanism That Restricts Aurora A (Thr-295) Phosphorylation during the Early Embryonic Cell Cycle

The mitotic kinase Aurora A (AurA) is regulated by a complex network of factors that includes co-activator binding, autophosphorylation, and dephosphorylation. Dephosphorylation of AurA by PP2A (human, Ser-51; Xenopus, Ser-53) destabilizes the protein, whereas mitotic dephosphorylation of its T-loop (human, Thr-288; Xenopus, Thr-295) by PP6 represses AurA activity. However, AurA(Thr-295) phosphorylation is restricted throughout the early embryonic cell cycle, not just during M-phase, and how Thr-295 is kept dephosphorylated during interphase and whether or not this mechanism impacts the cell cycle oscillator were unknown. Titration of okadaic acid (OA) or fostriecin into Xenopus early embryonic extract revealed that phosphatase activity other than PP1 continuously suppresses AurA(Thr-295) phosphorylation during the early embryonic cell cycle. Unexpectedly, we observed that inhibiting a phosphatase activity highly sensitive to OA caused an abnormal increase in AurA(Thr-295) phosphorylation late during interphase that corresponded with delayed cyclin-dependent kinase 1 (CDK1) activation. AurA(Thr-295) phosphorylation indeed influenced this timing, because AurA isoforms retaining an intact Thr-295 residue further delayed M-phase entry. Using mathematical modeling, we determined that one phosphatase would be insufficient to restrict AurA phosphorylation and regulate CDK1 activation, whereas a dual phosphatase topology best recapitulated our experimental observations. We propose that two phosphatases target Thr-295 of AurA to prevent premature AurA activation during interphase and that phosphorylated AurA(Thr-295) acts as a competitor substrate with a CDK1-activating phosphatase in late interphase. These results suggest a novel relationship between AurA and protein phosphatases during progression throughout the early embryonic cell cycle and shed new light on potential defects caused by AurA overexpression.