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.     

A putative myristoylated 2C-type protein phosphatase, PP2C74, interacts with SnRK1 in Arabidopsis

N-myristoylation is a lipid modification of many signaling proteins in which myristate is added to an N-terminal glycine residue. Here we show that PP2C74, a putative myristoylated 2C-type protein phosphatase (PP2C) in Arabidopsis, is transcribed in various tissues and has protein phosphatase activity. GFP-fused PP2C74 localized to the plasma membrane, but not when a glycine residue at position 2, which is the putative myristoylation site, was substituted with an alanine residue. Yeast two-hybrid analysis and GST pull-down analysis showed that PP2C74 interacts with AKIN10, the catalytic α subunit of the SnRK1 protein kinase complex, the β subunits of which are known targets of myristoylation.     

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.     

Myristoylation, phosphorylation, and subcellular distribution of the 80-kDa protein kinase C substrate in BC3H1 myocytes

Numerous reports have described a phosphoprotein with an apparent molecular mass of 68-87 kDa, often referred to as the 80K protein, which serves as a major specific substrate for protein kinase C in a wide variety of cell types. This protein has been shown to be myristoylated in macrophages, apparently in a stimulus-dependent manner. In the present study, we have defined the kinetics for myristoylation of the 80K protein in BC3H1 myocytes and have examined the subcellular distribution of the [3H]myristate and 32P-labeled forms of the protein before and after activation of protein kinase C by phorbol dibutyrate (PDBu). The 80K protein was identified in BC3H1 myocytes by apparent molecular mass of 68 kDa (consistent with the previously reported size of the murine homologue), isoelectric point of 4.6-4.8, PDBu-inducible phosphorylation, peptide mapping, and labeling with [3H]myristate. Incorporation of [3H]myristate by this protein occurred through an amide linkage and was abolished completely by cycloheximide. Pulse labeling of quiescent cells with [3H]myristate revealed no alteration in myristoylation of the 80K protein in either the crude membrane or soluble fractions after PDBu-induced phosphorylation. The subcellular distribution of this protein (approximately 80% membrane, approximately 20% cytosol) also was the same in control and PDBu-stimulated cells. Phosphorylation of both the membrane-bound and soluble forms was increased approximately 6-fold upon stimulation of cultures with PDBu; the soluble form was phosphorylated to a 4-fold higher stoichiometry than its membrane-bound counterpart. Together, these data demonstrate that the 80K protein is myristoylated cotranslationally in BC3H1 cells and that protein kinase C-dependent phosphorylation of the 80K protein does not alter its subcellular distribution or degree of myristoylation. The fact that 20% of total myristoylated 80K protein resides in the cytosol also indicates that myristoylation alone is not sufficient to target this protein to the plasma membrane.     

N-myristoylation of a peptide substrate for Src converts it into an apparent slow-binding bisubstrate-type inhibitor.

The conversion of a peptide substrate to a potent inhibitor by chemical modification is a promising approach in the development of inhibitors for protein tyrosine kinases. N-acylation of the synthetic peptide substrate NH2-Glu-Phe-Leu-Tyr-Gly-Val-Phe-Asp-CONH2 (EFLYGVFD) resulted in synergistic inhibition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was dependent upon the peptide sequence and the length of the acyl chain. The minimum length of the fatty acyl chain to synergistically inhibit Src was a lauryl (C11H23CO) group. N-myristoylated EFLYGVFD (myr-EFLYGVFD) inhibited the phosphorylation of poly E4Y by Src with an apparent Ki of 3 microm, whereas EFLYGVFD and myristic acid inhibited with Ki values of 260 and 35 microm, respectively. The nonacylated EFLYGVFD was a substrate for Src with Km and Vmax values of 100 microm and 400 nmol/min/mg protein, respectively. However, upon myristoylation, the peptide was no longer a substrate for Src. Both the acylated and non-acylated peptides were competitive inhibitors against the substrate poly E4Y. The non-acylated free peptide showed mixed inhibition against ATP while the myristoylated peptide was competitive against ATP. Myristic acid was uncompetitive against poly E4Y and competitive against ATP. Further analysis indicated that the myristoylated peptide acted as a reversible slow-binding inhibitor with two binding sites on Src. The myristoylated 8-mer peptide was reduced in size to a myristoylated 3-mer without losing the affinity or characteristics of a bisubstrate-type inhibitor. The conversion of a classical reversible inhibitor to a reversible slow-binding multisubstrate analogue has improved the potency of inhibition by the peptide.     

The N-terminal region of Pseudomonas type III effector AvrPtoB elicits Pto-dependent immunity and has two distinct virulence determinants

Resistance to bacterial speck disease in tomato is activated by the physical interaction of the host Pto kinase with either of the sequence-dissimilar type III effector proteins AvrPto or AvrPtoB (HopAB2) from Pseudomonas syringae pv. tomato. Pto-mediated immunity requires Prf, a protein with a nucleotide-binding site and leucine-rich repeats. The N-terminal 307 amino acids of AvrPtoB were previously reported to interact with the Pto kinase, and we show here that this region (AvrPtoB(1-307)) is sufficient for eliciting Pto/Prf-dependent immunity against P. s. pv. tomato. AvrPtoB(1-307) was also found to be sufficient for a virulence activity that enhances ethylene production and increases growth of P. s. pv. tomato and severity of speck disease on susceptible tomato lines lacking either Pto or Prf. Moreover, we found that residues 308-387 of AvrPtoB are required for the previously reported ability of AvrPtoB to suppress pathogen-associated molecular patterns-induced basal defenses in Arabidopsis. Thus, the N-terminal region of AvrPtoB has two structurally distinct domains involved in different virulence-promoting mechanisms. Random and targeted mutagenesis identified five tightly clustered residues in AvrPtoB(1-307) that are required for interaction with Pto and for elicitation of immunity to P. s. pv. tomato. Mutation of one of the five clustered residues abolished the ethylene-associated virulence activity of AvrPtoB(1-307). However, individual mutations of the other four residues, despite abolishing interaction with Pto and avirulence activity, had no effect on AvrPtoB(1-307) virulence activity. None of these mutations affected the basal defense-suppressing activity of AvrPtoB(1-387). Based on sequence alignments, estimates of helical propensity, and the previously reported structure of AvrPto, we hypothesize that the Pto-interacting domains of AvrPto and AvrPtoB(1-307) have structural similarity. Together, these data support a model in which AvrPtoB(1-307) promotes ethylene-associated virulence by interaction not with Pto but with another unknown host protein.     

The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity

Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch.     

Stimulation of cellular signaling and G protein subunit dissociation by G protein betagamma subunit-binding peptides

We previously developed peptides that bind to G protein betagamma subunits and selectively block interactions between betagamma subunits and a subset of effectors in vitro (Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, P., Gross, R. A., Taussig, R., and Smrcka, A. V. (2001) EMBO J. 20, 767-776). Here, we created cell-permeating versions of some of these peptides by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated betagamma-binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells caused rapid activation of extracellular signal-regulated kinase 1/2 in the absence of an agonist. This activation did not occur if the peptide lacked a myristate at the N terminus, if the peptide had a single point mutation to eliminate betagamma subunit binding, or if the cells stably expressed the C terminus of betaARK1. A human immunodeficiency virus TAT-modified peptide (TAT-SIRK) and a myristoylated version of a second peptide (mSCAR) that binds to the same site on betagamma subunits as mSIRK, also caused extracellular signal-regulated kinase activation. mSIRK also stimulated Jun N-terminal kinase phosphorylation, p38 mitogen-activated protein kinase phosphorylation, and phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified G protein subunits in vitro, SIRK promoted alpha subunit dissociation from betagamma subunits without stimulating nucleotide exchange. These data suggest a novel mechanism by which selective betagamma-binding peptides can release G protein betagamma subunits from heterotrimers to stimulate G protein pathways in cells.     

A myristoyl/phosphoserine switch controls cAMP-dependent protein kinase association to membranes

The cAMP-dependent protein kinase [protein kinase A (PKA)] mediates a myriad of cellular signaling events, and its activity is tightly regulated in both space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that, in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted byeither interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of the myristoylated C-subunit of PKA (PKA-C) steers the enzyme toward lipids independent of its regulatory subunit or an A-kinase anchoring protein, providing an additional mechanism to localize the enzyme near membrane-bound substrates.     

Phosphorylation of human N-myristoyltransferase by N-myristoylated SRC family tyrosine kinase members

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the amino terminal glycine residue of a number of important proteins especially the non-receptor tyrosine kinases whose activity is important for tumorigenesis. Human NMT was found to be phosphorylated by non-receptor tyrosine kinase family members of Lyn, Fyn and Lck and dephosphorylated by the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin. Deletion of 149 amino acids from the N-terminal end resulted in the absence of phosphorylation suggesting that the phosphorylation sites are located in the N-terminal end of NMT. Furthermore, a site-directed mutagenesis study indicated that substitution of tyrosine 100 with phenylalanine served NMT as a poor substrate for the Lyn kinase. A synthetic peptide corresponding to the amino-terminal region encompassing tyrosine 100 of NMT served as a good substrate for the Lyn and Fyn kinases. Our studies also indicated that NMT was found to interact with Lyn through its N-terminal end in a phosphorylation-dependent manner. This is the first study demonstrating the cross-talk between NMT and their myristoylated protein substrates in signaling pathways.     

Influence of the effector peptide of MARCKS-related protein on actin polymerization: a kinetic analysis

The members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with membranes, protein kinase C, and calmodulin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. This domain is also involved in the interaction between MARCKS/MRP and actin. In this article we show that a peptide corresponding to the effector domain of MRP, the effector peptide, strongly influences the dynamics of actin polymerization. Depending on the stoichiometric ratio of effector peptide to actin the peptide either accelerates or retards the actin polymerization process, which takes place in the presence of near-physiological salt concentrations. A model is developed in which this phenomenon is explained by two independent nucleation processes involving free actin monomers and peptide-bound actin monomers, respectively. As a control, a possible regulatory mechanism has been investigated: we show that calmodulin inhibits the actin polymerizing activity of the MRP effector peptide, thereby validating our model approach.     

Differential effects of acyl-CoA binding protein on enzymatic and non-enzymatic thioacylation of protein and peptide substrates

Both enzymatic and autocatalytic mechanisms have been proposed to account for protein thioacylation (commonly known as palmitoylation). Acyl-CoA binding proteins (ACBP) strongly suppress non-enzymatic thioacylation of cysteinyl-containing peptides by long-chain acyl-CoAs. At physiological concentrations of ACBP, acyl-CoAs, and membrane lipids, the rate of spontaneous acylation is expected to be too slow to contribute significantly to thioacylation of signaling proteins in mammalian cells (Leventis et al., Biochemistry 36 (1997) 5546-5553). Here we characterized the effects of ACBP on enzymatic thioacylation. A protein S-acyltransferase activity previously characterized using G-protein alpha-subunits as a substrate (Dunphy et al., J. Biol. Chem., 271 (1996) 7154-7159), was capable of thioacylating short lipid-modified cysteinyl-containing peptides. The minimum requirements for substrate recognition were a free cysteine thiol adjacent to a hydrophobic lipid anchor, either myristate or farnesyl isoprenoid. PAT activity displayed specificity for the acyl donor, efficiently utilizing long-chain acyl-CoAs, but not free fatty acid or S-palmitoyl-N-acetylcysteamine. ACBP only modestly inhibited enzymatic thioacylation of a myristoylated peptide or G-protein alpha-subunits under conditions where non-enzymatic thioacylation was reduced to background. Thus, protein S-acyltransferase remains active in the presence of physiological concentrations of ACBP and acyl-CoA in vitro and is likely to represent the predominant mechanism of thioacylation in vivo.     

Role of two conserved cytoplasmic threonine residues (T410 and T412) in CD5 signaling

CD5 is a transmembrane coreceptor that modulates activation and differentiation signals mediated by the Ag-specific receptor present on both T and B1a lymphocytes. CD5 lacks intrinsic catalytic activity, and its immunomodulatory properties result from intracellular interactions mediated by the CD5 cytoplasmic tail. The nature of these interactions is currently a matter of investigation. Here, we present a selective mutagenesis analysis of two conserved threonine residues (T410 and T412) located at the membrane-proximal cytoplasmic region of CD5. These residues are contained within consensus phosphorylation motifs for protein kinase C and are shown here to be critical for in vivo protein kinase C-mediated phosphorylation of CD5. Functional studies revealed that the integrity of T410 and T412 is also critical for CD5-mediated phosphatidylcholine-specific phospholipase C (PC-PLC) activation and phorbol ester-mediated inhibition of Ab-induced internalization of CD5. These results strongly argue in favor of a role for T410 and T412 in the signaling mediated by CD5.     

Thrombospondin-1 inhibits nitric oxide signaling via CD36 by inhibiting myristic acid uptake

Although CD36 is generally recognized to be an inhibitory signaling receptor for thrombospondin-1 (TSP1), the molecular mechanism for transduction of this signal remains unclear. Based on evidence that myristic acid and TSP1 each modulate endothelial cell nitric oxide signaling in a CD36-dependent manner, we examined the ability of TSP1 to modulate the fatty acid translocase activity of CD36. TSP1 and a CD36 antibody that mimics the activity of TSP1 inhibited myristate uptake. Recombinant TSP1 type 1 repeats were weakly inhibitory, but an anti-angiogenic peptide derived from this domain potently inhibited myristate uptake. This peptide also inhibited membrane translocation of the myristoylated CD36 signaling target Fyn and activation of Src family kinases. Myristate uptake stimulated cGMP synthesis via endothelial nitric-oxide synthase and soluble guanylyl cyclase. CD36 ligands blocked myristate-stimulated cGMP accumulation in proportion to their ability to inhibit myristate uptake. TSP1 also inhibited myristate-stimulated cGMP synthesis by engaging its receptor CD47. Myristate stimulated endothelial and vascular smooth muscle cell adhesion on type I collagen via the NO/cGMP pathway, and CD36 ligands that inhibit myristate uptake blocked this response. Therefore, the fatty acid translocase activity of CD36 elicits proangiogenic signaling in vascular cells, and TSP1 inhibits this response by simultaneously inhibiting fatty acid uptake via CD36 and downstream cGMP signaling via CD47.     

Mammalian reoviruses contain a myristoylated structural protein.

The structural protein mu 1 of mammalian reoviruses was noted to have a potential N-myristoylation sequence at the amino terminus of its deduced amino acid sequence. Virions labeled with [3H]myristic acid were used to demonstrate that mu 1 is modified by an amide-linked myristoyl group. A myristoylated peptide having a relative molecular weight (Mr) of approximately 4,000 was also shown to be a structural component of virions and was concluded to represent the 4.2-kDa amino-terminal fragment of mu 1 which is generated by the same proteolytic cleavage that yields the carboxy-terminal fragment and major outer capsid protein mu 1C. The myristoylated 4,000-Mr peptide was found to be present in reovirus intermediate subviral particles but to be absent from cores, indicating that it is a component of the outer capsid. A distinct large myristoylated fragment of the intact mu 1 protein was also identified in intermediate subviral particles, but no myristoylated mu-region proteins were identified in cores, consistent with the location of mu 1 in the outer capsid. Similarities between amino-terminal regions of the reovirus mu 1 protein and the poliovirus capsid polyprotein were noted. By analogy with other viruses that contain N-myristoylated structural proteins (particularly picornaviruses), we suggest that the myristoyl group attached to mu 1 and its amino-terminal fragments has an essential role in the assembly and structure of the reovirus outer capsid and in the process of reovirus entry into cells.     

Genetic and molecular requirements for function of the Pto/Prf effector recognition complex in tomato and Nicotiana benthamiana

The Pto gene of tomato (Solanum lycopersicum) confers specific recognition of the unrelated bacterial effector proteins AvrPto and AvrPtoB. Pto resides in a constitutive molecular complex with the nucleotide binding site-leucine rich repeats protein Prf. Prf is absolutely required for specific recognition of both effectors. Here, using stable transgenic lines, we show that expression of Pto from its genomic promoter in susceptible tomatoes was sufficient to complement recognition of Pseudomonas syringae pv. tomato (Pst) bacteria expressing either avrPto or avrPtoB. Pto kinase activity was absolutely required for specific immunity. Expression of the Pto N-myristoylation mutant, pto(G2A), conferred recognition of Pst (avrPtoB), but not Pst (avrPto), although bacterial growth in these lines was intermediate between resistant and susceptible lines. Overexpression of pto(G2A) complemented recognition of avrPto. Transgenic tomato plants overexpressing wild-type Pto exhibited constitutive growth phenotypes, but these were absent in lines overexpressing pto(G2A). Therefore, Pto myristoylation is a quantitative factor for effector recognition in tomato, but is absolutely required for overexpression phenotypes. Native expression of Pto in the heterologous species Nicotiana benthamiana did not confer resistance to P. syringae pv. tabaci (Pta) expressing avrPto or avrPtoB, but recognition of both effectors was complemented by Prf co-expression. Thus, specific resistance conferred solely by Pto in N. benthamiana is an artefact of overexpression. Finally, pto(G2A) did not confer recognition of either avrPto or avrPtoB in N. benthamiana, regardless of the presence of Prf. Thus, co-expression of Prf in N. benthamiana complements many but not all aspects of normal Pto function.     

Signal recognition and transduction mediated by the tomato Pto kinase: a paradigm of innate immunity in plants

Plant disease resistance is the result of an innate host defense mechanism, which relies on the ability of the plant to recognize pathogen invasion and to efficiently mount defense responses. In tomato, resistance to the pathogen Pseudomonas syringae is mediated by the specific interaction between the plant serine/threonine kinase Pto and the bacterial protein AvrPto. This article reviews molecular and biochemical properties that confer to Pto the capability to function as an intracellular receptor and to activate a signaling cascade leading to the induction of defense responses.     

Interaction between actin and the effector peptide of MARCKS-related protein. Identification of functional amino acid segments

It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity.