Plant and animal pathogen recognition receptors signal through non-RD kinases

Plants and animals mediate early steps of the innate immune response through pathogen recognition receptors (PRRs). PRRs commonly associate with or contain members of a monophyletic group of kinases called the interleukin-1 receptor-associated kinase (IRAK) family that include Drosophila Pelle, human IRAKs, rice XA21 and Arabidopsis FLS2. In mammals, PRRs can also associate with members of the receptor-interacting protein (RIP) kinase family, distant relatives to the IRAK family. Some IRAK and RIP family kinases fall into a small functional class of kinases termed non-RD, many of which do not autophosphorylate the activation loop. We surveyed the yeast, fly, worm, human, Arabidopsis, and rice kinomes (3,723 kinases) and found that despite the small number of non-RD kinases in these genomes (9%-29%), 12 of 15 kinases known or predicted to function in PRR signaling fall into the non-RD class. These data indicate that kinases associated with PRRs can largely be predicted by the lack of a single conserved residue and reveal new potential plant PRR subfamilies.     

The Drosophila tumor necrosis factor receptor-associated factor-1 (DTRAF1) interacts with Pelle and regulates NFkappaB activity

A member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family was identified in Drosophila. DTRAF1 contains 7 zinc finger domains followed by a TRAF domain, similar to mammalian TRAFs and other members of the family identified in data bases from Caenorhabditis elegans, Arabidopsis, and Dictyostelium. Analysis of DTRAF1 binding to different members of the human TNF receptor family showed that this protein can interact through its TRAF domain with the p75 neurotrophin receptor and weakly with the lymphotoxin-beta receptor. DTRAF1 can also self-associate and binds to human TRAF1, TRAF2, and TRAF4. Interestingly, DTRAF1 interacts with human cIAP-1 and cIAP-2 but not with Drosophila DIAP-1 and -2. By itself, DTRAF1 did not induce significant NFkappaB activation when overexpressed in mammalian cells, although it specifically increased NFkappaB induction by TRAF6. In contrast, TRAF2-mediated NFkappaB induction was partially inhibited by DTRAF1. Mutants of DTRAF1 lacking the N-terminal region inhibited NFkappaB induction by either TRAF2 or TRAF6. DTRAF1 specifically associated with the regulatory N-terminal domain of Pelle, a Drosophila homolog of the human kinase interleukin-1 receptor-associated kinase (IRAK). Interestingly, though Pelle and DTRAF1 individually were unable to induce NFkappaB in a human cell line, co-expression of Pelle and DTRAF1 resulted in significant NFkappaB activity. Interactions of DTRAF1 with human TRAF-, TNF receptor-, and IAP-family proteins imply strong evolutionary conservation of TRAF protein structure and function throughout Metazoan evolution.     

Cutting edge: molecular structure of the IL-1R-associated kinase-4 death domain and its implications for TLR signaling

IL-1R-associated kinase (IRAK) 4 is an essential component of innate immunity. IRAK-4 deficiency in mice and humans results in severe impairment of IL-1 and TLR signaling. We have solved the crystal structure for the death domain of Mus musculus IRAK-4 to 1.7 A resolution. This is the first glimpse of the structural details of a mammalian IRAK family member. The crystal structure reveals a six-helical bundle with a prominent loop, which among IRAKs and Pelle, a Drosophila homologue, is unique to IRAK-4. This highly structured loop contained between helices two and three, comprises an 11-aa stretch. Although innate immune domain recognition is thought to be very similar between Drosophila and mammals, this structural component points to a drastic difference. This structure can be used as a framework for future mutation and deletion studies and potential drug design.     

Natural history and functional divergence of protein tyrosine kinases

Cellular signaling is important for many biological processes including growth, differentiation, adhesion, motility and apoptosis. The protein tyrosine kinase (PTK) supergene family is the key mediator in cellular signaling in metazoans, directly associated with a variety of human diseases. All PTKs contain a highly conserved catalytic kinase domain, in spite of variable multi-domain structures. Within each PTK gene family, members exhibit functional divergence in substrate-specificity or temporal/tissue-specific expression, although their primary function is conserved. After conducting phylogenetic analysis on major PTK gene families, we found that the expanding of each PTK family was likely caused by gene or genome duplication event(s) that occurred before the emergence of teleosts but after the vertebrate-amphioxus split. We further investigated the evolutionary pattern of functional divergence after gene duplication in those gene families. Our results show that site-specific shifted evolutionary rate (altered functional constraint) is a common pattern in PTK gene family evolution.     

Molecular evolution of the NF-κB signaling system

The mechanisms of innate immunity in vertebrates show certain overall resemblances to immune mechanisms of insects. Two hypotheses have been proposed to explain these resemblances. (1) According to the evolutionary continuity hypothesis, innate immune mechanisms evolved in the common ancestor of vertebrates and insects and have been conserved since that time. (2) In the independent-evolution hypothesis, the mechanisms of innate immunity in vertebrates evolved independently from invertebrate immune mechanisms. Phylogenetic analysis of five gene families (Pelle, Rel, IkappaB, Toll, and TRAF) whose members are involved in NF-kappaB signaling in vertebrates and insects were used to decide between these hypotheses. The phylogenies of the Rel and TRAF families strongly supported independent evolution of immune functions in vertebrates and invertebrates, and, except for a possible case in the Pelle family, orthologous molecules having immune functions in both vertebrates and invertebrates were not found. The results suggest that NF-kappaB represents an ancient, generalized signaling system that has been co-opted for immune system roles independently in vertebrate and insect lineages.     

Pellino 1 is required for interleukin-1 (IL-1)-mediated signaling through its interaction with the IL-1 receptor-associated kinase 4 (IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6 (TRAF6) complex

The signaling pathway downstream of the mammalian interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) is evolutionally conserved with that mediated by the Drosophila Toll protein. Toll initiates its signal through the adapter molecule Tube and the serine-threonine kinase Pelle. Pelle is highly homologous to members of the IL-1R-associated kinase (IRAK) family in mammals. Recently, a novel Pelle-interacting protein called Pellino was identified in Drosophila. We now report a mammalian counterpart of Pellino, termed Pellino 1, which is required for NF kappa B activation and IL-8 gene expression in response to IL-1, probably through its signal-dependent interaction with IRAK4, IRAK, and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The Pellino 1-IRAK-IRAK4-TRAF6 signaling complex is likely to be intermediate, located between the IL-1 receptor complex and the TAK1 complex in the IL-1 pathway.     

Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members

Interleukin-1 receptor-associated kinase (IRAK) was first described as a signal transducer for the proinflammatory cytokine interleukin-1 (IL-1) and was later implicated in signal transduction of other members of the Toll-like receptor (TLR)/IL-1 receptor (IL-1R) family. In the meantime, four different IRAK-like molecules have been identified: two active kinases, IRAK-1 and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. All IRAKs mediate activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) pathways. Although earlier observations suggested that IRAKs have redundant functions, this hypothesis is now challenged by knockout studies. Furthermore, recent data imply a role for IRAK-1 in tumor necrosis factor receptor (TNFR) superfamily-induced signaling pathways as well. The scope of this review is to highlight the specific role of different IRAKs and to discuss several mechanisms that contribute to their activation and regulation.     

Evolutionary history of the vertebrate mitogen activated protein kinases family

Background

The mitogen activated protein kinases (MAPK) family pathway is implicated in diverse cellular processes and pathways essential to most organisms. Its evolution is conserved throughout the eukaryotic kingdoms. However, the detailed evolutionary history of the vertebrate MAPK family is largely unclear.

Methodology/Principal Findings

The MAPK family members were collected from literatures or by searching the genomes of several vertebrates and invertebrates with the known MAPK sequences as queries. We found that vertebrates had significantly more MAPK family members than invertebrates, and the vertebrate MAPK family originated from 3 progenitors, suggesting that a burst of gene duplication events had occurred after the divergence of vertebrates from invertebrates. Conservation of evolutionary synteny was observed in the vertebrate MAPK subfamilies 4, 6, 7, and 11 to 14. Based on synteny and phylogenetic relationships, MAPK12 appeared to have arisen from a tandem duplication of MAPK11 and the MAPK13-MAPK14 gene unit was from a segmental duplication of the MAPK11-MAPK12 gene unit. Adaptive evolution analyses reveal that purifying selection drove the evolution of MAPK family, implying strong functional constraints of MAPK genes. Intriguingly, however, intron losses were specifically observed in the MAPK4 and MAPK7 genes, but not in their flanking genes, during the evolution from teleosts to amphibians and mammals. The specific occurrence of intron losses in the MAPK4 and MAPK7 subfamilies might be associated with adaptive evolution of the vertebrates by enhancing the gene expression level of both MAPK genes.

Conclusions/Significance

These results provide valuable insight into the evolutionary history of the vertebrate MAPK family.     

Immune complex-mediated cell activation from systemic lupus erythematosus and rheumatoid arthritis patients elaborate different requirements for IRAK1/4 kinase activity across human cell types

IL-1R-associated kinases (IRAKs) are important mediators of MyD88-dependent signaling by the TLR/IL-1R superfamily and facilitate inflammatory responses. IRAK4 and IRAK1 function as active kinases and as scaffolds for protein-protein interactions. We report that although IRAK1/4 kinase activity is essential for human plasmacytoid dendritic cell (pDC) activation, it is dispensable in B, T, dendritic, and monocytic cells, which is in contrast with an essential active kinase role in comparable mouse cell types. An IRAK1/4 kinase inhibitor abrogated TLR7/9-induced IFN-α responses in both mouse and human pDCs, but other human immune cell populations activated via TLR7/9 or IL-1R were refractory to IRAK4 kinase inhibition. Gene ablation experiments using small interfering RNA demonstrated an essential scaffolding role for IRAK1 and IRAK4 in MyD88-dependent signaling. Finally, we demonstrate that autoimmune patient (systemic lupus erythematosus and rheumatoid arthritis) serum activates both pDC and B cells, but IRAK1/4 kinase inhibition affects only the pDC response, underscoring the differential IRAK1/4 functional requirements in human immune cells. These data reveal important species differences and elaborate cell type requirements for IRAK1/4 kinase activity.     

Molecular evolution and functional divergence of the Ca(2+) sensor protein in store-operated Ca(2+) entry: stromal interaction molecule

Receptor-mediated Ca(2+) signaling in many non-excitable cells initially induces Ca(2+) release from intracellular Ca(2+) stores, followed by Ca(2+) influx across the plasma membrane. Recent findings have suggested that stromal interaction molecules (STIMs) function as the Ca(2+) sensor to detect changes of Ca(2+) content in the intracellular Ca(2+) stores. Human STIMs and invertebrate STIM share several functionally important protein domains, but diverge significantly in the C-terminus. To better understand the evolutionary significance of STIM activity, phylogenetic analysis of the STIM protein family was conducted after extensive database searching. Results from phylogeny and sequence analysis revealed early adaptation of the C-terminal divergent domains in Urochordata, before the expansion of STIMs in Vertebrata. STIMs were subsequently subjected to one round of gene duplication as early as in the Euteleostomi lineage in vertebrates, with a second round of fish-specific gene duplication. After duplication, STIM-1 and STIM-2 molecules appeared to have undergone purifying selection indicating strong evolutionary constraints within each group. Furthermore, sequence analysis of the EF-hand Ca(2+) binding domain and the SAM domain, together with functional divergence studies, identified critical regions/residues likely underlying functional changes, and provided evidence for the hypothesis that STIM-1 and STIM-2 might have developed distinct functional properties after duplication.     

Identification and characterization of murine IRAK-2

Interleukin-1 receptor-associated kinases (IRAKs) are pivotal signaling elements of the Toll/IL-1 receptor (TIL) family, which play a role in innate immune responses by coordinating host defence mechanisms. Presently four different forms of human IRAK molecules are cloned (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, and hu-IRAK-4). In the murine system, only three genes have been identified so far, mouse Pelle-Like Kinase (mPLK), which corresponds to human IRAK-1, mu-IRAK-M, and mu-IRAK-4. Here we report the molecular cloning and characterization of murine IRAK-2 (mu-IRAK-2), a mouse homolog to human IRAK-2 (hu-IRAK-2). Murine and human IRAK-2 molecules show 67% sequence identity, they are ubiquitiously expressed, and both practically lack autophoshorylation kinase activity. The murine molecule reveals two remarkable differences to its human counterpart: it shows a C-terminal extension and it has no stimulatory effect on IL-1 induced NF-kappa B activation when compared to hu-IRAK-2, suggesting subtle functional differences in signaling by IRAK-2 in human and mouse cells.     

IRAK1: a critical signaling mediator of innate immunity

The innate immune system is equipped with sensitive and efficient machineries to provide an immediate, first line defense against infections. Toll-like receptors (TLRs) detect pathogens and the IL-1 receptor (IL-1R) family enables cells to quickly respond to inflammatory cytokines by mounting an efficient protective response. Interleukin-1 receptor activated kinases (IRAKs) are key mediators in the signaling pathways of TLRs/IL-1Rs. By means of their kinase and adaptor functions, IRAKs initiate a cascade of signaling events eventually leading to induction of inflammatory target gene expression. Due to this pivotal role, IRAK function is also highly regulated via multiple mechanisms. In this review, we focus on IRAK1, the earliest known and yet the most interesting member of this family. An overview on its structure, function and biology is given, with emphasis on the different novel mechanisms that regulate IRAK1 function. We also highlight several unresolved questions in this field and evaluate the potential of IRAK1 as a target for therapeutic intervention.     

Evolutionary Origin of the Mitochondrial Cholesterol Transport Machinery Reveals a Universal Mechanism of Steroid Hormone Biosynthesis in Animals

Steroidogenesis begins with the transport of cholesterol from intracellular stores into mitochondria via a series of protein-protein interactions involving cytosolic and mitochondrial proteins located at both the outer and inner mitochondrial membranes. In adrenal glands and gonads, this process is accelerated by hormones, leading to the production of high levels of steroids that control tissue development and function. A hormone-induced multiprotein complex, the transduceosome, was recently identified, and is composed of cytosolic and outer mitochondrial membrane proteins that control the rate of cholesterol entry into the outer mitochondrial membrane. More recent studies unveiled the steroidogenic metabolon, a bioactive, multimeric protein complex that spans the outer-inner mitochondrial membranes and is responsible for hormone-induced import, segregation, targeting, and metabolism of cholesterol by cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1) in the inner mitochondrial membrane. The availability of genome information allowed us to systematically explore the evolutionary origin of the proteins involved in the mitochondrial cholesterol transport machinery (transduceosome, steroidogenic metabolon, and signaling proteins), trace the original archetype, and predict their biological functions by molecular phylogenetic and functional divergence analyses, protein homology modeling and molecular docking. Although most members of these complexes have a history of gene duplication and functional divergence during evolution, phylogenomic analysis revealed that all vertebrates have the same functional complex members, suggesting a common mechanism in the first step of steroidogenesis. An archetype of the complex was found in invertebrates. The data presented herein suggest that the cholesterol transport machinery is responsible for steroidogenesis among all vertebrates and is evolutionarily conserved throughout the entire animal kingdom.     

Diversity, classification and function of the plant protein kinase superfamily

Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants.     

Phylogenetic and evolutionary analysis of the septin protein family in metazoan

Septins, a conserved family of cytoskeletal GTP-binding proteins, were presented in diverse eukaryotes. Here, a comprehensive phylogenetic and evolutionary analysis for septin proteins in metazoan was carried out. First, we demonstrated that all septin proteins in metazoan could be clustered into four subgroups, and the representative homologue of every subgroup was presented in the non-vertebrate chordate Ciona intestinalis, indicating that the emergence of the four septin subgroups should have occurred prior to divergence of vertebrates and invertebrates, and the expansion of the septin gene number in vertebrates was mainly by the duplication of pre-existing genes rather than by the appearance of new septin subgroup. Second, the direct orthologues of most human septins existed in zebrafish, which suggested that human septin gene repertoire was mainly formed by as far as before the split between fishes and land vertebrates. Third, we found that the evolutionary rate within septin family in mammalian lineage varies significantly, human SEPT1, SEPT 10, SEPT 12, and SEPT 14 displayed a relative elevated evolutionary rate compared with other septin members. Our data will provide new insights for the further function study of this protein family.     

All in the family: evolutionary and functional relationships among death receptors

Over the last decade, significant progress has been made towards identifying the signaling pathways within mammalian cells that lead to apoptosis mediated by death receptors. The simultaneous expression of more than one death receptor in many, if not all, cell types suggests that functional innovation has driven the divergence of these receptors and their cognate ligands. To better understand the physiological divergence of the death receptors, a phylogenetic analysis of vertebrate death receptors was conducted based upon amino-acid sequences encoding the death domain regions of currently known and newly identified members of the family. Evidence is presented to indicate an ancient radiation of death receptors that predates the emergence of vertebrates, as well as ongoing divergence of additional receptors both within several receptor lineages as well as modern taxonomic lineages. We speculate that divergence among death receptors has led to their functional specialization. For instance, some receptors appear to be primarily involved in mediating the immune response, while others play critical roles during development and tissue differentiation. The following represents an evolutionary approach towards an understanding of the complex relationship among death receptors and their proposed physiological functions in vertebrate species.     

On the Origin and Evolution of Plant Brassinosteroid Receptor Kinases

Brassinosteroid (BR) signaling pathway is so far the best-understood receptor-kinase signaling pathway in plants. In Arabidopsis, the activation of this pathway requires binding of BRs to the receptor kinase BRASSINOSTEROID-INSENSITIVE I (AtBRI1). Although the function of AtBRI1 has been extensively studied, it is not known when the binding function emerged and how this important component of BR signaling pathway and related genes (the BRI1–BRL gene family) have evolved in plants. We define BRI1–BRL genes in sequenced plant genomes, construct profiles for critical protein domains, scan them against all accessible plant gene/EST resources, and reveal the evolution of domain configuration of this family. We also investigate its evolutionary pattern through phylogenetic analysis. The complete BR receptor domain configuration originates through two domain gain events in the ancestral receptor-like kinase: first juxtamembrane domain gained during the early diversification of land plants, and then island domain (ID) acquired in the common ancestor of angiosperms and gymnosperms after its divergence from spike moss. The 70 amino acid ID has characteristic sequences of BRI1–BRL family and this family keeps relative stable copy numbers during the history of angiosperms and the majority of duplications and losses have occurred in terminal taxa in current taxon sampling. This study reveals important events shaping structural and functional characteristics of plant BR receptors. It answers the question of how and when BR receptors originates, which provide insights into the origin and evolution of the BR signaling pathway.     

Phylogenetic analysis and evolution of aromatic amino acid hydroxylase

A study was performed to investigate the phylogenetic relationship among AAAH members and to statistically evaluate sequence conservation and functional divergence. In total, 161 genes were identified from 103 species. Phylogenetic analysis showed that well-conserved subfamilies exist. Exon-intron structure analysis showed that the gene structures of AAAH were highly conserved across some different lineage species, while some species-specific introns were also found. The dynamic distribution of ACT domain suggested one gene fusion event has occurred in eukaryota. Significant functional divergence was found between some subgroups. Analysis of the site-specific profiles revealed critical amino acid residues for functional divergence. This study highlights the molecular evolution of this family and may provide a starting point for further experimental verifications.