Molecular dissection of alleles of the osmotic-1 locus of Neurospora crassa

Two-component histidine kinases of bacteria, plants, and fungi are involved in the regulation of intracellular events in response to changes in external environmental conditions. Fungal histidine kinases play important roles in osmoregulation, in vivo and in planta virulence, and sensitivity to certain classes of antifungals. The osmotic-1 (OS-1) locus of Neurospora crassa encodes a predicted protein with homology to histidine kinases and appears to be an osmosensor. Mutants of the OS-1 locus are hypersensitive to salt and are strongly resistant to dicarboximide antifungals. Molecular analysis of each of eight OS-1 mutants revealed that seven resulted from amino acid changes in a domain of the protein known as the linker region. These results indicate that the linker region of fungal two-component histidine kinases is essential for proper functioning of the kinase.     

SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ

The Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeS^L but causing activation in strains containing SaeS^P. Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/phosphatase activity.     

Design and Signaling Mechanism of Light-Regulated Histidine Kinases

Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by ∼ 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40°-60° rotational movement within an α-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by α-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.     

A cytoplasmic coiled-coil domain is required for histidine kinase activity of the yeast osmosensor, SLN1

The yeast histidine kinase, Sln1p, is a plasma membrane-associated osmosensor that regulates the activity of the osmotic stress MAP kinase pathway. Changes in the osmotic environment of the cell influence the autokinase activity of the cytoplasmic kinase domain of Sln1p. Neither the nature of the stimulus, the mechanism by which the osmotic signal is transduced nor the manner in which the kinase is regulated is currently clear. We have identified several mutations located in the linker region of the Sln1 kinase (just upstream of the kinase domain) that cause hyperactivity of the Sln1 kinase. This region of histidine kinases is largely uncharacterized, but its location between the transmembrane domains and the cytoplasmic kinase domain suggests that it may have a potential role in signal transduction. In this study, we have investigated the Sln1 linker region in order to understand its function in signal transduction and regulation of Sln1 kinase activity. Our results indicate that the linker region forms a coiled-coil structure and suggest a mechanism by which alterations induced by osmotic stress influence kinase activity by altering the alignment of the phospho-accepting histidine with respect to the catalytic domain of the kinase.     

The hybrid histidine kinase Slr1759 of the cyanobacterium Synechocystis sp. PCC 6803 contains FAD at its PAS domain

The cyanobacterium Synechocystis sp. PCC 6803 harbours 47 histidine kinases (Hiks). Among these are hybrid histidine kinases with one or two response regulator domains as well as numerous Hiks with several sensory domains. One example is the hybrid histidine kinase Slr1759 (Hik14) that has two PAS domains arranged in tandem linked to a predicted GAF domain. Here, we show that a Slr1759 derivative recombinantly expressed in Escherichia coli has a flavin cofactor. Using truncated Slr1759 variants, it is shown that the flavin associates with the first PAS domain. The cofactor reconstitutes the activity of d-amino acid oxidase apoprotein from pig kidney, indicating that the flavin derivative is FAD. Furthermore, the Slr1759 histidine kinase domain indeed undergoes autophosphorylation in vitro. The phosphorylated product of a recombinant Slr1759 derivative is sensitive to acids, pointing to a histidine residue as the phosphate-accepting group.     

Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses

The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.     

Genetic analysis of the catalytic domain of the chemotaxis-associated histidine kinase CheA.

Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis. The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemotaxis-associated response regulators, CheY and CheB. CheA(L) first autophosphorylates amino acid His-48 before its phosphoryl group is transferred to these response regulators. The short form, CheA(S), lacks the amino-terminal 97 amino acids of CheA(L) and therefore does not possess the site of phosphorylation. The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, highly conserved among histidine kinases of the family of two-component signal transduction systems. On the basis of sequence similarity to highly conserved regions of certain eukaryotic kinases, the G1 and G2 regions are purported to be involved in the binding and hydrolysis of ATP. We report here that alleles mutated in the G1, G2, or F region synthesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo. We also show that in vitro, the nonphosphorylatable CheA(S) protein mediates transphosphorylation of a CheA(L) variant defective in both G1 and G2. In contrast, CheA(L) variants defective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all. These results are consistent with a mechanism by which the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that dimer.     

Branched-chain alpha-ketoacid dehydrogenase kinase. Molecular cloning, expression, and sequence similarity with histidine protein kinases.

A cDNA for branched-chain alpha-ketoacid dehydrogenase kinase was cloned from a rat heart cDNA library. The cDNA had an open reading frame encoding a protein of 382 amino acid residues with a calculated molecular weight of 43,280. The clone codes for the branched-chain alpha-ketoacid dehydrogenase kinase based on the following: 1) the deduced amino acid sequence contained the partial sequence of the kinase determined by direct sequencing; 2) expression of the cDNA in Escherichia coli resulted in synthesis of a 43,000-Da protein that was recognized specifically by kinase antibodies; and 3) enzyme activity that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex was found in extracts of E. coli expressing the protein. Northern blot analysis indicated the mRNA for the branched-chain alpha-ketoacid dehydrogenase kinase was more abundant in rat heart than in rat liver, as expected from the relative amounts of kinase activity expressed in these tissues. The deduced sequence of the kinase aligned with a high degree of similarity within subdomains characteristic of procaryotic histidine protein kinases. This first mitochondrial protein kinase to be cloned appears more closely related in sequence to procaryotic histidine protein kinases than to eucaryotic serine/threonine protein kinases.     

Effect of linker sequence on the stability of circularly permuted variants of ribonuclease T1

Circularly permuted variants of ribonuclease T1 were constructed with a library of residues covalently linking the original amino and carboxyl terminal ends of the wild-type protein. The library of linking peptides consisted of three amino acids containing any combination of proline, aspartate, asparagine, serine, threonine, tyrosine, alanine, and histidine. Forty two unique linker sequences were isolated and 10 of these mutants were further characterized with regard to catalytic activity and overall thermodynamic stability. The 10 mutants with the different linking sequences (HPD, TPH, DTD, TPD, PYH, PAT, PHP, DSS, SPP, and TPS), in addition to GGG and GPG, were 4.0-6.2 kcal/mol less stable than the wild-type ribonuclease T1. However, these circular permuted variants were only 0.4-2.6 kcal/mol less stable than the direct parent protein that is missing the disulfide bond connecting residues 2 and 10. The most stable linking peptide was HPD.     

Characterization of sporulation histidine kinases of Bacillus anthracis

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals. Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.     

A novel functional assay for fungal histidine kinases group III reveals the role of HAMP domains for fungicide sensitivity

Signal transduction systems comprising histidine kinases are suggested as new molecular targets of antibiotics. The important human fungal pathogen Candida albicans possesses three histidine kinases, one of which is the type III histidine kinase CaNik1, which activates the MAP kinase Hog1. We established a screening system for inhibitors of this class of histidine kinases by functional expression of the CaNIK1 gene in S. cerevisiae. This transformant was susceptible to fungicides to which the wild type strain was resistant, such as fludioxonil and ambruticin. Growth inhibition correlated with phosphorylation of Hog1 and was dependent on an intact Hog1 pathway. At the N-terminus the histidine kinase CaNik1 has four amino acid repeats of 92 amino acids each and one truncated repeat of 72 amino acids. Within these repeats we identified 9 HAMP domains with a paired structure. We constructed mutants in which one or two pairs of these domains were deleted. S. cerevisiae transformants expressing the full-length CaNIK1 showed the highest sensitivity to the fungicides, any truncation reduced the susceptibility of the transformants to the fungicides. This indicates that the HAMP domains are decisive for the mode of action of the antifungal compounds.     

Cloning and characterization of the histidine kinase gene<Emphasis Type="Italic"> Dic1</Emphasis> from<Emphasis Type="Italic"> Cochliobolus heterostrophus</Emphasis> that confers dicarboximide resistance and osmotic adaptation

A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.Communicated by C. A. M. J. J. van den Hondel     

Small molecule functional discrimination of the kinases required for the microbial synthesis of threonine and isoleucine

The biosynthesis of l-threonine and l-isoleucine in bacteria and in fungi requires the action of 2 amino acid kinases: aspartate kinase and homoserine kinase. Although these kinases bind similar substrates and catalyze analogous phosphotransfer chemistry, they do not show high amino acid sequence homology. We show that despite this difference, both kinases form a ternary complex consisting of enzyme- adenosine triphosphate- amino acid to accomplish phosphoryl transfer. With this similarity in mind, we set out to identify molecules that could lead to inhibitors with activity against both kinases in the pathway. We synthesized a series of aspartic acid-adenosine bisubstrate compounds separated by a variable length alkyl linker that we hypothesized could bind to these kinases. These bisubstrate compounds only inhibited the bacterial aspartate kinase. These results reveal unexpected differences in small molecule interactions among these functionally similar enzymes.     

Th1 and Th2 deviation of myelin-autoreactive T cells by altered peptide ligands is associated with reciprocal regulation of Lck, Fyn, and ZAP-70

Th0 clones recognizing an immunodominant peptide of myelin basic protein (residues 83-99) were derived from patients with multiple sclerosis. We demonstrate that analogue peptides with alanine substitution at Val86 and His88 had a unique partial agonistic property in inducing Th0 -->Th1 and Th0 -->Th2 deviation of the myelin basic protein-reactive T cell clones, respectively. Th0 to Th1 deviation induced by peptide 86V-->A correlated with up-regulation of Fyn and ZAP-70 kinase activities. Conversely, Th0 to Th2 deviation induced by peptide 88H-->A was associated with complete failure to activate Fyn and ZAP-70 kinases. The observed Th1 and Th2 shift also correlated, to a lesser extent, with Lck kinase activity that was down-regulated with Th1 deviation and increased with Th2 deviation in some T cell clones. We demonstrated that the Th1 and Th2 shift induced by the analogue peptides was a reversible process, as the T cell clones previously exposed to either 86V-->A or 88H-->A peptide could revert to an opposite phenotype when rechallenged reciprocally with a different analogue peptide. The study has important implications in our understanding of regulation of TCR-associated tyrosine kinases by altered peptide ligands and its role in cytokine regulation of autoreactive T cells.     

Mapping the functional domains of elongation factor-2 kinase

A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., et al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, A. G., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that are not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R45]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation. eEF-2 was the only known substrate for eEF-2 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36".     

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.     

Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells

In this paper, we describe how a dynamic regulatory process is necessary to restrict microvilli to the apical aspect of polarized epithelial cells. We found that local phosphocycling regulation of ezrin, a critical plasma membrane–cytoskeletal linker of microvilli, was required to restrict its function to the apical membrane. Proteomic approaches and ribonucleic acid interference knockdown identified lymphocyte-oriented kinase (LOK) and SLK as the relevant kinases. Using drug-resistant LOK and SLK variants showed that these kinases were sufficient to restrict ezrin function to the apical domain. Both kinases were enriched in microvilli and locally activated there. Unregulated kinase activity caused ezrin mislocalization toward the basolateral domain, whereas expression of the kinase regulatory regions of LOK or SLK resulted in local inhibition of ezrin phosphorylation by the endogenous kinases. Thus, the domain-specific presence of microvilli is a dynamic process requiring a localized kinase driving the phosphocycling of ezrin to continually bias its function to the apical membrane.