PII,the key regulator of nitrogen metabolism in the cyanobacteria

PII proteins are a protein family important to signal transduction in bacteria and plants. PII plays a critical role in regulation of carbon and nitrogen metabolism in cyanobacteria. Through conformation change and covalent modification, which are regulated by 2-oxoglutarate, PII interacts with different target proteins in response to changes of cellular energy status and carbon and nitrogen sources in cyanobacteria and regulates cellular metabolism. This article reports recent progress in PII research in cyanobacteria and discusses the mechanism of PII regulation of cellular metabolism.     

PII,the key regulator of nitrogen metabolism in the cyanobacteria

PII proteins are a protein family important to signal transduction in bacteria and plants. PII plays a critical role in regulation of carbon and nitrogen metabolism in cyanobacteria. Through conformation change and covalent modification, which are regulated by 2-oxoglutarate, PII interacts with different target proteins in response to changes of cellular energy status and carbon and nitrogen sources in cyanobacteria and regulates cellular metabolism. This article reports recent progress in PII research in cyanobacteria and discusses the mechanism of PII regulation of cellular metabolism .     

Molecular properties of the putative nitrogen sensor PII from Arabidopsis thaliana

Although the signal sensing protein PII is well known to play a central role in bacterial nitrogen metabolism, the structure and function of PII in plants remains only partially understood. Comparative modeling was undertaken based on the high degree of amino acid identity between Escherichia coli and Arabidopsis PII. The mature Arabidopsis PII predicted structure superimposes very well onto the E. coli PII structure (Calpha root mean square deviation < 0.4 A). The model of the highly conserved T-loop suggests a molecular mechanism by which the plant PII may regulate putative post-translational modification in response to metabolite binding. Consistent with the presence of key conserved residues necessary for trimer formation, gel filtration showed the oligomeric structure of Arabidopsis thaliana PII to be a homotrimer. We have demonstrated that Arabidopsis PII binds to the small molecules, ATP, ADP, 2KG, and with lesser affinity to OAA, using isothermal titration calorimetry. We have determined the metabolite dissociation constants and compared these with known physiological concentrations of these metabolites in the plant to identify the Arabidopsis PII effector molecules and their possible roles. We predict that the plant PII is likely continually bound by ATP, and its ligand-bound state only varying with respect to the degree of 2KG binding. Based on our in vitro binding studies, the function of plant PII as a 2KG sensor is suggested.     

The GlnD and GlnK homologues of Streptomyces coelicolor A3(2) are functionally dissimilar to their nitrogen regulatory system counterparts from enteric bacteria

Glutamine synthetase I (GSI) enzyme activity in Streptomyces coelicolor is controlled post-translationally by the adenylyltransferase (GlnE) as in enteric bacteria. Although other homologues of the Escherichia coli Ntr system (glnK, coding for a PII family protein; and glnD, coding for an uridylyltransferase) are found in the S. coelicolor genome, the regulation of the GSI activity was found to be different. The functions of glnK and glnD were analysed by specific mutants. Surprisingly, biochemical assay and two-dimensional PAGE analysis showed that modification of GSI by GlnE occurs normally in all mutant strains, and neither GlnK nor GlnD are required for the regulation of GlnE in response to nitrogen stimuli. Analysis of the post-translational regulation of GlnK in vivo by two-dimensional PAGE and mass spectrometry indicated that it is subject to both a reversible and a non-reversible modification in a direct response to nitrogen availability. The irreversible modification was identified as removal of the first three N-terminal amino acid residues of the protein, and the reversible modification as adenylylation of the conserved tyro-sine 51 residue that is known to be uridylylated in E. coli. The glnD insertion mutant expressing only the N-terminal half of GlnD was capable of adenylylating GlnK, but was unable to perform the reverse deadenylylation reaction in response to excess ammonium. The glnD null mutant completely lacked the ability to adenylylate GlnK. This work provides the first example of a PII protein that is modified by adenylylation, and demonstrates that this reaction is performed by a homologue of GlnD, previously described only as a uridylyltransferase enzyme.     

Nucleotidylation, not phosphorylation, is the major source of the phosphotyrosine detected in enteric bacteria.

The majority of the phosphotyrosine recovered from partial acid hydrolysates of 32P-labeled Escherichia coli is derived from a single prominent protein. We show here by biochemical, genetic, and immunological criteria that this protein is actually glutamine synthetase adenylylated (not phosphorylated) at tyrosine. Furthermore, all of the phosphotyrosine detectable in partial acid hydrolysates of 32P-labeled Salmonella typhimurium was eliminated in a strain deficient in both glutamine synthetase and uridylyltransferase, an enzyme which uridylylates the regulatory protein PII at a tyrosine residue. These results suggest that protein-tyrosine phosphorylation represents a rare modification in eubacterial cells.     

Targeted mutation of serine 697 in the Ret tyrosine kinase causes migration defect of enteric neural crest cells

The RET receptor tyrosine kinase plays a critical role in the development of the enteric nervous system (ENS) and the kidney. Upon glial-cell-line-derived neurotrophic factor (GDNF) stimulation, RET can activate a variety of intracellular signals, including the Ras/mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K)/AKT, and RAC1/JUN NH(2)-terminal kinase (JNK) pathways. We recently demonstrated that the RAC1/JNK pathway is regulated by serine phosphorylation at the juxtamembrane region of RET in a cAMP-dependent manner. To determine the importance of cAMP-dependent modification of the RET signal in vivo, we generated mutant mice in which serine residue 697, a putative protein kinase A (PKA) phosphorylation site, was replaced with alanine (designated S697A mice). Homozygous S697A mutant mice lacked the ENS in the distal colon, resulting from a migration defect of enteric neural crest cells (ENCCs). In vitro organ culture showed an impaired chemoattractant response of the mutant ENCCs to GDNF. JNK activation by GDNF but not ERK, AKT and SRC activation was markedly reduced in neurons derived from the mutant mice. The JNK inhibitor SP600125 and the PKA inhibitor KT5720 suppressed migration of the ENCCs in cultured guts from wild-type mice to comparable degrees. Thus, these findings indicated that cAMP-dependent modification of RET function regulates the JNK signaling responsible for proper migration of the ENCCs in the developing gut.     

Crystal structure of Arabidopsis PII reveals novel structural elements unique to plants

The 1.9 A resolution crystal structure of PII from Arabidopsis thaliana reveals for the first time the molecular structure of a widely conserved regulator of carbon and nitrogen metabolism from a eukaryote. The structure provides a framework for understanding the arrangement of highly conserved residues shared with PII proteins from bacteria, archaea, and red algae as well as residues conserved only in plant PII. Most strikingly, a highly conserved segment at the N-terminus that is found only in plant PII forms numerous interactions with the alpha2 helix and projects from the surface of the homotrimer opposite to that occupied by the T-loop. In addition, solvent-exposed residues near the T-loop are highly conserved in plants but differ in prokaryotes. Several residues at the C-terminus that are also highly conserved only in plants contribute part of the ATP-binding site and likely participate in an ATP-induced conformational change. Structures of PII also reveal how citrate and malonate bind near the triphosphate binding site occupied by ATP in bacterial and archaeal PII proteins.     

PII is important in regulation of nitrogen metabolism but not required for heterocyst formation in the Cyanobacterium Anabaena sp. PCC 7120

PII is an important signal protein for regulation of nitrogen metabolism in bacteria and plants. We constructed a mutant of glnB, encoding PII, in a heterocystous cyanobacterium, Anabaena sp. PCC 7120, with a cre-loxP system. The mutant (MP2alpha) grew more slowly than the wild type under all nitrogen regimens. It excreted a large amount of ammonium when grown on nitrate due to altered activities of glutamine synthetase and nitrate reductase. MP2alpha had a low nitrogenase activity but was able to form heterocysts under diazotrophic conditions, suggesting that PII is not required for heterocyst differentiation. Analysis of the PII with mass spectroscopy found tyrosine nitration at Tyr-51 under diazotrophic conditions while no phosphorylation at Ser-49 was detected. The strains 51F and 49A, which have PII with mutations of Y51F and S49A, respectively, were constructed to analyze the functions of the two key residues on the T-loop. Like MP2alpha, they had low nitrogenase activity and grew slowly under diazotrophic conditions. 49A was also impaired in nitrate uptake and formed heterocysts in the presence of nitrate. The up-regulation of ntcA after nitrogen step-down, which was present in the wild type, was not observed in 51F and 49A. While our results showed that the Ser-49 residue is important to the function of PII in Anabaena sp. PCC 7120, evidence from the PII pattern of the wild type and 49A in non-denaturing gel electrophoresis suggested that Ser-49 is not modified. The possible physiological roles of tyrosine nitration of PII are discussed.     

Microbial elicitors induce activation and dual phosphorylation of the Arabidopsis thaliana MAPK 6

Protein kinases related to the family of mitogen-activated kinases (MAPKs) have been established as signal transduction components in a variety of processes in plants. For Arabidopsis thaliana, however, although one of the genetically best studied plant species, biochemical data on activation of mitogen-activated protein kinases are lacking. A. thaliana MAPK 6 (AtMPK6) is the Arabidopsis orthologue of a tobacco MAPK termed salicylate-induced protein kinase, which is activated by general and race-specific elicitors as well as by physical stress. Using a C terminus-specific antibody, we show that AtMPK6 is activated in elicitor-treated cell cultures of A. thaliana. Four different elicitors from bacteria, fungi, and plants lead to a rapid and transient activation of AtMPK6, indicating a conserved signaling pathway. The induction was equally rapid as medium alkalinization, one of the earliest elicitor response observed in cell cultures. A similarly rapid activation of AtMPK6 was observed in elicitor-treated leaf strips, demonstrating that recognition of the elicitors and activation of the MAPK pathway occurs also in intact plants. We demonstrate by in vivo labeling that AtMPK6 is phosphorylated on threonine and tyrosine residues in elicited cells.     

The essential role of hypusine in eukaryotic translation initiation factor 4D (eIF-4D). Purification of eIF-4D and its precursors and comparison of their activities

Eukaryotic translation initiation factor 4D (eIF-4D) is the only protein known to contain the amino acid, hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. This unusual amino acid is formed post-translationally by modification of a single specific lysine residue in an eIF-4D precursor protein. Two separate eIF-4D precursors, each of which contains a lysine residue in place of the hypusine residue and each of which thereby serves as a protein substrate for the hypusine modification, were purified from DL-2-difluoromethylornithine-treated Chinese hamster ovary cells by means of a five-step procedure. These two precursors termed PI and PII both have apparent molecular masses of approximately 17 kDa, indistinguishable from that of eIF-4D, but exhibit more acidic isoelectric points (5.1 and 5.25 for PI and PII, respectively, compared with 5.37 for eIF-4D). These physical characteristics, together with other properties, indicate that eIF-4D differs from PII only in possessing the hypusine residue in place of a lysine residue, whereas an additional structural difference exists between PI and eIF-4D. eIF-4D from CHO cells provides a significant enhancement of methionyl-puromycin synthesis, a model assay for translation initiation. Neither PI nor PII stimulates this in vitro system. These findings are the first direct evidence that hypusine is essential for the biological activity of eIF-4D.     

Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including beta-catenin and the NF-kappaB inhibitor IkappaB-alpha. Ubiquitination of these proteins as well as others is catalyzed by the SCF(betaTrCP) ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-kappaB and beta-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin-proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.     

Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK.

PII-like proteins are signal transduction proteins found in bacteria, archaea and eukaryotes. They mediate a variety of cellular responses. A second PII-like protein, called GlnK, has been found in several organisms. In the diazotroph Herbaspirillum seropedicae, PII protein is involved in sensing nitrogen levels and controlling nitrogen fixation genes. In this work, the crystal structure of the unliganded H. seropedicae PII was solved by X-ray diffraction. H. seropedicae PII has a Gly residue, Gly108 preceding Pro109 and the main-chain forms a beta turn. The glycine at position 108 allows a bend in the C-terminal main-chain, thereby modifying the surface of the cleft between monomers and potentially changing function. The structure suggests that the C-terminal region of PII proteins may be involved in specificity of function, and nonenteric diazotrophs are found to have the C-terminal consensus XGXDAX(107-112). We are also proposing binding sites for ATP and 2-oxoglutarate based on the structural alignment of PII with PII-ATP/GlnK-ATP, 5-carboxymethyl-2-hydroxymuconate isomerase and 4-oxalocrotonate tautomerase bound to the inhibitor 2-oxo-3-pentynoate.     

The PII protein in the cyanobacterium Synechococcus sp. strain PCC 7942 is modified by serine phosphorylation and signals the cellular N-status.

The glnB gene product (PII protein) from Synechococcus sp. has previously been identified among 32P-labeled proteins, and its modification state has been observed to depend on both the nitrogen source and the spectral light quality (N. F. Tsinoremas, A. M. Castets, M. A. Harrison, J. F. Allen, and N. Tandeau de Marsac, Proc. Natl. Acad. Sci. USA 88:4565-4569, 1991). As shown in this study, modification of the PII protein primarily responds to the N-status of the cell, and its light-dependent variations are are mediated through nitrate metabolism. Modification of the PII protein results in the appearance of three isomeric forms with increasing negative charge. Unlike its homolog counterparts characterized so far, PII in Synechococcus sp. is modified by phosphorylation on a serine residue, which represents a unique kind of protein modification in bacterial nitrogen signalling pathways.     

Cell-type specific modification of PII is involved in the regulation of nitrogen metabolism in the cyanobacterium Anabaena PCC 7120

In the heterocystous cyanobacterium Anabaena PCC 7120, the modification state of the signalling PII protein is regulated according to the nitrogen regime of the cells, as already observed in some unicellular cyanobacteria. However, during the adaptation to diazotrophic growth conditions, PII is phosphorylated in vegetative cells while unphosphorylated in heterocysts. Isolation of mutants affected on PII modification state and analysis of their phenotypes allow us to show the implication of PII in the regulation of molecular nitrogen assimilation and more specifically, the requirement of unmodified state of PII in the formation of polar nodules of cyanophycin in heterocysts.     

Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942.

The PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942 signals the cellular N status by being phosphorylated or dephosphorylated at a seryl residue. Here we show that the PII-modifying system responds to the activity of ammonium assimilation via the glutamine synthase-glutamate synthase pathway and to the state of CO2 fixation. To identify possible functions of PII in this microorganism, a PII-deficient mutant was created and its general phenotype was characterized. The analysis shows that the PII protein interferes with the regulation of enzymes required for nitrogen assimilation, although ammonium repression is still detectable in the PII-deficient mutant. We suggest that the phosphorylation and dephosphorylation of PII are part of a complex signal transduction network involved in global nitrogen control in cyanobacteria. In this regulatory process, PII might be involved in mediating the tight coordination between carbon and nitrogen assimilation.