Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two α:β tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser¹⁶ or Ser⁶³, and doubly phosphorylated at Ser²⁵ and Ser³⁸, on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser¹⁶ or Ser⁶³ strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser¹⁶ and Ser⁶³ and support the hypothesis that selective targeting by Ser¹⁶-specific or Ser⁶³-specific kinases provides complimentary mechanisms for regulating microtubule function.Stathmin is an 18-kDa ubiquitously expressed microtubule-destabilizing phosphoprotein whose activity is modulated by phosphorylation of its four serine residues, Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³ (1–7). Several classes of kinases have been identified that phosphorylate stathmin, including kinases associated with cell growth and differentiation such as members of the mitogen-activated protein kinase (MAPK)2 family, cAMP-dependent protein kinase (1–5, 8–11), and kinases associated with cell cycle regulation such as cyclin-dependent kinase 1 (3, 12–14). Phosphorylation of stathmin is required for cell cycle progression through mitosis and for proper assembly/function of the mitotic spindle (3, 13–16). Inhibition of stathmin phosphorylation produces strong mitotic phenotypes characterized by disassembly and disorganization of mitotic spindles and abnormal chromosome distributions (3, 13–14).Stathmin is known to destabilize microtubules in two ways. One is by binding to soluble tubulin and forming a stable complex that cannot polymerize into microtubules, consisting of one molecule of stathmin and two molecules of tubulin (T2S complex) (17–24). Addition of stathmin to microtubules in equilibrium with soluble tubulin results in sequestration of the tubulin and a reduction in the level of microtubule polymer (17–18, 22, 25–28). In addition to reducing the amount of assembled polymer, tubulin sequestration by stathmin has been shown to increase the switching frequency at microtubule plus ends from growth to shortening (called the catastrophe frequency) as the microtubules relax to a new steady state (17, 29). The second way is by binding directly to microtubules (27–30). The direct binding of stathmin to microtubules increases the catastrophe frequency at both ends of the microtubules and considerably more strongly at minus ends than at plus ends (27). Consistent with its strong catastrophe-promoting activity at minus ends, stathmin increases the treadmilling rate of steady-state microtubules in vitro (27). These results have led to the suggestion that stathmin might be an important cellular regulator of minus-end microtubule dynamics (27).Phosphorylation of stathmin diminishes its ability to regulate microtubule polymerization (3, 14, 25–26). Phosphorylation of Ser¹⁶ or Ser⁶³ appears to be more critical than phosphorylation of Ser²⁵ and Ser³⁸ for the ability of stathmin to bind to soluble tubulin and to inhibit microtubule assembly in vitro (3, 25). Inhibition of stathmin phosphorylation induces defects in spindle assembly and organization (3, 14) suggesting that not only soluble tubulin-microtubule levels are regulated by phosphorylation of stathmin, but the dynamics of microtubules could also be regulated in a phosphorylation-dependent manner.It is not known how phosphorylation at any of the four serine residues of stathmin affects its ability to regulate microtubule dynamics and, specifically, its ability to increase the catastrophe frequency at plus and minus ends due to its direct interaction with microtubules. Thus, we determined the effects of stathmin individually phosphorylated at either Ser¹⁶ or Ser⁶³ and doubly phosphorylated at both Ser²⁵ and Ser³⁸ on dynamic instability at plus and minus ends in vitro at microtubule polymer steady state and physiological pH (pH 7.2). We find that phosphorylation of Ser¹⁶ strongly reduces the direct catastrophe-promoting activity of stathmin at plus ends and abolishes it at minus ends, whereas phosphorylation of Ser⁶³ abolishes the activity at both ends. The effects of phosphorylation of individual serines correlated well with stathmin''s reduced abilities to form stable T2S complexes, to inhibit microtubule polymerization, and to bind to microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not alter the ability of stathmin to modulate dynamic instability at the microtubule ends, its ability to form a stable T2S complex, or its ability to bind to microtubules. The data further support the hypotheses that phosphorylation of stathmin on either Ser¹⁶ or Ser⁶³ plays a critical role in regulating microtubule polymerization and dynamics in cells.     

Queuosine Formation in Eukaryotic tRNA Occurs via a Mitochondria-localized Heteromeric Transglycosylase

tRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNA^Asp, tRNA^Asn, tRNA^His, and tRNA^Tyr; an almost universal event in eubacterial and eukaryotic species. Despite extensive characterization of the eubacterial TGT the eukaryotic activity has remained undefined. Our search of mouse EST and cDNA data bases identified a homologue of the Escherichia coli TGT and three spliced variants of the queuine tRNA guanine transglycosylase domain containing 1 (QTRTD1) gene. QTRTD1 variant_1 (Qv1) was found to be the predominant adult form. Functional cooperativity of TGT and Qv1 was suggested by their coordinate mRNA expression in Northern blots and from their association in vivo by immunoprecipitation. Neither TGT nor Qv1 alone could complement a tgt mutation in E. coli. However, transglycosylase activity could be obtained when the proteins were combined in vitro. Confocal and immunoblot analysis suggest that TGT weakly interacts with the outer mitochondrial membrane possibly through association with Qv1, which was found to be stably associated with the organelle.Queuosine (Q³; (7-{[(4,5-cis-dihydroxy-2-cyclo-penten-1-yl)-amino]methyl}-7-deazaguanosine) is a modified 7-deazaguanosine molecule found at the wobble position of transfer RNA that contains a GUN anticodon sequence: tRNA^Tyr, tRNA^Asn, tRNA^His, and tRNA^Asp (1). The Q-modification is widely distributed in nature in the tRNA of eubacteria, plants, and animals; a notable exception being yeast and plant leaf cells (2, 3). Interestingly, Q-modification has also been detected in aspartyl tRNA from mitochondria of rat (4) and opossum (5). In most eukaryotes, the Q molecule can be further modified by the addition of a mannosyl group to Q-tRNA^Asp and a galactosyl group to Q-tRNA^Tyr (1).Eubacteria are unique in their ability to synthesize Q. As part of this biosynthetic process, the eubacterial tRNA guanine transglycosylase (TGT) enzyme inserts the Q precursor molecule, 7-aminomethyl-7-deazaguanine (preQ₁) into tRNA, which is then converted to Q by two further enzymatic steps at the tRNA level (6). Eukaryotes by contrast salvage queuosine from food and enteric bacteria either as the free base (referred to as queuine) or as queuosine 5′-phosphate subsequent to normal tRNA turnover (7). A Q-related molecule, archaeosine, is found at position 15 of the D loop of most archaeal tRNA, where it functions to stabilize the tRNA structure (8). The enzyme involved in archaeosine biosynthesis is structurally and mechanistically related to the eubacterial TGT but with adaptations necessitated by the differences imposed by its unique substrate and tRNA specificity (9, 10).The crystal structure of the Zymononas mobilis (Z. mobilis) TGT has been determined and revealed the enzyme to be an irregular (β/α)₈ TIM barrel with a C-terminal zinc-binding subdomain (11). Insight into the residues involved in catalysis came from mutational and kinetic analysis of the recombinant Escherichia coli enzyme (12) and from the Z. mobilis TGT structure as an RNA-bound intermediate complexed to the final preQ₁-modified RNA product (13). This work showed the essential role of Asp-280 (Z. mobilis numbering) as the active site nucleophile. Asp-102, which was originally ascribed the role of active site nucleophile, functions as a general acid/base during catalysis (12, 10). Although, the E. coli and Z. mobilis TGT enzymes are monomeric in solution (14), at high protein concentrations the E. coli enzyme can oligomerize (15), and structural data from the Z. mobilis TGT has shown the formation of a 2:1 complex with tRNA; a possible functional requirement for catalysis (10).In contrast to the eubacterial enzyme, which is a single protein species, purification of the eukaryotic TGT suggested that the catalytically active enzyme is a heterodimeric molecule: subunits of 60 and 43 kDa in rabbit erythrocytes (16), 66 and 32 kDa in bovine liver (17), 60 and 34.5 kDa in rat liver (18), and a homodimer of two 68-kDa proteins in wheat germ (16, 19). A partial amino acid sequence was recovered from two of these active enzyme preparations. The identity of the proteins from bovine liver (17) could not be assigned at the time of publication. However, our searches show that the peptides from the larger 65-kDa subunit are identical to asparaginyl tRNA synthetase, and those of the smaller 32-kDa subunit correspond to 2,4-dienoyl CoA reductase. A highly pure preparation from rabbit reticulocytes (20) gave peptides with homology to the immunophilin p59, human elongation factor 2 (EF2), and a deubiquitinating enzyme, USP14. It is noteworthy that none of the peptide sequences obtained showed similarity to the eubacterial TGT. The results do suggest, however, that in eukaryotes the TGT activity could be embedded in a multisubunit complex.Most recently, Deshpande and Katze (21) identified a cDNA clone encoding a putative TGT catalytic subunit. Cloning the cDNA into a mammalian expression plasmid reconstituted TGT activity in GC₃/c1 cells, which are known to be naturally deficient in Q-containing tRNA (22). In this study, we identify for the first time the composition of the eukaryotic tRNA guanine transglycosylase, reconstitute the catalytic activity in vitro, and examine the intracellular distribution of the active subunits.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.     

Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag SDS-PAGE

Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, spine formation, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes down-regulation of Cdk5, whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser⁸ and Thr¹³⁸ as major phosphorylation sites using metabolic labeling of cultured cells followed by two-dimensional phosphopeptide mapping and phosphospecific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the phosphorylation states of p35 in neuronal culture and brain. Using Phos-tag acrylamide, the electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We found a novel phosphorylation site at Ser⁹¹, which was phosphorylated by Ca²⁺-calmodulin-dependent protein kinase II in vitro. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states and quantified Ser⁸, Ser⁹¹, and Thr¹³⁸ phosphorylation. The highest level of p35 phosphorylation was observed in embryonic brain; Ser⁸ was phosphorylated in all p35 molecules, whereas Ser⁹¹ was phosphorylated in 60% and Thr¹³⁸ was phosphorylated in ∼12% of p35 molecules. These are the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.Phosphorylation is a major post-translational modification of proteins, modulating a variety of cellular functions (1, 2). Because most phosphorylation occurs in a highly site-specific manner, identification of phosphorylation sites has been a subject of intense investigation. Several analytical methods have been utilized to identify phosphorylation sites, including mass spectrometry, amino acid sequencing, and radioisotope phosphate labeling of proteins with mutation(s) at putative phosphorylation site(s) (3, 4). Phosphorylation site-specific antibodies are frequently used to detect phosphorylation at target sites (5, 6). Many phosphospecific antibodies are now commercially available. These phosphospecific antibodies are convenient and useful tools for examining site-specific phosphorylation both in vivo and in vitro. However, they are not appropriate for estimating quantitative ratios of phosphorylation states. Electrophoretic mobility shift on SDS-PAGE is also often used to observe phosphorylation (7–10), but this method is not always applied to site-specific phosphorylation.Phos-tag is a newly developed dinuclear metal complex that can be used to provide phosphate-binding sites when conjugated to analytical materials such as acrylamide and biotin (11). In SDS-PAGE using Phos-tag acrylamide, phosphorylated proteins are trapped by the Phos-tag sites, delaying their migration and thus separating them from unphosphorylated proteins. Subsequent immunoblot analysis with phosphorylation-independent antibodies reveals both the phosphorylated and unphosphorylated bands. Because the migration of the phosphorylated proteins is greatly delayed compared with migration in Laemmli SDS-PAGE, it is easy to identify the phosphorylated proteins from observed positions on blots. In the past 3 years, this method has been used to detect phosphorylation states for many proteins such as ERK1/2, cdc37, myosin light chain, eIF2α, protein kinase D, β-casein, SIRT7, and dysbindin-1 (12–21).Cyclin-dependent kinase 5 (Cdk5)¹ is a proline-directed serine/threonine kinase that is expressed predominantly in postmitotic neurons and regulates various neuronal events such as neuronal migration, spine formation, synaptic activity, and cell death (22–24). Cdk5 is activated by binding to activation subunit p35 and inactivated by proteasomal degradation of p35 (25). In addition, Cdk5 activity is deregulated by cleavage of p35 to p25 with calpain, resulting in abnormal activation and ultimately causing neuronal cell death (26–29). Proteolysis of p35, either by proteasomal degradation or cleavage by calpain, is regulated by phosphorylation of p35 by Cdk5 (30–33). Therefore, phosphorylation of p35 is essential for proper regulation of Cdk5 activity and function. We previously identified Ser⁸ and Thr¹³⁸ as major p35 phosphorylation sites (33). We also showed that phosphorylation of p35 decreased during brain development and proposed its relationship to age-dependent vulnerability of neurons to stress stimuli (32). Thus, to understand the in vivo regulation of Cdk5 activity, it is critical to analyze the phosphorylation states of p35 in brain. However, there is no convenient method to analyze the precise in vivo phosphorylation status of the endogenous proteins.In this study, we applied the Phos-tag SDS-PAGE method to analyze the phosphorylation states of p35 in vivo and in cultured neurons. We constructed standard band profiles of phosphorylated p35 by Phos-tag SDS-PAGE using Ala mutants at Ser⁸ and/or Thr¹³⁸. From these experiments, we observed an unidentified in vivo phosphorylation site at Ser⁹¹. We quantified the phosphorylation at each site in cultured neurons and brain, providing the first quantitative estimate of the in vivo phosphorylation states of p35. We discuss the usefulness of Phos-tag SDS-PAGE to analyze the in vivo phosphorylation states of proteins.     

O-Linked N-Acetylglucosamine Modification of Insulin Receptor Substrate-1 Occurs in Close Proximity to Multiple SH2 Domain Binding Motifs

Insulin receptor substrate-1 (IRS-1) is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Ser/Thr kinases impact the metabolic and mitogenic effects elicited by insulin and IGF-1 through feedback and feed forward regulation at the level of IRS-1. Ser/Thr residues of IRS-1 are also O-GlcNAc-modified, which may influence the phosphorylation status of the protein. To facilitate the understanding of the functional effects of O-GlcNAc modification on IRS-1-mediated signaling, we identified the sites of O-GlcNAc modification of rat and human IRS-1. Tandem mass spectrometric analysis of IRS-1, exogenously expressed in HEK293 cells, revealed that the C terminus, which is rich in docking sites for SH2 domain-containing proteins, was O-GlcNAc-modified at multiple residues. Rat IRS-1 was O-GlcNAc-modified at Ser⁹¹⁴, Ser¹⁰⁰⁹, Ser¹⁰³⁶, and Ser¹⁰⁴¹. Human IRS-1 was O-GlcNAc-modified at Ser⁹⁸⁴ or Ser⁹⁸⁵, at Ser¹⁰¹¹, and possibly at multiple sites within residues 1025–1045. O-GlcNAc modification at a conserved residue in rat (Ser¹⁰⁰⁹) and human (Ser¹⁰¹¹) IRS-1 is adjacent to a putative binding motif for the N-terminal SH2 domains of p85α and p85β regulatory subunits of phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2 (PTPN11). Immunoblot analysis using an antibody generated against human IRS-1 Ser¹⁰¹¹ GlcNAc further confirmed the site of attachment and the identity of the +203.2-Da mass shift as β-N-acetylglucosamine. The accumulation of IRS-1 Ser¹⁰¹¹ GlcNAc in HEPG2 liver cells and MC3T3-E1 preosteoblasts upon inhibition of O-GlcNAcase indicates that O-GlcNAcylation of endogenously expressed IRS-1 is a dynamic process that occurs at normal glucose concentrations (5 mm). O-GlcNAc modification did not occur at any known or newly identified Ser/Thr phosphorylation sites and in most cases occurred simultaneously with phosphorylation of nearby residues. These findings suggest that O-GlcNAc modification represents an additional layer of posttranslational regulation that may impact the specificity of effects elicited by insulin and IGF-1.Insulin receptor substrate-1 (IRS-1)1 is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Many of the metabolic and mitogenic effects elicited by insulin and IGF-1 are mediated and modulated by posttranslational modifications of IRS-1, and tight regulation at the posttranslational level is crucial for maintaining insulin sensitivity and controlling growth factor-induced proliferation. Following hormonal stimulation, IRS-1 is phosphorylated by the receptor tyrosine kinases creating SH2 domain docking sites for downstream binding partners including the p85 regulatory subunits of phosphatidylinositol 3-kinase, Grb2, and the tyrosine phosphatase SHP2 (PTPN11) (1). Binding of p85 phosphatidylinositol 3-kinase and Grb2 activate the PI3K/Akt and Ras-MAPK pathways, respectively, whereas binding of SHP2 results in tyrosine dephosphorylation and signal attenuation (2). Positive and negative feedback regulation by Ser/Thr kinases, such as Akt (3), c-Jun N-terminal kinase (JNK) (4), S6K (5), and ERK (6), impact the interactions of IRS-1 with SH2 domain proteins and the receptor thereby affecting the duration and outcome of the signal. IRS-1 has been described as a central node for the integration of information regarding the nutrient and stress status of the cell (7). This information is encoded by site-specific phosphorylation by a number of kinases that regulate the specificity of effects that are elicited following receptor stimulation. Many sites of Ser/Thr phosphorylation have been identified on IRS-1, and cross-talk among Tyr and Ser/Thr phosphorylations at specific residues is evidence of dynamic and complex posttranslational regulation (8, 9). Inappropriate phosphorylation of IRS-1 resulting in the disruption of interactions of IRS-1 with binding partners is implicated in the development of insulin resistance (10) and altered IGF-1 signaling in breast cancer tissue (11, 12).In addition to phosphorylation, Ser/Thr residues in IRS-1 are also dynamically modified by GlcNAc in a nutrient-responsive manner. As opposed to a negatively charged phosphate group, O-GlcNAcylation imparts a bulky, hydrophilic, electrostatically neutral moiety to Ser/Thr residues. The enzymes responsible for the incorporation and removal of the monosaccharide from proteins, O-GlcNAc-transferase and O-GlcNAcase, respectively, are localized in the cytoplasm and the nucleus of all eukaryotic cells (13, 14). Recent studies suggest that the activity of O-GlcNAc-transferase is regulated by insulin (15) and that localization of O-GlcNAc-transferase to the membrane is driven by direct association with phosphatidylinositide 3-phosphate (16). The abundance of O-GlcNAc modification on many proteins in the insulin signaling pathway increases with sustained high glucose and chronic insulin stimulation, and elevated O-GlcNAc modification of IRS-1 correlates with the development of insulin resistance in multiple cell types including 3T3-L1 adipocytes (17, 18), MIN6 pancreatic beta cells (19), Fao rat hepatoma cells (16), human aortic endothelial cells (20), and skeletal muscle (21). The impact of O-GlcNAcylation on insulin signaling and diabetic complications was reviewed recently (22, 23). The direct effect of O-GlcNAc modification on signaling via IRS-1 is not known because conditions that mimic those in the uncontrolled diabetic patient may also result in phosphorylation of IRS-1 at inhibitory sites (16, 24) and O-GlcNAc modification of other proteins in the insulin signaling pathway, such as the insulin receptor, Akt (18), FoxO (25), AMP-activated protein kinase (26), and β-catenin (17).To elucidate site-specific effects of O-GlcNAc modification on IRS-1-mediated signal transduction, we identified the sites of O-GlcNAc modification of rat and human IRS-1 by tandem mass spectrometry. To facilitate detection of the O-GlcNAc-modified peptides and assign the sites of modification, CID coupled with neutral loss-triggered MS3 and electron transfer dissociation (ETD) (27) tandem spectrometric approaches were used. Fragmentation of O-GlcNAc-modified peptides by ETD did not destroy the labile O-linkage (28) permitting direct detection of these peptides by the database searching algorithm ProteinProspector2 (29). O-GlcNAc modification occurred in close proximity to multiple SH2 domain binding motifs and within a region of IRS-1 shown previously to interact with the insulin and IGF-1 receptors (30).     

Analysis of PTEN Complex Assembly and Identification of Heterogeneous Nuclear Ribonucleoprotein C as a Component of the PTEN-associated Complex

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is well characterized for its role in antagonizing the phosphoinositide 3-kinase pathway. Previous studies using size-exclusion chromatography demonstrated PTEN recruitment into high molecular mass complexes and hypothesized that PTEN phosphorylation status and PDZ binding domain may be required for such complex formation. In this study, we set out to test the structural requirements for PTEN complex assembly and identify the component(s) of the PTEN complex(es). Our results demonstrated that the PTEN catalytic function and PDZ binding domain are not absolutely required for its complex formation. On the other hand, PTEN phosphorylation status has a significant impact on its complex assembly. Our results further demonstrate enrichment of the PTEN complex in nuclear lysates, suggesting a mechanism through which PTEN phosphorylation may regulate its complex assembly. These results prompted further characterization of other protein components within the PTEN complex(es). Using size-exclusion chromatography and two-dimensional difference gel electrophoresis followed by mass spectrometry analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a novel protein recruited to higher molecular mass fractions in the presence of PTEN. Further analysis indicates that endogenous hnRNP C and PTEN interact and co-localize within the nucleus, suggesting a potential role for PTEN, alongside hnRNP C, in RNA regulation.Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)⁴ was cloned in 1997 (1–3) and has been well characterized for its tumor-suppressive role by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol 4,5-bisphosphate and antagonizing the phosphoinositide 3-kinase pathway (4–7). PTEN also regulates cell migration, cell cycle progression, DNA damage response, and chromosome stability independently of its lipid phosphatase activity through its potential protein phosphatase activity and/or protein-protein interaction (8–11) (for recent reviews, see 12–14).PTEN is composed of an N-terminal catalytic domain and a C-terminal regulatory domain. The catalytic domain contains a conserved signature motif (HCXXGXXR) found in dual-specific protein phosphatases, and mutations within this catalytic domain, including the C124S mutation, are known to abrogate PTEN catalytic activity (4). The C terminus of PTEN contains a PDZ (PDS-95/Disc-large/Zo-1) binding domain, which interacts with PDZ-containing proteins such as MAGI-1b, MAGI-2, MAGI-3, hDLG, hMAST and NHERF (15–19). In addition to the PDZ binding domain, several key serine and threonine phosphorylation sites (Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵) at the PTEN C terminus are reported to play an important role in regulating its stability, localization, and activity (20–26).Recent studies suggest that PTEN may function within higher molecular mass complexes. Through size-exclusion chromatography, Vazquez et al. (27) demonstrated that PTEN can be separated into two populations: a monomeric hyperphosphorylated subpopulation and a higher molecular mass hypophosphorylated subpopulation. It was hypothesized that PTEN in its dephosphorylated form can interact with PDZ-containing proteins such as hDLG and be recruited into a higher molecular mass complex. Although the components within PTEN complex(es) have not been systematically studied and purified, MAGI-2, hDLG (27), NHERF2, PDGFR (19), NEP (28), and MVP (29) have been identified as potential components of the PTEN complex using the same size-exclusion chromatography methodology.In this paper, we aim to (i) investigate the essential elements of PTEN required for its complex formation and (ii) dissect the components of the PTEN-associated complex(es). Our results indicate that PTEN catalytic activity or its PDZ binding domain is not absolutely required for complex assembly. PTEN phosphorylation status on amino acids Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵, on the other hand, has a significant role in complex formation. In addition, we demonstrate that the PTEN complex is enriched in nuclear lysates, which suggests a mechanism through which phosphorylation can regulate complex assembly. Using two-dimensional difference gel electrophoresis (DIGE) analysis and comparing proteins present in higher molecular mass fractions in the presence and absence of PTEN followed by mass spectrometry analysis, we have identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a potential component within the PTEN complex. PTEN and hnRNP C are shown here to interact and co-localize in the nucleus. We hypothesize that the PTEN and hnRNP C complex may play a role in RNA regulation.     

Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase

Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD₂)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-β chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the β-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys⁶⁵ residue and its network of hydrogen bonds with the polar residues Ser⁴⁵, Thr⁶⁷, and Ser⁸¹, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys⁶⁵ residue is activated by its interaction with Ser⁴⁵ and Thr⁶⁷ and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.Prostaglandin (PG)⁶ D synthase (PGDS; PGH₂ d-isomerase (EC 5.3.99.2)) (1, 2) produces PGD₂, having 9α-hydroxy and 11-keto groups, from PGH₂, which bears the chemically labile 9,11-endoperoxide group and is produced as a common intermediate of all prostanoids by the action of cyclooxygenase (PGH₂ synthase). Two distinct types of PGDS have evolved from phylogenetically distinct protein families (2, 3). One is hematopoietic PGDS (H-PGDS), which belongs to the σ class of GSH S-transferases (4, 5), and the other is lipocalin type PGDS (L-PGDS), a member of the lipocalin family (6, 7). L-PGDS is the only enzyme in the lipocalin family and is identical to β-trace, a major protein in human cerebrospinal fluid (8, 9). Although H-PGDS and L-PGDS catalyze the same reaction, their amino acid sequences and tertiary structures are quite different from each other, indicating that these enzymes are a new example of functional convergence (2, 3).L-PGDS is expressed in the heart, central nervous system, and male genital organs of various mammals and is involved in various physiological and pathological functions (reviewed in Refs. 6 and 7). In the brain, L-PGDS produces PGD₂, which is involved in the regulation of pain and non-rapid eye movement sleep, as was shown in studies using gene knock-out mice (10, 11) and human enzyme transgenic mice (12). L-PGDS is regulated by SOX9 and is involved in the differentiation of male genital organs (13–15). This enzyme is also expressed in adipocytes (16), vascular smooth muscle cells (17), and myocardial cells (18, 19) and is involved in adipocyte differentiation, the progression of arteriosclerosis (20), and the protection against hypoxemia (18) or ischemia/reperfusion injury (19). L-PGDS binds various lipophilic compounds, such as retinoids (21), bilirubin, biliverdin (22), gangliosides (23), and amyloid-β peptides (24, 25), with high affinity, acting as an extracellular transporter of these compounds and serving as an endogenous amyloid-β chaperone to prevent amyloid deposition in vivo (24).Although many biochemical and physiological studies suggest important roles of PGD₂ and L-PGDS/β-trace in the regulation of sleep and other biological functions, the crystal structure of L-PGDS has not been resolved. In this study, we determined the crystal structures of two different forms of the Δ^1–24-C65A mutant of mouse L-PGDS in both open and closed conformations. L-PGDS was shown to possess a typical lipocalin fold, the β-barrel, which is a unique structural component specific to L-PGDS and comprises a mobile E-F loop and a large central cavity with two compartments. By performing site-directed mutagenesis of Δ^1–24-L-PGDS and the Δ^1–24-C65A mutant, we found that the Cys⁶⁵ surrounded by the hydroxyl side chains of Ser⁴⁵, Thr⁶⁷, and Ser⁸¹ was activated to contribute to the catalysis by L-PGDS.     

Dual Targeting of a tRNAAsp Requires Two Different Aspartyl-tRNA Synthetases in Trypanosoma brucei

The mitochondrion of the parasitic protozoon Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by partial import of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single copy genes, suggesting the use of the same enzyme in the cytosol and in the mitochondrion. However, the T. brucei genome encodes two distinct genes for eukaryotic aspartyl-tRNA synthetase (AspRS), although the cell has a single tRNA^Asp isoacceptor only. Phylogenetic analysis showed that the two T. brucei AspRSs evolved from a duplication early in kinetoplastid evolution and also revealed that eight other major duplications of AspRS occurred in the eukaryotic domain. RNA interference analysis established that both Tb-AspRS1 and Tb-AspRS2 are essential for growth and required for cytosolic and mitochondrial Asp-tRNA^Asp formation, respectively. In vitro charging assays demonstrated that the mitochondrial Tb-AspRS2 aminoacylates both cytosolic and mitochondrial tRNA^Asp, whereas the cytosolic Tb-AspRS1 selectively recognizes cytosolic but not mitochondrial tRNA^Asp. This indicates that cytosolic and mitochondrial tRNA^Asp, although derived from the same nuclear gene, are physically different, most likely due to a mitochondria-specific nucleotide modification. Mitochondrial Tb-AspRS2 defines a novel group of eukaryotic AspRSs with an expanded substrate specificity that are restricted to trypanosomatids and therefore may be exploited as a novel drug target.In most animal and fungal mitochondria, the total set of tRNAs required for translation is encoded on the mitochondrial genome and thus of bacterial evolutionary origin. The aminoacyl-tRNA synthetases (aaRSs)² responsible for charging of mitochondrial tRNAs are always nuclear encoded and need to be imported into mitochondria. We therefore expect to find two sets of aaRSs, one for cytosolic aminoacyl-tRNA synthesis and a second one, of bacterial evolutionary origin, for aminoacylation of mitochondrial tRNAs (1, 2).In most cells, however, some aaRSs are targeted to both the cytosol as well as to mitochondria (3). In Saccharomyces cerevisiae, for example, four aaRSs are double-targeted to both compartments, indicating that they are able to aminoacylate tRNAs of both eukaryotic and bacterial evolutionary origin (4–6). In plants, the situation is more complex, since protein synthesis occurs in three compartments: the cytosol, the mitochondria, and the plastids. A recent analysis in Arabidopsis has shown that, rather than having three unique sets of aaRSs specific for the three translation systems, more than 15 aaRSs were dually targeted to the mitochondria and the plastid (7). Moreover, there is at least one aaRS that is shared between all three compartments. In summary, these examples indicate that the overlap between the different sets of aaRSs used in the various translation systems is variable and can be extensive.Most eukaryotes, except many animals and fungi, lack a variable number of mitochondrial tRNA genes. Mitochondrial translation in these organisms depends on import of a small fraction of the corresponding nucleus-encoded cytosolic tRNAs (8–10). As a consequence, imported tRNAs are always of eukaryotic evolutionary origin. An intriguing situation is found in trypanosomatids (such as Trypanosoma brucei and Leishmania spp.), where all mitochondrial tRNA genes have apparently been lost and all mitochondrial tRNAs are imported from the cytosol. In these organisms, all mitochondrial tRNAs derive from cytosolic tRNAs (11). It is therefore reasonable to assume that trypanosomal aaRSs are dually targeted to the cytosol and the mitochondrion. For the T. brucei glutaminyl-tRNA synthetase (GlnRS) and the glutamyl-tRNA synthetase, the dual localization has been shown experimentally (12). Moreover, dual targeting of essentially all aaRSs is suggested by the fact that the genome of T. brucei and other trypanosomatids encodes only 23 distinct aaRSs, fewer than any other eukaryote that has a mitochondrial translation system (13). Unexpectedly, two distinct genes were found for the tryptophanyl-tRNA synthetase (TrpRS), the lysyl-tRNA synthetase and the aspartyl-tRNA synthetase (AspRS). A recent study has shown that the two trypanosomal TrpRSs are required for cytosolic and mitochondrial tryptophanyl-tRNA formation (14). Trypanosomal tRNA^Trp is imported to the mitochondria, where it undergoes C to U editing at the wobble nucleotide and is thiolated at position 33. The RNA editing is required to decode the reassigned mitochondrial tryptophan codon UGA (14–16). Both nucleotide modifications are antideterminants for the cytosolic TrpRS (14). As we concluded previously (14), the presence of a second TrpRS with expanded substrate specificity is required to efficiently aminoacylate imported, mature tRNA^Trp in trypanosomal mitochondria.The present study focuses on the characterization and functional analysis of another pair of duplicated trypanosomal aaRSs, the AspRSs. We show that the two enzymes are individually essential for normal growth of insect stage T. brucei. We also demonstrate that the two trypanosomal AspRSs are of eukaryotic evolutionary origin and that the aminoacylation of the cytosolic and mitochondrial tRNA^Asp species requires these two distinct AspRSs.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Catalytic Properties of RNase BN/RNase Z from Escherichia coli: RNase BN IS BOTH AN EXO- AND ENDORIBONUCLEASE*

Processing of the 3′ terminus of tRNA in many organisms is carried out by an endoribonuclease termed RNase Z or 3′-tRNase, which cleaves after the discriminator nucleotide to allow addition of the universal -CCA sequence. In some eubacteria, such as Escherichia coli, the -CCA sequence is encoded in all known tRNA genes. Nevertheless, an RNase Z homologue (RNase BN) is still present, even though its action is not needed for tRNA maturation. To help identify which RNA molecules might be potential substrates for RNase BN, we carried out a detailed examination of its specificity and catalytic potential using a variety of synthetic substrates. We show here that RNase BN is active on both double- and single-stranded RNA but that duplex RNA is preferred. The enzyme displays a profound base specificity, showing no activity on runs of C residues. RNase BN is strongly inhibited by the presence of a 3′-CCA sequence or a 3′-phosphoryl group. Digestion by RNase BN leads to 3-mers as the limit products, but the rate slows on molecules shorter than 10 nucleotides in length. Most interestingly, RNase BN acts as a distributive exoribonuclease on some substrates, releasing mononucleotides and a ladder of digestion products. However, RNase BN also cleaves endonucleolytically, releasing 3′ fragments as short as 4 nucleotides. Although the presence of a 3′-phosphoryl group abolishes exoribonuclease action, it has no effect on the endoribonucleolytic cleavages. These data suggest that RNase BN may differ from other members of the RNase Z family, and they provide important information to be considered in identifying a physiological role for this enzyme.Maturation of tRNA precursors requires the removal of 5′ and 3′ precursor-specific sequences to generate the mature, functional tRNA (1). In eukaryotes, archaea, and certain eubacteria, the 3′-processing step is carried out by an endoribonuclease termed RNase Z or 3′-tRNase (2–6). However, in some bacteria, such as Escherichia coli, removal of 3′ extra residues is catalyzed by any of a number of exoribonucleases (7, 8). The major determinant for which mode of 3′-processing is utilized appears to be whether or not the universal 3′-terminal CCA sequence is encoded (2, 9). Thus, for those tRNA precursors in which the CCA sequence is absent, endonucleolytic cleavage by RNase Z right after the discriminator nucleotide generates a substrate for subsequent CCA addition by tRNA nucleotidyltransferase (1–3, 10). In view of this role for RNase Z in 3′-tRNA maturation, it is surprising that E. coli, an organism in which the CCA sequence is encoded in all tRNA genes (2), nevertheless contains an RNase Z homologue (11), because its action would appear not to be necessary. In fact, the physiological function of this enzyme in E. coli remains unclear, because mutants lacking this protein have no obvious growth phenotype (12). Hence, there is considerable interest in understanding the enzymatic capabilities of this enzyme.The E. coli RNase Z homologue initially was identified as a zinc phosphodiesterase (11) encoded by the elaC gene (now called rbn) (13). Subsequent work showed that the protein also displayed endoribonuclease activity on certain tRNA precursors in vitro (6, 14). However, more recent studies revealed that this protein actually is RNase BN, an enzyme originally discovered in 1983 and shown to be essential for maturation of those bacteriophage T4 tRNA precursors that lack a CCA sequence (15, 16). Using synthetic mimics of these T4 tRNA precursors, RNase BN was found to remove their 3′-terminal residue as a mononucleotide to generate a substrate for tRNA nucleotidyltransferase. Based on these reactions RNase BN was originally thought to be an exoribonuclease (13, 15, 17). However, subsequent work by us and others showed that it can act as an endoribonuclease on tRNA precursors (13, 18). RNase BN is required for maturation of tRNA precursors in E. coli mutant strains devoid of all other 3′-tRNA maturation exoribonucleases, although it is the least efficient RNase in this regard (7, 19). Thus, under normal circumstances, it is unlikely that RNase BN functions in maturation of tRNA in vivo except in phage T4-infected cells (15, 16).To obtain additional information on what types of RNA molecules might be substrates for RNase BN and to clarify whether it is an exo- or endoribonuclease, we have carried out a detailed examination of its catalytic properties and substrate specificity. We show here that RNase BN has both exo- and endoribonuclease activity and that it can act on a wide variety of RNA substrates. These findings suggest that E. coli RNase BN may differ from other members of the RNase Z family of enzymes.