Carboxyl Domain Research Articles

P27Kip1 and p21Cip1 collaborate in the regulation of transcription by recruiting cyclin-Cdk complexes on the promoters of target genes.

Transcriptional repressor complexes containing p130 and E2F4 regulate the expression of genes involved in DNA replication. During the G1 phase of the cell cycle, sequential phosphorylation of p130 by cyclin-dependent kinases (Cdks) disrupts these complexes allowing gene expression. The Cdk inhibitor and tumor suppressor p27Kip1 associates with p130 and E2F4 by its carboxyl domain on the promoters of target genes but its role in the regulation of transcription remains unclear. We report here that p27Kip1 recruits cyclin D2/D3–Cdk4 complexes on the promoters by its amino terminal domain in early and mid G1. In cells lacking p27Kip1, cyclin D2/D3–Cdk4 did not associate to the promoters and phosphorylation of p130 and transcription of target genes was increased. In late G1, these complexes were substituted by p21Cip1-cyclin D1–Cdk2. In p21Cip1 null cells cyclin D1–Cdk2 were not found on the promoters and transcription was elevated. In p21/p27 double null cells transcription was higher than in control cells and single knock out cells. Thus, our results clarify the role of p27Kip1 and p21Cip1 in transcriptional regulation of genes repressed by p130/E2F4 complexes in which p27Kip1 and p21Cip1 play a sequential role by recruiting and regulating the activity of specific cyclin–Cdk complexes on the promoters.

Structure of the WD40 domain of SCAP from fission yeast reveals the molecular basis for SREBP recognition.

The sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP) are central players in the SREBP pathway, which control the cellular lipid homeostasis. SCAP binds to SREBP through their carboxyl (C) domains and escorts SREBP from the endoplasmic reticulum to the Golgi upon sterol depletion. A conserved pathway, with the homologues of SREBP and SCAP being Sre1 and Scp1, was identified in fission yeast Schizosaccharomyces pombe. Here we report the in vitro reconstitution of the complex between the C domains of Sre1 and Scp1 as well as the crystal structure of the WD40 domain of Scp1 at 2.1 Å resolution. The structure reveals an eight-bladed β-propeller that exhibits several distinctive features from a canonical WD40 repeat domain. Structural and biochemical characterization led to the identification of two Scp1 elements that are involved in Sre1 recognition, an Arg/Lys-enriched surface patch on the top face of the WD40 propeller and a 30-residue C-terminal tail. The structural and biochemical findings were corroborated by in vivo examinations. These studies serve as a framework for the mechanistic understanding and further functional characterization of the SREBP and SCAP proteins in fission yeast and higher organisms.

Mechanisms of inhibition of Rhizobium etli pyruvate carboxylase by L-aspartate.

l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that l-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. l-Aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of l-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of l-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. l-Aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, l-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and l-aspartate) are spatially distinct but connected by a network of allosteric interactions.

Physiological roles of regulated Ire1 dependent decay.

Inositol-requiring enzyme 1 (Ire1) is an important transducer of the unfolded protein response (UPR) that is activated by the accumulation of misfolded proteins in the endoplamic reticulum (ER stress). Activated Ire1 mediates the splicing of an intron from the mRNA of Xbp1, causing a frame-shift during translation and introducing a new carboxyl domain in the Xbp1 protein, which only then becomes a fully functional transcription factor. Studies using cell culture systems demonstrated that Ire1 also promotes the degradation of mRNAs encoding mostly ER-targeted proteins, to reduce the load of incoming ER “client” proteins during ER stress. This process was called RIDD (regulated Ire1-dependent decay), but its physiological significance remained poorly characterized beyond cell culture systems. Here we review several recent studies that have highlighted the physiological roles of RIDD in specific biological paradigms, such as photoreceptor differentiation in Drosophila or mammalian liver and endocrine pancreas function. These studies demonstrate the importance of RIDD in tissues undergoing intense secretory function and highlight the physiologic role of RIDD during UPR activation in cells and organisms.

Calorimetric studies of the interactions of linker histone H10 and its carboxyl (H10–C) and globular (H10–G) domains with calf-thymus DNA

Histone H1 is a chromatin protein found in most eukaryotes. ITC and CD have been used to study the binding of H10 and its C-terminal, H10–C, and globular, H10–G, domains to a highly polymerized DNA. ITC results indicate that H10 and H10–C bind tightly to DNA (Ka≈1×107), with an unfavorable ΔH (ΔH≈+22kcal/mol) and a favorable ΔS (−TΔS≈−30kcal/mol). Binding H10–G to DNA at 25°C is calorimetrically silent. A multiple independent site model fits the ITC data, with the anomaly in the data near saturation attributed to rearrangement of bound H1, maximizing the number of binding sites. CD experiments indicate that H10/DNA and H10–C/DNA complexes form with little change in protein structure but with some DNA restructuring. Salt dependent ITC experiments indicate that the electrostatic contribution to binding H10 or H10–C is small ranging from 6% to 17% of the total ΔG.

Influence of solvent on the supramolecular architectures in molecular gels

Elucidating the molecular structures, responsible for promoting self-assembly of low-molecular weight organogelators (LMOG) into supramolecular fibers, has been an extensive area of study. Although this has been a fruitful endeavor, this study illustrates that the chemical nature of the solvent and solvent–gelator interactions are equally important. The nanostructure, microstructure and supramolecular structures, of 12HSA molecules gels, are all influenced by the chemical nature of the solvent, which correlate to the hydrogen-bonding Hansen solubility parameter (∂h). Depending on the solvent employed, the polymorphic form, arrangement of the carboxylic acid dimers, domain size, fiber morphology, microstructure, thermal properties and visual appearance of the gel all differ. Solvents that have δh 4.4 MPa1/2.

The Minimal Deneddylase Core of the COP9 Signalosome Excludes the Csn6 MPN− Domain

The COP9 signalosome (CSN) is a eukaryotic protein complex, which regulates a wide range of biological processes mainly through modulating the cullin ubiquitin E3 ligases in the ubiquitin-proteasome pathway. The CSN possesses a highly conserved deneddylase activity that centers at the JAMM motif of the Csn5 subunit but requires other subunits in a complex assembly. The classic CSN is composed of 8 subunits (Csn1–8), yet in several Ascomycota, the complex is smaller and lacks orthologs for a few CSN subunits, but nevertheless contains a conserved Csn5. This feature makes yeast a powerful model to determine the minimal assemblage required for deneddylation activity. Here we report, that Csi1, a diverged S. cerevisiae CSN subunit, displays significant homology with the carboxyl terminal domain of the canonical Csn6, but lacks the amino terminal MPN- domain. Through the comparative and experimental analyses of the budding yeast and the mammalian CSNs, we demonstrate that the MPN− domain of the canonical mouse Csn6 is not part of the CSN deneddylase core. We also show that the carboxyl domain of Csn6 has an indispensable role in maintaining the integrity of the CSN complex. The CSN complex assembled with the carboxyl fragment of Csn6, despite its lack of an MPN− domain, is fully active in deneddylation of cullins. We propose that the budding yeast Csi1 is a functional equivalent of the canonical Csn6, and thus the composition of the CSN across phyla is more conserved than hitherto appreciated.

Get5 Carboxyl-terminal Domain Is a Novel Dimerization Motif That Tethers an Extended Get4/Get5 Complex

Tail-anchored trans-membrane proteins are targeted to membranes post-translationally. The proteins Get4 and Get5 form an obligate complex that catalyzes the transfer of tail-anchored proteins destined to the endoplasmic reticulum from Sgt2 to the cytosolic targeting factor Get3. Get5 forms a homodimer mediated by its carboxyl domain. We show here that a conserved motif exists within the carboxyl domain. A high resolution crystal structure and solution NMR structures of this motif reveal a novel and stable helical dimerization domain. We additionally determined a solution NMR structure of a divergent fungal homolog, and comparison of these structures allows annotation of specific stabilizing interactions. Using solution x-ray scattering and the structures of all folded domains, we present a model of the full-length Get4/Get5 complex.

Human cell-death-inducing DFF45-like effector C induces apoptosis via caspase-8

Human cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector C (CIDEC) is a potent apoptotic inducer. Previous studies have indicated that the Fat-specific protein 27 (Fsp27), a mouse homolog of CIDEC, induces apoptosis via caspase-3, -7, and -9 and triggers the release of cytochrome c from mitochondria, which implies that the mitochondrial pathway is involved in Fsp27-induced apoptosis. In the current study, we found that CIDEC-induced apoptosis was mediated by caspase-8. The caspase inhibitor assay showed that CIDEC-induced apoptosis was dramatically reduced in the presence of the general caspase inhibitor, the caspase-3 inhibitor, and the caspase-8 inhibitor, whereas the caspase-9 inhibitor only weakly inhibited CIDEC-induced apoptosis. These results confirmed that the activation of caspase-3 and caspase-8 were involved in CIDEC-induced apoptosis. Moreover, in caspase-3- or caspase-8-deficient cells, CIDEC-induced apoptosis were dramatically decreased, which demonstrated that CIDEC-induced apoptosis might require the activation of caspase-3 and caspase-8. Because caspase-8 in general is a key effecter of death-receptor pathway and activated by Fas-Associated protein with Death Domain (FADD), we examined whether FADD was involved in CIDEC-induced apoptosis. Our results demonstrated that CIDEC-induced apoptosis was independent of FADD, suggesting that CIDEC-induced apoptosis might be in a death-receptor-independent, caspase-8-dependent manner. It was also found that the region of amino acid 168-200 in carboxyl domain of CIDEC was critical for its crucial pro-apoptotic function.

Luminal-Applied Flagellin Is Internalized by Polarized Intestinal Epithelial Cells and Elicits Immune Responses via the TLR5 Dependent Mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.

High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli

The carboxylic domain of the Clostridium botulinum neurotoxin heavy chain (BoNT/A-HC), which has been reported as a vaccine candidate, contains the principle protective antigenic determinants. In this study, the high level expression of the BoNT/A-Hc was achieved by high cell density cultivation of recombinant Escherichia coli in a 2-l batch stirred-tank bioreactor. In order to maximize protein expression, post-induction time and IPTG inducer concentration were optimized by the Taguchi statistical design method. Results showed that the middle of the logarithmic phase and an IPTG concentration of 1mM presented the optimum conditions for the maximum expression of BoNT/A-HC. High cell density cultivation was subsequently carried out as an effective strategy for the high level expression of recombinant BoNT/A-Hc. Consequently, soluble BoNT/A-Hc was produced at the maximum level of 486mgl(-1), at 3h post-induction, which was approximately 9.3 and 7.8 times higher than the levels produced by the shake flask and batch culturing methods, respectively.

KCNE2 uses More Domains than KCNE1 to Modulate KCNQ1 Channel Function

KCNE1 & KCNE2 are both single membrane-passing peptides with amino- & carboxyl ends in extra- & intra-cellular compartments. They also share high sequence homology in the transmembrane (TM) and juxtamembranous regions. Yet the two have distinctly different effects when associated with the KCNQ1 channel. Understanding the structural basis for differential KCNQ1 modulation by the 2 KCNE subunits is prerequisite to drug design targeting such interactions. We use the techniques of NMR, cysteine (Cys)-scanning mutagenesis and voltage clamping to explore this issue. We learn the following: (1) KCNE2 differs from KCNE1 in the extracellular juxtamembranous region (helix vs unstructured loop). Cys substitution in this region has distinct impact on the gating kinetics and/or pore conductance of the KCNQ1/KCNE2 channel complex, but has little or no effects on the KCNQ1/KCNE1 channel function. (2) KCNE2 & KCNE1 sequences diverge in the carboxyl end. Truncating this region of KCNE1 (93 −129) does not interfere with its ability to modulate KCNQ1, while truncating the corresponding region of KCNE2 (98-123) abolishes its function as a KCNQ1 modulator. Intracellular application of a peptide corresponding to KCNE2 aa 98-123 reduces currents through KCNQ1, increases currents through KCNQ1/KCNE2, but has no effects on currents through KCNQ1/KCNE1. (3) Cys substitution along the TM helices of KCNE1 and KCNE2 affects the gating kinetics and/or pore conductance of the KCNQ1/KCNE channel complexes. Structural alignment suggests a rotation of KCNE2 relative to KCNE1 in terms of helical faces interacting with the pore domain and voltage-sensing domain of the KCNQ1 channel. We propose that while both KCNE subunits utilize their TM helices to interact with KCNQ1, the more rigid helical structure of KCNE2 allows it to allosterically modulate the KCNQ1 gating and ion permeation properties by the extracellular juxtamembranous and cytoplasmic carboxyl domains.

Roles of the α1A-adrenergic receptor carboxyl tail in protein kinase C-induced phosphorylation and desensitization

Noradrenaline- and tetradecanoyl phorbol acetate (TPA)-induced phosphorylation and functional desensitization of the following receptors were studied: (1) wild-type bovine α(1A)- and hamster α(1B)-adrenergic receptors (ARs), (2) chimeric ARs in which the carboxyl terminus tails were exchanged (α(1AB)- and α(1BA)-ARs), and (3) carboxyl terminus-truncated α(1A)-ARs fussed to enhanced green fluorescent protein. Noradrenaline and TPA pronouncedly increased α(1B)-AR phosphorylation while TPA markedly desensitized these receptors. In contrast, TPA-induced desensitization and TPA- and noradrenaline-induced phosphorylation of α(1A)-ARs were clearly of lesser magnitude. Chimeric ARs with exchanged carboxyl terminus tails showed that the extent of phosphorylation reflected the carboxyl domain rather than the receptor core. Surprisingly, there was no correlation between phosphorylation and functional desensitization, i.e., activation of protein kinase C clearly desensitized both chimeric receptors to a similar extent. Interestingly, TPA and noradrenaline increased carboxyl terminus-truncated α(1A)-AR phosphorylation and TPA also induced receptor desensitization. We were unable to detect carboxyl terminus-truncated α(1A)-AR internalization after 5-min stimulations with noradrenaline or TPA. Our results suggest the following: (a) the α(1A)-AR carboxyl terminus tail was not essential for signaling or desensitization; (b) carboxyl terminus tail exchange "transplanted" the phosphorylation pattern of the receptors, but the functional consequences of such a transplant were very limited; (c) α(1A)-AR desensitization was not associated to receptor internalization.

Structure of a fucose transporter in an outward-open conformation

The major facilitator superfamily (MFS) transporters are an ancient and widespread family of secondary active transporters. In Escherichia coli, the uptake of l-fucose, a source of carbon for microorganisms, is mediated by an MFS proton symporter, FucP. Despite intensive study of the MFS transporters, atomic structure information is only available on three proteins and the outward-open conformation has yet to be captured. Here we report the crystal structure of FucP at 3.1 Å resolution, which shows that it contains an outward-open, amphipathic cavity. The similarly folded amino and carboxyl domains of FucP have contrasting surface features along the transport path, with negative electrostatic potential on the N domain and hydrophobic surface on the C domain. FucP only contains two acidic residues along the transport path, Asp 46 and Glu 135, which can undergo cycles of protonation and deprotonation. Their essential role in active transport is supported by both in vivo and in vitro experiments. Structure-based biochemical analyses provide insights into energy coupling, substrate recognition and the transport mechanism of FucP.

Optimization of the BoNT/A‐Hc expression in recombinant Escherichia coli using the Taguchi statistical method

The use of the recombinant BoNT/A-Hc (carboxylic domain of the Clostridium botulinum neurotoxin heavy chain) has been proposed as a vaccine candidate for botulism. This fragment contains the principle protective antigenic determinant. In the present study, in order to maximize recombinant protein expression, after verification of recombinant BoNT/A-Hc by Western blotting, modified M9 medium was selected as a simple medium, and the operational and medium-composition variables together with their interactions were optimized by using the Taguchi statistical method. ANOVA for the obtained data indicated that 3.5 g, 15 g, 30 g, 15 g, 4 g, 0.7 mM, 1.5 ml per litre of medium, 30 degrees C and 15 h represented optimum values of (NH(4))(2)SO(4), glucose, K(2)HPO(4), KH(2)PO(4), MgSO(4) * 7H(2)O, isopropyl beta-D-thiogalactoside concentration, trace-elements solution, temperature and post-induction time respectively. Consequently, under these optimum conditions, 52.1 mg/l of soluble BoNT/A-Hc was obtained in shake flask culture.

Allosteric Mechanism in Neurophysin

Neurophysins are small two- domain proteins that exhibit ligand-facilitated dimerization and its reciprocal-stronger binding by dimer than by monomer. We have previously reported evidence of conformational differences between unliganded monomer and dimer, but had not established their identity, their origin, or how they affect binding affinity.In the present study we have determined the relative structures of a neurophysin monomer and its dimer by NMR spectroscopy. The structures show that a number of neurophysin residues that interact with the essential tyrosine of the ligand peptide move from locations in the unliganded monomer that are unfavorable for binding to positions in the unliganded dimer that more closely resemble those of the liganded state, thereby contributing to the stronger binding by dimer. We have also identified the origin of the dimerization-induced conformational change; i.e., the same residues that form the principal inter-domain bonds in the monomer via side-chain interactions form the principal inter-subunit bonds in the dimer via backbone hydrogen bonding. Steric crowding upon dimerization leads to a modification of inter-domain interactions, a resultant change in the relative orientation of the two domains and associated changes in intra-domain interactions. The principal residues responsible for these interactions are the Cys residues of the 28-34 disulfide bridge (amino domain) and 74-79 disulfide bridge (carboxyl domain). Strong inter-domain van der Waals interactions between sulfur atoms appear to play important roles in determining the relative domain orientation.(Supported by grant GM-17528 from NIH).

Crystal structure and mutagenic analysis of GDOsp, a gentisate 1,2‐dioxygenase from Silicibacter Pomeroyi

Dioxygenases catalyze dioxygen incorporation into various organic compounds and play a key role in the complex degradation pathway of mono- and polycyclic aromatic and hetero-aromatic compounds. Here we report the crystal structure of gentisate 1,2-dioxygenase from Silicibacter pomeroyi (GDOsp) at a 2.8 A resolution. The enzyme possessed a conserved three-dimensional structure of the bicupin family, forming a homotetramerization. However, each subunit of GDOsp unusually contained two ferrous centers that were located in its two homologous cupin domains, respectively. Further mutagenic analysis indicated that the enzyme activity of GDOsp depends on the microenvironment in both metal-binding sites. Moreover, homologous structural comparison and functional study on GDOsp variants unveiled a group of functionally essential residues and suggested that the active site of the enzyme is located in the amino-terminal domain, but could be influenced by changes in the carboxyl domain. Therefore, GDOsp may provide a working model for studying long-distance communication within a protein (or its complex).

TSPY and its X-encoded homologue interact with cyclin B but exert contrasting functions on cyclin-dependent kinase 1 activities

Testis-specific protein Y-encoded (TSPY) is the putative gene for the gonadoblastoma locus on the Y chromosome (GBY). TSPY and an X-homologue, TSPX, harbor a conserved domain, designated as SET/NAP domain, but differ at their C termini. Ectopic expression of TSPY accelerates cell proliferation by abbreviating the G(2)/M stage, whereas overexpression of TSPX retards cells at the same stage of the cell cycle. Previous studies demonstrated that the SET oncoprotein is capable of binding to cyclin B. Using various protein interaction techniques, we demonstrated that TSPY and TSPX indeed bind competitively to cyclin B at their SET/NAP domains in vitro and in vivo. TSPY colocalizes with cyclin B1 during the cell cycle, particularly on the mitotic spindles at metaphase. TSPY enhances while TSPX represses the cyclin B1-CDK1 phosphorylation activity. The inhibitory effect of TSPX on the cyclin B1-CDK1 complex has been mapped to its carboxyl acidic domain that is absent in TSPY, suggesting that TSPX could serve a normal function in modulating cell-cycle progression at the G(2)/M stage, whereas TSPY has acquired a specialized function in germ cell renewal and differentiation. Epigenetic dysregulation of TSPY in incompatible germ or somatic cells could promote cell proliferation and predispose susceptible cells to tumorigenesis.

생쥐 Wee1 인산화효소들의 각 도메인의 역할에 관한 연구

Wee1 인산화효소는 세포주기 조절의 핵심 단백질인 cdc2/cyclinB 복합체를 인산화하여 활성을 억제, 조절하는 주요한 효소이다. 지금까지 포유동물에서는 Wee1A, Wee1B 그리고 Myt1의 세 가지 효소가 발견되었다. Wee1 인산화효소의 조절기작을 연구하기 위하여 생쥐의 Wee1A와 Wee1B를 발톱개구리의 난자에 주사한 후 단백질의 변형을 관찰하였다. 이 세포주기 과정에서 두 효소는 모두 인산화 되었으며, Wee1A단백질은 분해되는 것을 관찰 할 수 있었다. 또한 세포외 인산화 방법을 통하여 Wee1A가 PKA와 Akt에 의해 인산화됨을 확인하였다. 이러한 Wee1 인산화효소의 인산화와 단백질 안정성에 영향을 미치는 단백질 내의 부위를 살펴보고자, Wee1A와 Wee1B의 아미노 도메인과 카르복실 도메인을 서로 치환한 단백질을 제조하여 개구리 난자에 주사하고 인산화 정도와 단백질의 안정성을 조사하였을 때, Wee1A의 아미노 도메인이 단백질의 인산화와 안정화에 중요한 영향을 미친다는 것을 규명하였다. 그리고 Wee1B의 아미노 도메인과 Wee1A의 카르복실 도메인은 효소의 활성을 조절하는 역할을 한다. The molecular machinery controlling cell cycle is centered around the regulation of the activity of maturation-promoting factor (MPF), a complex composed of a catalytic Cdc2 and the cyclinB regulatory subunit. Cdc2 kinase is inactivated by phosphorylation of inhibitory kinase, Wee1. It has been known that there are three different Wee1 kinases in the mammalian cell, Wee1A, Wee1B and Myt1. To investigate the regulatory mechanism of Wee1 kinases, the phosphorylation and degradation of Wee1A and Wee1B were checked in the Xenopus oocyte cell cycle. When Wee1 kinases were injected into frog oocyte, Wee1B was more stable than Wee1A. Wee1A and Wee1B kinase were phosphorylated by many kinases such as PKA and Akt. The roles of amino or carboxyl terminal in mouse Wee1A or Wee1B kinase were investigated using chimeric constructs. The degree of protein phosphorylation, degradation and cell cycle progression were different between chimeric constructs. The amino domain of Wee1A was implicated in the protein phosphorylation and degradation while amino domain of Wee1B and carboxyl domain of Wee1A were involved in the activity regulation. These results suggested that the domains of Wee1 kinase have different and significant roles in regulating the Wee1 kinases in the cell cycle progression.

Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme

Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillus thermodenitrificans enzyme the biotin is covalently attached to K1112. A mutant form of the enzyme (K1112A) has been prepared which is not biotinylated. This mutant did not catalyse the complete reaction, but did catalyse ATP-cleavage and the carboxylation of free biotin. Oxaloacetate decarboxylation was not catalysed, even in the presence of free biotin, suggesting that only the biotin carboxylation domain of the enzyme is accessible to free biotin. This mutant allowed the study of ATP-cleavage both coupled and not coupled to biotin carboxylation. Kinetic analyses of these reactions indicate that the major effect of the enzyme activator, acetyl CoA, is to promote the carboxylation of biotin. Acetyl CoA reduces the Kms for both MgATP and biotin. In addition, pH profiles of the ATP-cleavage reaction in the presence and absence of free biotin revealed the involvement of several ionisable residues in both ATP-cleavage and biotin carboxylation. K1112A also catalyses the phosphorylation of ADP from carbamoyl phosphate. Stopped-flow studies using the fluorescent ATP analogue, formycin A-5′-triphosphate, in which nucleotide binding to the holoenzyme was compared to K1112A indicated that the presence of biotin enhanced binding. Attempts to trap the putative carboxyphosphate intermediate in K1112A using diazomethane were unsuccessful.