Cloning and sequence analysis of the human cDNA encoding the synaptoporin (Δ), a highly conservative synaptic vesicle protein

Synaptoporin is a membrane protein of synaptic vesicles, which belongs to the synaptophysin family. It was first isolated from rat brain. Here we report the cloning and characterization of the human cDNA that encodes the human homologue of synaptoporin. It has two splicing variants, which is in accordance with its ortholog in chicken. Its deduced amino acid sequence displays 97% and 81% identity with the synaptoporin of rat and chicken, respectively. This gene was mapped to the chromosome 3p14.3 by matching against the Human Genome Sequence Database. 16-tissue RT-PCR analysis shows that human synaptoporin is specifically expressed in the brain.     

Assignment of the gene encoding the catalytic subunit Cβ of CAMP-dependent protein kinase to the p36 band on chromosome

Summary A cDNA for the human catalytic subunit (C) of cAMP-dependent protein kinase (PKA) has been cloned from a testis cDNA library. In the present study, we have determined the chromosomal localization of this gene using a cDNA for C as a probe. Southern blot analysis of genomic DNA from human/mouse cell hybrids revealed that the presence or absence of a 20-kbXbaI fragment, which hybridized with the C probe, was concordant with the presence of human chromosome 1.In situ hybridization to metaphase chromosome confirmed the somatic cell hybrid data and regionally mapped the C gene of PKA to the p36 band on chromosome 1.     

Structure and function of the human sperm-specific isoform of protein kinase A (PKA) catalytic subunit Cα2

Protein kinase A (PKA) exists as several tissue-specific isoforms that through phosphorylation of serine and threonine residues of substrate proteins act as key regulators of a number of cellular processes. We here demonstrate that the human sperm-specific isoform of PKA named Cα2 is important for sperm motility and thus male fertility. Furthermore, we report on the first three-dimensional crystal structure of human apo Cα2 to 2.1 ?. Apo Cα2 displays an open conformation similar to the well-characterized apo structure of murine Cα1. The asymmetric unit contains two molecules and the core of the small lobe is rotated by almost 13° in the A molecule relative to the B molecule. In addition, a salt bridge between Lys72 and Glu91 was observed for Cα2 in the apo-form, a conformation previously found only in dimeric or ternary complexes of Cα1. Human Cα2 and Cα1 share primary structure with the exception of the amino acids at the N-terminus coded for by an alternative exon 1. The N-terminal glycine of Cα1 is myristoylated and this aliphatic chain anchors the N-terminus to an intramolecular hydrophobic pocket. Cα2 cannot be myristoylated and the crystal structure revealed that the equivalent hydrophobic pocket is unoccupied and exposed. Nuclear magnetic resonance (NMR) spectroscopy further demonstrated that detergents with hydrophobic moieties of different lengths can bind deep into this uncovered pocket. Our findings indicate that Cα2 through the hydrophobic pocket has the ability to bind intracellular targets in the sperm cell, which may modulate protein stability, activity and/or cellular localization.     

Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

Protein kinase CK2 (formerly “casein kinase 2”) is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement the limited biochemical knowledge about the second paralog (CK2α′), we developed a well-soluble catalytically active full-length mutant of human CK2α′, characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity of CK2α′ for CK2β is about 12 times lower than that of CK2α and is less driven by enthalpy. This result fits the observation that the β4/β5 loop, a key element of the CK2α/CK2β interface, adopts an open conformation in CK2α′, while in CK2α, it opens only after assembly with CK2β. The open β4/β5 loop in CK2α′ is stabilized by two elements that are absent in CK2α: (1) the extension of the N-terminal β-sheet by an additional β-strand, and (2) the filling of a conserved hydrophobic cavity between the β4/β5 loop and helix αC by a tryptophan residue. Moreover, the interdomain hinge region of CK2α′ adopts a fully functional conformation, while unbound CK2α is often found with a nonproductive hinge conformation that is overcome only by CK2β binding. Taken together, CK2α′ exhibits a significantly lower affinity for CK2β than CK2α; moreover, in functionally critical regions, it is less dependent on CK2β to obtain a fully functional conformation.     

Tissue-specific differences of the mitochondrial protein import machinery: in vitro import,processing and degradation of the pre-F1β subunit of the ATP synthase in spinach leaf and root mitochondria

In this study we report the first comparison of the mitochondrial protein import and processing events in two different tissues from the same organism. Both spinach leaf and root mitochondria were able to import and process the in vitro transcribed and translated Neurospora crassa F₁ subunit of ATP synthase to the mature size product. Temperature optimum for protein import, 20 °C, was considerably lower than that found in other systems. In spinach leaf mitochondria, the processing peptidase has been shown to constitute an integral part of the bc₁ complex of the respiratory chain. In accordance with these results, the majority of the processing activity in root mitochondria was also localized in the membrane. However, although the same amount of the processing peptidase was present per mg of membrane protein in both leaf and root mitochondria, as determined immunologically, the specific processing activity was several-fold higher in roots. Furthermore, in contrast to the processing enzyme in leaf, a portion of the processing activity could be disassociated from the root membrane with relatively weak salt treatment. The processing event in both the leaf and root membranes was always accompanied by a degradation of the F₁ precursor. The degradation activity was found to be several-fold higher in roots than in leaves and was also partially dissociated from the membrane after salt treatment. Both the processing and degradation activities were inhibited by orthophenanthroline, a known metalloprotease inhibitor. These results show tissue-specific differencies of the processing event catalyzed by the bc₁ complex and indicate the presence of two populations of the processing peptidase in root mitochondria.     

Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2

CK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2α catalytic subunit in the hinge/αD region, the p-loop and the β4β5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/αD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the β regulatory subunits.     

Cloning and sequence of the cDNA encoding the β4 integrin subunit in rat peripheral nerve.

β4 and α6 integrin subunits dimerize to form an adhesion receptor that is necessary to nucleate hemidesmosomes and to anchor epithelial cells to their basal laminae. β4 is also expressed in Schwann cells (which do not contain hemidesmosomes) in peripheral nerve, where it may function in the formation or maintenance of myelin. The cDNA for β4 integrin has been cloned from epithelia-derived human and mouse tissues. We cloned cDNAs encoding β4 integrin from libraries derived from rat peripheral nerve, and determined the complete nucleotide sequence encoding the signal peptide and mature protein. Comparison of the deduced amino acid (aa) sequence revealed 95.1% and 87.5% identity with the mouse and human epithelia-derived sequences, respectively. The amino acid sequence of postulated signal transduction domains in β4 was 100% identical among rat, mouse, and human. Our cDNA clones included two of the four postulated alternatively spliced variants previously described in epithelial clones. Despite the potentially diverse functions of β4 integrin in Schwann cells and keratinocytes, the cDNAs for nerve-derived β4 integrin are highly similar to those cloned from epithelia.     

Mapping of the regulatory type Iα and catalytic β subunits of cAMP-dependent protein kinase and interleukin 1α and 1β in the pig

The genes coding for the regulatory type I subunit (PRKAR1A) and the catalytic subunit (PRKACB) of cAMP-dependent protein kinase and the genes for interleukin 1 (IL1A) and interleukin 1 (IL1B) were localized in the pig by means of radioactive in situ hybridization. PRKAR1A was mapped to 12p1.4 and PRKARB to 6q3.1 q3.3. The genes for IL1A and IL1B were both assigned to Chromosome (Chr) 3, in the region q1.2 q1.3 and q1.1 q1.4, respectively. The cDNA nucleotide sequences of these porcine genes were compared with those of human, mouse, and cattle. The location of the genes was discussed in relation to the position of their homologous loci in these mammalian species.     

Overexpression of nuclear protein kinase CK2 α catalytic subunit (CK2α) as a poor prognosticator in human colorectal cancer

Background

Colorectal cancer (CRC) is one of the most common malignancies but the current therapeutic approaches for advanced CRC are less efficient. Thus, novel therapeutic approaches are badly needed. The purpose of this study is to investigate the involvement of nuclear protein kinase CK2 α subunit (CK2α) in tumor progression, and in the prognosis of human CRC.

Methodology/Principal Findings

Expression levels of nuclear CK2α were analyzed in 245 colorectal tissues from patients with CRC by immunohistochemistry, quantitative real-time PCR and Western blot. We correlated the expression levels with clinicopathologic parameters and prognosis in human CRC patients. Overexpression of nuclear CK2α was significantly correlated with depth of invasion, nodal status, American Joint Committee on Cancer (AJCC) staging, degree of differentiation, and perineural invasion. Patients with high expression levels of nuclear CK2α had a significantly poorer overall survival rate compared with patients with low expression levels of nuclear CK2α. In multi-variate Cox regression analysis, overexpression of nuclear CK2α was proven to be an independent prognostic marker for CRC. In addition, DLD-1 human colon cancer cells were employed as a cellular model to study the role of CK2α on cell growth, and the expression of CK2α in DLD-1 cells was inhibited by using siRNA technology. The data indicated that CK2α-specific siRNA treatment resulted in growth inhibition.

Conclusions/Significance

Taken together, overexpression of nuclear CK2α can be a useful marker for predicting the outcome of patients with CRC.     

AMP-activated protein kinase-��1 as an activating kinase of TGF-��-activated kinase 1 has a key role in inflammatory signals

Although previous studies have proposed plausible mechanisms of the activation of transforming growth factor-β-activated kinase 1 (TAK1) in inflammatory signals, including Toll-like receptors (TLRs), its activating kinase still remains to be unclear. In the present study, we have provided evidences that AMP-activated protein kinase (AMPK)-α1 has a pivotal role for activating TAK1, and thereby regulate NF-κB-dependent gene expressions in inflammatory signaling mediated by TLR4 and TNF-α stimulation. AMPK-α1 specifically interacts with TAK1 and reciprocally regulates their kinase activities. Upon the stimulation of lipopolysaccharide, AMPK-α1-knockdown (AMPK-α1^KD) or TAK1-knockdown human monocytic THP-1 cells exhibit a dramatic reduction in the TAK1 or AMPK-α1 kinase activity, respectively, and subsequent suppressions of its downstream signaling cascades, which further leads to inhibitions of NF-κB and thereby productions of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Importantly, the microarray analysis of AMPK-α1^KD cells revealed a dramatic reduction in the NF-κB-dependent genes induced by TLR4 and TNF-α stimulation, and the observation was in significant correlation with the results of quantitative real-time PCR. Moreover, AMPK-α1^KD cells are highly sensitive to the TNF-α-induced apoptosis, which is accompanied with dramatic reductions in the NF-κB-dependent and anti-apoptotic genes. As a result, our data demonstrate that AMPK-α1 as an activating kinase of TAK1 has a key role in mediating inflammatory signals triggered by TLR4 and TNF-α.     

Cloning and analysis of the cDNA encoding the rod G-protein transducin α, β1 and γ1 subunits from the canine retina

The canine (Canis familiaris) retinal rod transducin (G_T) α, β1 and γ1 subunits were sequenced. Cloning of the cDNAs was accomplished by polymerase chain reaction (PCR) using degenerate and wild type retinal cDNA libraries as templates. The deduced amino acid sequences were highly similar to rod transducins from other species: G_Tα differed by 5 amino acids from the corresponding human sequence, whereas β1 and γ1 were identical to human sequences. The coding sequence of rod transducin was evaluated as a possible cause for the recessively inherited retinal rod-cone degeneration: there were no nucleotide differences between the wild type and retinal degenerate strains.     

Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase α

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase α (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.     

Purification and functional analysis of protein kinase G-1α using a bacterial expression system

3',5' Cyclic guanosine monophosphate (cGMP)-dependent protein kinase G-1α (PKG-1α) is an enzyme that is a target of several anti-hypertensive and erectile dysfunction drugs. Binding of cGMP to PKG-1α produces a conformational change that leads to enzyme activation. Activated PKG-1α performs important roles both in blood vessel vasodilation and in maintaining the smooth muscle cell in a differentiated contractile state. Recombinant PKG-1α has been expressed and purified using Sf9-insect cells. However, attempts at purifying full length protein in a soluble and active form in prokaryotes have thus far been unsuccessful. These attempts have been hampered by the lack of proper eukaryotic protein folding machinery in bacteria. In this study, we report the successful expression and purification of PKG-1α using a genetically engineered Escherichia coli strain, Rosetta-gami 2(DE3), transduced with full-length human PKG-1α cDNA containing a C-terminal histidine tag. PKG-1α was purified to homogeneity using sequential nickel affinity chromatography, gel filtration and ion exchange MonoQ columns. Protein identity was confirmed by immunoblot analysis. N-terminal sequencing using Edman degradation demonstrated that the purified protein was full length. Analysis of enzyme kinetics, using a nonlinear regression curve, identified that, at constant cGMP levels (10μM) and varying ATP concentrations, PKG-1α had a maximal velocity (V(max)) of 5.02±0.25pmol/min/μg and a Michaelis-Menten constant (K(m)) of 11.78±2.68μM ATP. Recent studies have suggested that endothelial function can be attenuated by oxidative and/or nitrosative stress but the role of PKG-1α under these conditions is unclear. We found that PKG-1α enzyme activity was attenuated by exposure to the NO donor, spermine NONOate, hydrogen peroxide, and peroxynitrite but not by superoxide, suggesting that the attenuation of PKG-1α activity may be an under-appreciated mechanism underlying the development of endothelial dysfunction in a number of cardiovascular diseases.     

Modification and identification of glutamate residues at the arginine-recognition site in the catalytic subunit of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

It has been proposed that the active centre of cyclic AMP-dependent protein kinase contains an arginine-recognition site, which is considered to be essential for the function of the catalytic subunit of the kinase [Matsuo, Huang & Huang (1978) Biochem. J.173, 441-447]. The catalytic subunit can be inactivated by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide and glycine ethyl ester at pH6.5. The enzyme can be protected from inactivation by preincubation with histone, a protein substrate of the enzyme. On the other hand, ATP, which also serves as a protein kinase substrate, does not afford protection. Polyarginine, a competitive inhibitor of protein kinase, which is known from kinetic studies to interact specifically with the arginine-recognition site, partially protects the catalytic subunit from inactivation by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide. These results lead to the conclusion that the site of modification by carbodi-imide/glycine ethyl ester is most likely located at the arginine-recognition site of the active centre. A value of 1.7+/-0.2 (mean+/-s.d.) mol of carboxy groups per mol of catalytic subunit has been obtained for the number of essential carboxy groups for the function of protein kinase; a complete chemical modification of these essential carboxy groups results in total loss of catalytic activity. Finally, we have identified the essential carboxy group in the catalytic subunit of cyclic AMP-dependent protein kinase as being derived from glutamate residues. This is achieved by a three-step procedure involving an extensive proteolytic digestion of the [1-(14)C]glycine ethyl ester-modified enzyme and two successive high-voltage electrophoreses of the hydrolysate. It is concluded that 1.7mol of glutamyl carboxy groups per mol of catalytic subunit may be considered a component of the arginine-recognition site in the active centre of cyclic AMP-dependent protein kinase.