Cytoplasmic Creatine Kinase Research Articles

Seasonal expression of cytoplasmic creatine kinase in the epididymal epithelium of Pelodiscus sinensis

ABSTRACT During hibernation of Pelodiscus sinensis, sperm mature and are stored in the epididymis. We investigated seasonal changes in the morphology of epithelial cells of the epididymis of P. sinensis and changes in expression of cytoplasmic creatine kinase (CK). We found that the epididymal epithelium proliferates rapidly to form multiple layers from June to September, while the epididymal epithelial cells are arranged in a single layer from October to May. From the March before the mating period to the end of the mating period in September, a large amount of neutral glycoprotein is secreted in the epididymal epithelium and in the sperm aggregation area; after October, the glycoprotein in the epididymis decreases. At sperm maturation, cytoplasmic CK is expressed abundantly in the villous epithelium, which is formed by proliferation of epididymal epithelial cells. During hibernation and reproduction, the epididymal epithelium of P. sinensis exhibits different proliferation and secretion patterns as the animal adapts to two types of sperm storage. Cytoplasmic CK may participate in regulating the energy metabolism of the epididymal epithelium; it is an important enzyme for regulating sperm maturation.

Structure and function of resistance arteries from BB-creatine kinase and ubiquitous Mt-creatine kinase double knockout mice

Increasing evidence indicates that the enzyme creatine kinase (CK) is intimately involved in microvascular contractility. The mitochondrial isoenzyme catalyses phosphocreatine synthesis from ATP, while cytoplasmic CK, predominantly the BB isoenzyme in vascular tissue, is tightly bound near myosin ATPase, where it favours ATP production from phosphocreatine to metabolically support vascular contractility. However, the effect of CK gene inactivation on microvascular function is hitherto unknown. We studied functional and structural parameters of mesenteric resistance arteries isolated from 5 adult male mice lacking cytoplasmic BB-CK and ubiquitous mitochondrial CK (CK-/-) vs 6 sex/age-matched controls. Using a Mulvany Halpern myograph, we assessed the acute maximum contractile force with 125mMK+ and 10-5M norepinephrine, and the effect of two inhibitors, dinitrofluorobenzene, which inhibits phosphotransfer enzymes (0.1μM), and the specific adenylate kinase inhibitor P1, P5-di(adenosine 5') pentaphosphate (10-6 to 10-5M). WT and CK-/- did not significantly differ in media thickness, vascular elasticity parameters, or acute maximum contractile force. CK-/- arteries displayed greater reduction in contractility after dinitrofluorobenzene 38%; vs 14% in WT; and after AK inhibition, 14% vs 5.5% in WT, and displayed abnormal mitochondria, with a partial loss of the inner membrane. Thus, CK-/- mice display a surprisingly mild phenotype in vascular dysfunction. However, the mitochondrial abnormalities and greater effect of inhibitors on contractility may reflect a compromised energy metabolism. In CK-/- mice, compensatory mechanisms salvage energy metabolism, as described for other CK knock-out models.

Creatine is Neuroprotective to Retinal Neurons In Vitro But Not In Vivo.

To investigate the neuroprotective properties of creatine in the retina using in vitro and in vivo models of injury. Two different rat retinal culture systems (one containing retinal ganglion cells [RGC] and one not) were subjected to either metabolic stress, via treatments with the mitochondrial complex IV inhibitor sodium azide, or excitotoxic stress, via treatment with N-methyl-D-aspartate for 24 hours, in the presence or absence of creatine (0.5, 1.0, and 5.0 mM). Neuronal survival was assessed by immunolabeling for cell-specific antigens. Putative mechanisms of creatine action were investigated in vitro. Expression of creatine kinase (CK) isoenzymes in the rat retina was examined using Western blotting and immunohistochemistry. The effect of oral creatine supplementation (2%, wt/wt) on retinal and blood creatine levels was determined as well as RGC survival in rats treated with N-methyl-D-aspartate (NMDA; 10 nmol) or high IOP-induced ischemia reperfusion. Creatine significantly prevented neuronal death induced by sodium azide and NMDA in both culture systems. Creatine administration did not alter cellular adenosine triphosphate (ATP). Inhibition of CK blocked the protective effect of creatine. Retinal neurons, including RGCs, expressed predominantly mitochondrial CK isoforms, while glial cells expressed exclusively cytoplasmic CKs. In vivo, NMDA and ischemia reperfusion caused substantial loss of RGCs. Creatine supplementation led to elevated blood and retinal levels of this compound but did not significantly augment RGC survival in either model. Creatine increased neuronal survival in retinal cultures; however, no significant protection of RGCs was evident in vivo, despite elevated levels of this compound being present in the retina after oral supplementation.

A diverse array of creatine kinase and arginine kinase isoform genes is present in the starlet sea anemone Nematostella vectensis, a cnidarian model system for studying developmental evolution

Phosphagen (guanidino) kinases (PK) constitute a family of homologous phosphotransferases catalyzing the reversible transfer of the high-energy phosphoryl group of ATP to naturally occurring guanidine compounds. Prior work has shown that PKs can be phylogenetically separated into two distinct groups— an arginine kinase (AK) subfamily and a creatine kinase (CK) subfamily. The latter includes three CK isoforms— cytoplasmic CK (CyCK), mitochondrial CK (MiCK) and three-domain flagellar CK (fCK). In the present study we identified six unique PK genes from the draft genome sequence of the starlet sea anemone Nematostella vectensis, a well-known model organism for understanding metazoan developmental evolution. Using reverse transcription polymerase chain reaction (RTPCR) methods, full length cDNAs were amplified for all of these PKs. These cDNAs were cloned and expressed in Escherichia coli as 6x His-tagged fusion proteins. The six PKs were identified as the three typical CK isoforms (CyCK, MiCK and fCK), two unusual AKs (a two-domain AK (2DAK) and a three-domain AK (3DAK)) and a PK which phosphorylated arginine. The latter enzyme had a very low AK activity (its apparent Vmax value being less than 0.2% that of 3DAK), lacks several key residues necessary for AK enzyme activity, and was tentatively designated as AK1. As far as we know, this constitutes the first report of an AK with the three fused AK domains. The Bayesian tree suggested that the third domain of 3DAK likely evolved from the gene for domain 2 of typical two-domain AK found widely in cnidarians. Construction of phylogenetic trees and comparison of exon–intron organizations of their respective genes indicated that the N. vectensis three-domain fCK and 3DAK evolved independently, and both enzymes are likely to be targeted to cell membranes since they have a myristoylation signal at their respective N-termini. These results complement prior work on other basal invertebrates showing that multiple CK and AK isoform genes were present at the dawn of the radiation of metazoans. The presence of isoform diversity in an organism lacking in structural complexity reflects an early imperative for targeting of PKs to particular cellular contexts such as muscle fibers, neurons, ciliated/flagellated epithelia and spermatozoa.

RANKL Up-regulates Brain-type Creatine Kinase via Poly(ADP-ribose) Polymerase-1 during Osteoclastogenesis

Receptor activator of nuclear factor κB ligand (RANKL) is the key regulator for osteoclast formation and function. During osteoclastogenesis, RANKL-stimulated signals differentially modulate expression of a large number of proteins. Using proteomics approaches, we identified that brain-type cytoplasmic creatine kinase (Ckb) was greatly induced in mature osteoclasts. Ckb has been shown to contribute to osteoclast function. However, the mechanisms of Ckb regulation and the contribution of other isoforms of creatine kinase during RANKL-induced osteoclastogenesis are unknown. We found that Ckb was the predominant isoform of creatine kinase during osteoclastogenesis. Real-time PCR confirmed that RANKL induced ckb mRNA expression by over 40-fold in primary mouse bone marrow macrophages and Raw 264.7 cells. The RANKL-responsive region was identified within the -0.4- to -0.2-kb 5'-flanking region of the ckb gene. Affinity binding purification followed by mass spectrometry analysis revealed that poly(ADP-ribose) polymerase-1 (PARP-1) bound to the -0.4/-0.2-kb fragment that negatively regulated expression of ckb in response to RANKL stimulation. Electrophoretic mobility shift assays with PARP-1-specific antibody located the binding site of PARP-1 to the TTCCCA consensus sequence. The expression of PARP-1 was reduced during RANKL-induced osteoclastogenesis, concurrently with increased expression of Ckb. Consistently, knockdown of PARP-1 by lentivirus-delivered shRNA enhanced ckb mRNA expression. The activity of PARP-1 was determined to be required for its inhibitory effect on the ckb expression. In summary, we have demonstrated that PARP-1 is a negative regulator of the ckb expression. Down-regulation of PARP-1 is responsible for the up-regulation of ckb during RANKL-induced osteoclastogenesis.

Enzymatic Characteristics of Creatine Kinase System under Conditions of Cerebral Circulatory Disorders

Changes in the catalytic properties of cytoplasmic and mitochondrial creatine kinase were studied at various periods of cerebral circulatory disorders. Cerebral ischemia was accompanied by activation of free radical processes, changes in the dimer/octamer ratio for mitochondrial creatine kinase, and appearance of new catalytic properties of isoenzymes.

Social isolation in rats inhibits oxidative metabolism, decreases the content of mitochondrial K-Ras and activates mitochondrial hexokinase

Recent observations have suggested that Ras signaling includes combinations of extracellular-signal-regulated Ras activation at the plasma membrane and endomembranes, and translocation of Ras from the plasma membrane to intracellular compartments. In this study we have shown that social isolation of rat decreases the content of Bcl-2-associated K-Ras in hippocampal mitochondria, whereas the amount of H-Ras is increased in the microsomal fraction. Furthermore, we have found that galectin 1, a binding partner of activated Ras, was increased in the soluble fractions. The redistribution of Ras isoforms was accompanied by acceleration in mitochondrial hexokinase and inhibition of mitochondrial aconitase, succinate dehydrogenase, and creatine kinase, whereas the activity of aldolase, as well as cytoplasmic creatine kinase was not changed. Our data suggest that inhibition of mitochondrial oxidative metabolism by reactive oxygen species (ROS) and compensatory elevation of glycolysis in hippocampus occurs during social isolation of rats and Ras trafficking could play an important role in switching of impaired oxidative phosphorylation to anaerobic glycolysis.

The role of Arg-96 in Danio rerio creatine kinase in substrate recognition and active center configuration

In creatine kinases (CKs), the amino acid residue-96 is a strictly conserved arginine. This residue is not directly associated with substrate binding, but it is located close to the binding site of the substrate creatine. On the other hand, the residue-96 is known to be involved in expression in the substrate specificity of various other phosphagen (guanidino) kinases, since each enzyme has a specific residue at this position: arginine kinase (Tyr), glycocyamine kinase (Ile), taurocyamine kinase (His) and lombricine kinase (Lys). To gain a greater understanding of the role of residue-96 in CKs, we replaced this residue in zebra fish Danio rerio cytoplasmic CK with other 19 amino acids, and expressed these constructs in Escherichia coli. All the twenty recombinant enzymes, including the wild-type, were obtained as soluble form, and their activities were determined in the forward direction. Compared with the activity of wild-type, the R96K mutant showed significant activity (8.3% to the wild-type), but 10 mutants (R96Y, A, S, E, H, T, F, C, V and N) showed a weak activity (0.056–1.0%). In the remaining mutants (R96Q, G, M, P, L, W, D and I), the activity was less than 0.05%. Our mutagenesis studies indicated that Arg-96 in Danio CK can be substituted for partially by Lys, but other replacements caused remarkable loss of activity. From careful inspection of the crystal structures (transition state analog complex (TSAC) and open state) of Torpedo cytoplasmic CK, we found that the side chain of R96 forms hydrogen bonds with A339 and D340 only in the TSAC structure. Based on the assumption that CKs consist of four dynamic domains (domains 1–3, and fixed domain), the above hydrogen bonds act to link putative domains 1 and 3 in TSAC structure. We suggest that residue-96 in CK and equivalent residues in other phosphagen kinases, which are structurally similar, have dual roles: (1) one involves in distinguishing guanidino substrates, and (2) the other plays a key role in organizing the hydrogen-bond network around residue-96 which offers an appropriate active center for the high catalytic turnover. The mode of development of the network appears to be unique each phosphagen kinase, reflecting evolution of each enzyme.

A novel arginine kinase from the shrimp Neocaridina denticulata: The fourth arginine kinase gene lineage

Arginine kinases (AK) evolved independently at least three times: first at an early stage of phosphagen kinase evolution (typical AK), second from the cytoplasmic creatine kinase (CK) gene later in metazoan evolution ( Stichopus AK) and third from the mitochondrial CK gene in the course of annelid radiation ( Sabellastarte AK). Here, we present a possible fourth AK lineage. We amplified cDNA encoding three AKs (AK1, AK2 and AK3) from the shrimp Neocaridina denticulata, and determined their amino acid sequences (355–356 residues). Each cDNA sequence was cloned in a pET plasmid and the corresponding recombinant kinase was expressed in E. coli. The AKs showed monomeric nature similar to typical AKs on an analytical gel filtration column. While the amino acid sequence of AK2 corresponded to that of typical AK, containing the conserved key residues established in Limulus AK for the substrate binding site, those of AK1 and AK3 lacked some of these key residues, indicating a similar evolution to Stichopus and Sabellastarte AKs. Moreover, phylogenetic analysis of phosphagen kinases indicated that Neocaridina AK1 and AK3 diverged at the deepest branching point close to the root of the tree and formed a distinct cluster outside the typical AK cluster, which included Neocaridina AK2. Kinetic constants of Neocaridina AKs were similar to those of other AKs. However, activation energy ( E a) for the transition state of AK1 and AK3 was about 1.5-fold larger than that of AK2. The Δ H o‡ values for AK1 and AK3 were also about 1.5-fold larger than that for AK2, but all three Δ G o‡ values were very similar (71–72 kJ/mol); this results in similar reaction velocities for the three AK reactions due to a lower decrease in entropy in the activated complexes of AK1 and AK3 reactions compared to that of AK2. These findings suggest that Neocaridina AK1 and AK3 are unique from the known three AK gene lineages and therefore comprises a fourth AK lineage.

Expression And Purification Of A Stable, Monomeric Creatine Kinase

Three isoform gene families of creatine kinase (CK) are present in animals. Two of these, mitochondrial and cytoplasmic CKs, are obligate oligomers . There is substantial evidence for functional interaction between subunits. Attempts at generating active, monomeric CKs have failed or in one case produced ephemerally active but unstable monomers. A third CK isoform, the so-called flagellar CK, is monomeric but is composed of three complete, contiguous CK domains. Each of these domains is catalytically competent but there is clear interaction between active sites (Hoffman et al., FEBS J 275: 646-654 [2008]). In the present effort, we have used a flagellar CK expression construct as a platform to engineer, express and purify a single domain, monomeric CK. Boundaries between the three domains (D1, D2 and D3) were identified by comparison of key catalytic residues and predicted secondary structural elements. A cDNA coding for D2 was amplified by PCR and inserted into an expression vector. Subsequent expression and purification yielded a recombinant CK which was stable as evidenced by the retention of activity over several weeks. Size exclusion chromatography showed that this CK was monomeric as expected, with a mass similar to the predicted Mr based on the amino acid composition. The engineering of a monomeric CK in the present effort clearly shows that oligomerization is not required for catalysis. Conventional wisdom supports the view that CKs evolved from a related phosphotransferase, arginine kinase (AK). AKs are typically monomeric. It seems likely that oligomerization occurred later in the evolution of CKs perhaps due to the selective pressure for targeting to and binding in intracellular compartments. (Supported by NSF grant IOB-0542236 to WRE).

Evolution of the diverse array of phosphagen systems present in annelids

Annelids as a group express a variety of phosphagen kinases including creatine kinase (CK), glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and a unique arginine kinase (AK) restricted to annelids. In prior work, we have determined and compared the intron/exon organization of the annelid genes for cytoplasmic GK, LK, AK, and mitochondrial TK and LK (MiTK and MiLK, respectively), and found that these annelid genes, irrespective of cytoplasmic or mitochondrial, have the same 8-intron/9-exon organization strikingly similar to mitochondrial CK (MiCK) genes. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids. To gain a greater understanding of the evolutionary processes leading to the diversity of annelid phosphagen kinases, we determined for the first time the intron/exon organization of a cytoplasmic CK gene from a polychaete as well as that of another polychaete MiCK gene. These gene structures, coupled with a phylogenetic analyses of annelid enzymes and assessment of the fidelity of substrate specificity of some these phosphagen kinases, provide insight into the pattern of radiation of the annelid enzymes. Annelid phosphagen kinases appeared to have diverged in the following order (earliest first): (1) cytoplasmic AK, LK and TK, (2) GK, and (3) mitochondrial MiLK and MiTK. Interestingly, phylogenetic analyses showed that the above phosphagen kinases appear to be basal to all CK isoforms (mitochondrial, cytoplasmic and flagellar CKs). This somewhat paradoxical placement of CKs most likely reflects a higher rate of evolution and radiation of the annelid-specific LK, TK and GK genes than the CK isoform genes.

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-kappaB ligand in cooperation with macrophage colony-stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb-/- mice were similarly affected. In vivo studies showed that Ckb-/- mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.

Topically applied growth factors change skin cytoplasmic creatine kinase activity and distribution and produce abnormal keratinocyte differentiation in murine skin

The skin has a functional and active phosphocreatine (PCR)/creatine kinase (CPK) system that regenerates adenosine triphosphate energy reserves during periods of ischemia. The objective of this study was to evaluate how topically applied growth factors affect CPK activity and distribution, and what histological changes growth factors induce in murine skin. Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha) and suramin (growth factor inhibitor) were applied to murine skin for nine days before mice were sacrificed and CPK level and distributions were measured. TGF-alpha considerably increased CPK activity. Both EGF and TGF-alpha induced a CPK MM to CPK BB transition and histologically induced abnormal differentiation of keratinocytes. The skin PCR/CPK system is affected by growth factors. Furthermore, this system appears to play an important role, both in the normal physiology of skin and pathophysiological conditions such as psoriasis and carcinogenesis.

Evolution of the Cytoplasmic and Mitochondrial Phosphagen Kinases Unique to Annelid Groups

Creatine kinase (CK) is a member of a group of phosphoryl transfer enzymes called phosphagen kinases that play a key role in cellular energy transactions in animals. Three CK isoform gene families are known-cytoplasmic CK (CK), flagellar CK (fCK), and mitochondrial CK (MiCK). Each of the isoforms has a unique gene structure (intron/exon organization). A broad array of other phosphagen kinases is present in animals. Some of these enzymes are found only in annelids and closely related groups including glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK), and a unique arginine kinase (AK) restricted to annelids. Phylogenetic analyses of these annelid phosphagen kinases indicate that they appear to have evolved from a CK-like ancestor. To gain a greater understanding of the relationship of the CK isoforms to the annelid enzymes, we have determined the intron/exon organization of the genes for the following phosphagen kinases: Eisenia LK, Sabellastarte AK, and Arenicola mitochondrial TK (MiTK). Analysis of genomic database for the polychaete Capitella sp. yielded two putative LK genes [cytoplasmic LK and mitochondrial LK (MiLK)]. The intron/exon organization of these genes was compared with available data for cytoplasmic and mitochondrial CKs, and an annelid GK. Surprisingly, these annelid genes, irrespective of whether they are cytoplasmic (LK, AK, and GK) or mitochondrial (MiTK and MiLK), had the same 8-intron/9-exon organization and were strikingly similar to MiCK genes sharing seven of eight splice junctions. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids.

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.

MURF-1 and MURF-2 Target a Specific Subset of Myofibrillar Proteins Redundantly: Towards Understanding MURF-dependent Muscle Ubiquitination

MURF-1, MURF-2 and MURF-3 are a specific class of RING finger proteins that are expressed in striated muscle tissues. MURF-1 has been suggested to act as an ubiquitin ligase, thereby controlling proteasome-dependent degradation of muscle proteins. Here, we performed yeast two-hybrid (YTH) screens of skeletal muscle cDNA libraries with MURF-1 baits to identify potential myocellular targets of MURF-1-dependent ubiquitination. This identified eight myofibrillar proteins as binding partners of MURF-1: titin, nebulin, the nebulin-related protein NRAP, troponin-I (TnI), troponin-T (TnT), myosin light chain 2 (MLC-2), myotilin and T-cap. YTH mating studies with MURF-1,2,3 baits indicated that these eight myofibrillar proteins are all targeted redundantly by both MURF-1 and MURF-2. Western blot studies on cardiac tissues from wild-type and MURF-1-deficient mice suggested that titin and nebulin were ubiquitinated at similar levels, and MLC-2 and TnI at reduced levels in MURF-1 KO mice. Mapping of the TnI and titin binding sites on MURF-1 peptide scans demonstrated their binding to motifs highly conserved between MURF-1 and MURF-2. Our data are consistent with a model in which MURF-1 and MURF-2 together target a specific set of myofibrillar proteins redundantly, most likely to control their ubiquitination-dependent degradation. Finally, our YTH screens identified the interaction of MURF-1 with 11 enzymes required for ATP/energy production in muscle including the mitochondrial ATP synthase and cytoplasmic creatine kinase. These data raise the possibility that MURF-1 may coordinately regulate the energy metabolism of mitochondrial and cytoplasmic compartments.

Interdependent Regulation of Insulin Receptor Kinase Activity by ADP and Hydrogen Peroxide

Insulin signaling requires autophosphorylation of the insulin receptor kinase (IRK) domain. Using purified recombinant IRK fragments and the isolated intact insulin receptor, we show here that autophosphorylation is inhibited by ADP and that this effect is essentially reversed by hydrogen peroxide. Autophosphorylation was inhibited by hydrogen peroxide (60 microM) in the absence of ADP but enhanced in the presence of inhibitory concentrations of ADP (67 microM). Enhancement by hydrogen peroxide required direct interaction of hydrogen peroxide with the kinase domain and was not seen in insulin receptor mutants C1245A and C1308A. A similar enhancement was obtained in intact cells in the absence of insulin upon treatment with 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea, indicating that IRK activity can be alternatively enhanced by a shift in the thiol/disulfide redox status. Molecular modeling of the IRK domain indicated that the ATP-binding site becomes distorted after releasing the nucleotide unless the IRK domain is oxidatively derivatized at Cys1245. Recent clinical studies suggest that these effects may play a role in obesity due to the fact that cytoplasmic creatine kinase in combination with phosphocreatine normally ensures rapid removal of ADP in muscle cells but not in fat cells.

Isolation, characterization, and cDNA-derived amino acid sequence of glycocyamine kinase from the tropical marine worm Namalycastis sp.

We isolated cytoplasmic glycocyamine kinase (GK) and creatine kinase (CK) from the tropical marine worm Namalycastis sp. by ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-5PW chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the isolated GK is highly purified and appears to be a heterodimer of two distinct subunits, α and β, with molecular masses of ∼40 kDa. The complete nucleotide sequences of the cDNAs for Namalycastis GKα and GKβ were 1527 (encoding 374 amino acids) and 1579 bp (encoding 390 amino acids), respectively. The predicted amino acid sequences differ only in the N-terminal 50 residues. This is consistent with the characteristics of Neanthes GKα and GKβ chains, which we have previously shown to be generated by alternative splicing. The recombinant enzymes GKα, GKβ, and CK from Namalycastis were successfully expressed in Escherichia coli as maltose-binding protein fusion proteins. In contrast to the stable GKβ enzyme, GKα was quite unstable, and its activity decreased remarkably with time. Thus, the N-terminal 50 residues appear to play a key role in enzyme stability. The kinetic parameters for the native GK heterodimer were similar to GKβ, suggesting that GKα would have an activity similar to GKβ if part of a heterodimer. This is the first report of precise kinetic parameters for GK. Finally, based on our results, we present a model for pluriphosphagen function in Namalycastis wherein cytoplasmic GK and CK and mitochondrial CK function together with phosphocreatine and phosphoglycocyamine to enable cells to respond quickly to a sudden large energy requirement.

Cloning and expression of mitochondrial and protoflagellar creatine kinases from a marine sponge: implications for the origin of intracellular energy transport systems

Creatine kinase (CK) plays a central role in energy transactions in cells displaying high and variable rates of ATP turnover. Cytoplasmic and mitochondrial CK genes code for isoforms which are targeted to distinct intracellular compartments often in close physical proximity to sites of ATP hydrolysis or synthesis. In certain lower groups a third CK gene is present which codes for a flagellar CK isoform consisting of three complete, fused CK domains. Recent work has shown that cytoplasmic, mitochondrial, and flagellar CKs are present in protochordates and in deuterostome and protostome invertebrates. We report here that the marine sponge Tethya aurantia, a representative of the oldest of all multi-cellular animal groups, expresses three unique CK transcripts. One of these CK transcripts codes for protein that has a mitochondrial targeting sequence and in a phylogenetic analysis is positioned at the base of the cluster containing mitochondrial CK sequences from invertebrates, protochordates, and vertebrates; it is clearly a mitochondrial CK. When expressed in Escherichia coli the mitochondrial form from T. aurantia was found to be dimeric unlike all other mitochondrial CKs which are typically octameric. The other two T. aurantia transcripts code for proteins that appear to be more closely related to flagellar CKs. These protoflagellar CKs were found to be dimers when expressed in Escherichia coli. Sponges last shared a common ancestor with higher animals as long as one billion years ago. The antiquity of intracellular localization, as evidenced by the presence of a true mitochondrial CK and protoflagellar CKs in the sponge T. aurantia, indicates that physical constraints on cellular energy transport were key, early driving forces in the evolution of this key enzyme system.

Elements of the major myofibrillar binding peptide motif are present in the earliest of true muscle type creatine kinases

Most vertebrates possess two genes for cytoplasmic creatine kinase (CK) coding for muscle (M-CK) and brain (B-CK) isoforms which assemble into homo-dimeric (MM, BB) and hetero-dimeric (MB) active enzymes. In mammals and birds, a significant fraction of MM-CK is bound to the myofibrillar M-line where it is thought to facilitate energy buffering and transport. Myofibrillar binding is mediated by major and minor lysine charge clamp motifs (K104/K115 [major] and K8/K24 [minor] in chicken M-CK) located in the N-terminal region [J. Cell Biol. 149 (2000) 1225]. We have obtained the cDNA and deduced amino acid sequences for cytoplasmic CKs from two hagfish, Myxine glutinosa and Eptatretus stoutii, non-vertebrate craniates, and the sequences for two cytoplasmic CKs from the lamprey Lampetra japonica, a jawless true vertebrate. All four cDNAs code for CKs consisting of approximately 380 residues. Phylogenetic analyses showed that the hagfish and lamprey CKs are coded for by genes which are clearly muscle type (M) creatine kinases. Two of these four M-CKs have the K104/K115-equivalent residues of the major myofibrillar binding region while the other two have the K115 equivalent but lack the corresponding K104 residue. All four M-CKs lack the K8/K24 equivalent elements of the minor myofibrillar binding region. Comparison of these sequences to corresponding sequences of cytoplasmic CKs from two protochordates (tunicate, amphioxus) and M- and B-CKs from true fish and above reveal a pattern of acquisition (and loss) of key lysine residues consistent with the physiological context in which these enzymes operate.