Mitochondrial kinases and their molecular interaction with cardiolipin

Mitochondrial isoforms of creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D) are not phylogenetically related but share functionally important properties. They both use mitochondrially generated ATP with the ultimate goal of maintaining proper nucleotide pools, are located in the intermembrane/cristae space, have symmetrical oligomeric structures, and show high affinity binding to anionic phospholipids, in particular cardiolipin. The structural basis and functional consequences of the cardiolipin interaction have been studied and are discussed in detail in this review. They mainly result in a functional interaction of MtCK and NDPK-D with inner membrane adenylate translocator, probably by forming proteolipid complexes. These interactions allow for privileged exchange of metabolites (channeling) that ultimately regulate mitochondrial respiration. Further functions of the MtCK/membrane interaction include formation of cardiolipin membrane patches, stabilization of mitochondria and a role in apoptotic signaling, as well as in case of both kinases, a role in facilitating lipid transfer between two membranes. Finally, disturbed cardiolipin interactions of MtCK, NDPK-D and other proteins like cytochrome c and truncated Bid are discussed more generally in the context of apoptosis and necrosis.     

Metabolism of deoxypyrimidines and deoxypyrimidine antiviral analogs in isolated brain mitochondria

The goal of this project was to characterize deoxypyrimidine salvage pathways used to maintain deoxynucleoside triphosphate pools in isolated brain mitochondria and to determine the extent that antiviral pyrimidine analogs utilize or affect these pathways. Mitochondria from rat brains were incubated in media with labeled and unlabeled deoxynucleosides and deoxynucleoside analogs. Products were analyzed by HPLC coupled to an inline UV monitor and liquid scintillation counter. Isolated mitochondria transported thymidine and deoxycytidine into the matrix, and readily phosphorylated both of these to mono-, di-, and tri-phosphate nucleotides. Rates of phosphorylation were much higher than rates observed in mitochondria from heart and liver. Deoxyuridine was phosphorylated much more slowly than thymidine and only to dUMP. 3'-azido-3'-deoxythymidine, zidovudine (AZT), an antiviral thymidine analog, was phosphorylated to AZT-MP as readily as thymidine was phosphorylated to TMP, but little if any AZT-DP or AZT-TP was observed. AZT at 5.5 ± 1.7 μM was shown to inhibit thymidine phosphorylation by 50%, but was not observed to inhibit deoxycytidine phosphorylation except at levels > 100 μM. Stavudine and lamivudine were inert when incubated with isolated brain mitochondria. The kinetics of phosphorylation of thymidine, dC, and AZT were significantly different in brain mitochondria compared to mitochondria from liver and heart.     

Induction of deoxypyrimidine kinase activity in human embryonic lung cells infected with varicella-zoster virus.

Deoxypyrimidine kinase (deoxythymidine [TdR] kinase and deoxycytidine kinase) activity was induced in human embryonic lung cells after infection with varicella-zoster virus (VZ virus). Increased enzyme activity was also produced by using cell-associated virus as inoculum instead of cell-free virus. Anti-VZ virus serum inhibited both the appearance of cytopathic effect and the induction of enzyme activity. The induced TdR kinase activity was more thermostable than that induced by herpes simplex virus type 1. Also, the TdR kinase activity of VZ virus-infected cells was inhibited by dTTP less than in mock-infected cells and more than in herpes simplex virus type 1-infected cells.     

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.     

G-protein-coupled receptor kinases.

Rhodopsin kinase and the beta-adrenergic receptor kinase (beta ARK) catalyse the phosphorylation of the activated forms of the G-protein-coupled receptors, rhodopsin and the beta 2-adrenergic receptor (beta 2AR), respectively. The interaction between receptor and kinase is independent of second messengers and appears to involve a multipoint attachment of kinase and substrate with the specificity being restricted by both the primary amino acid sequence and conformation of the substrate. Kinetic, functional and sequence information reveals that rhodopsin kinase and beta ARK are closely related, suggesting they may be members of a family of G-protein-coupled receptor kinases.     

Mitochondrial beta-oxidation.

Mitochondrial beta-oxidation is a complex pathway involving, in the case of saturated straight chain fatty acids of even carbon number, at least 16 proteins which are organized into two functional subdomains; one associated with the inner face of the inner mitochondrial membrane and the other in the matrix. Overall, the pathway is subject to intramitochondrial control at multiple sites. However, at least in the liver, carnitine palmitoyl transferase I exerts approximately 80% of control over pathway flux under normal conditions. Clearly, when one or more enzyme activities are attenuated because of a mutation, the major site of flux control will change.     

Characterization and comparison of membrane-associated and cytosolic cAMP-dependent protein kinases. Studies on human erythrocyte protein kinases.

Cyclic AMP-dependent protein kinase from human erythrocyte plasma membranes was solubilized with Triton X-100, partially purified, and systematically characterized by a series of physicochemical studies. Sedimentation and gel filtration experiments showed that the 6.6 S holoenzyme had a Stokes radius (a) of 5.7 nm and was dissociated into native 4.8 S cAMP-binding (a = 4.5 nm) and 3.2 S catalytic (a = 2.6 nm) subunits. A minimum subunit molecular weight of 48,000 was established for the regulatory subunit by photoaffinity labeling with 8-azido[32P]cAMP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. These data suggest an asymmetric tetrameric (R2C2) structure (Mr approximately equal to 160,000) for the membrane-derived enzyme. Membrane-derived protein kinase was characterized as a type I enzyme on the basis of its R subunit molecular weight, pI values (R, 4.9; holoenzyme, 5.75 and 5.95), dissociation by 0.5 M NaCl and 50 microgram/ml of protamine, 20-fold reduced affinity for cAMP in the presence of 0.3 mM MgATP, elution from DEAE-cellulose at low ionic strength, and kinetic and cAMP-binding properties. The physicochemical properties of the membrane protein kinase closely parallel the characteristics of erythrocyte cytosolic protein kinase I but are clearly dissimilar from those of the soluble type II enzyme. Moreover, regulatory subunits of the membrane-associated and cytosolic type I kinases were indistinguishable in size, shape, subunit molecular weight, charge, binding and reassociation properties, and peptide maps of the photoaffinity-labeled cAMP-binding site, suggesting a high degree of structural and functional homology in this pair of enzymes. In view of the predominant occurrence of particulate type II protein kinases in rabbit heart and bovine cerebral cortex, the present results suggest that the distribution of membrane-associated protein kinases may be tissue- or species-specific, but not isoenzyme-specific.     

MAP kinases bite back.

Recent studies in a model system challenge our understanding of how signal transmission through a MAP kinase cascade proceeds and how signaling specificity may be achieved.     

Phytochromes are Pr-ipatetic kinases.

A series of new studies reveal how the red/far-red light photoreceptors called phytochromes act. Phytochrome A and phytochrome B each move to the nucleus when activated by light, and phytochrome A is a kinase. Phytochrome-interacting proteins provide candidate signal transduction components and a recent physiological study suggests how phyA may mediate responses to far-red light. Regulation of phytochrome nuclear localization and kinase activities creates multiple phytochrome species, which may each have different regulatory activities.     

Mitochondrial DNA and genetic disease.

Since the human mitochondrial genome was characterised and sequenced in 1981, it has been viewed as the likely site of genetic diseases showing a maternal inheritance pattern and associated with defects of the respiratory chain, such as the mitochondrial myopathies (MMs). The properties that make it a candidate for the source of such conditions are that it encodes polypeptides involved in electron transport and that it is maternally inherited. However, several of the mtDNA diseases only fulfill one or other of these criteria: the first group of mtDNA diseases showed only sporadic deletions, and the first point mutation in Leber's Hereditary Optic Neuropathy (LHON) is not associated with a clear biochemical defect. Furthermore, it is now clear that both autosomal dominant and probably recessive nuclear genes can cause abnormalities of mtDNA. Each of these major groups will be considered in turn.     

The nucleoside diphosphate kinase from mimivirus: a peculiar affinity for deoxypyrimidine nucleotides

The first viral Nucleoside Diphosphate Kinase was recently identified in the giant double-stranded DNA virus Acanthamoeba polyphag a Mimivirus (ApM). Here we report its expression and detailed biochemical characterization. NDK_apm exhibits unique features such as a shorter Kpn-loop, a structural motif previously reported to be part of the active site and involved in oligomer formation. Enzymatic activity measurements on the recombinant NDK_apm revealed its preferential affinity for deoxypyrimidine nucleotides. This property might represent an adaptation of NDK_apm to the production of the limiting TTP deoxynucleotide required for the replication of the large A+T rich (72%) viral genome. The NDK_apm might also assume a role in dUTP detoxification to compensate for the surprising absence of Mimivirus dUTPase (deoxyuridine triphosphate pyrophosphatase) an important enzyme conserved in most viruses. Although the phylogenetic analysis of NDK sequences sampled through organisms from the three domains of life is only partially informative, it favors an ancestral origin for NDK_apm over a recent acquisition from a eukaryotic organism by horizontal gene transfer.     

Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.

Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. We have identified a new subfamily of murine serine/threonine kinases, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases, Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates their in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells.