Levels and activity of cyclic guanosine monophosphate-dependent protein kinase in nurse and forager honeybees.

Age-dependent division of labour in honeybees was shown to be connected to sensory response thresholds. Foragers show a higher gustatory responsiveness than nurse bees. It is generally assumed that nutrition-related signalling pathways underlie this behavioural plasticity. Here, one important candidate gene is the foraging gene, which encodes a cyclic guanosine monophosphate-dependent protein kinase (PKG). Several roles of members of this enzyme family were analysed in vertebrates. They own functions in important processes such as growth, secretion and neuronal adaptation. Honeybee foraging messenger RNA expression is upregulated in the brain of foragers. In vivo activation of PKG can modulate gustatory responsiveness. We present for the first time PKG protein level and activity data in the context of social behaviour and feeding. Protein level was significantly higher in brains of foragers than in those of nurse bees, substantiating the role of PKG in behavioural plasticity. However, enzyme activity did not differ between behavioural roles. The mediation of feeding status appears independent of PKG signalling. Neither PKG content nor enzyme activity differed between starved and satiated individuals. We suggest that even though nutrition-related pathways are surely involved in controlling behavioural plasticity, which involves changes in PKG signalling, mediation of satiety itself is independent of PKG.

Properties of a new calcium-permeable single channel from tracheal microsomes.

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.

Distribution of cGMP-dependent protein kinase type I and its isoforms in the mouse brain and retina.

Nitric oxide (NO) modulates a variety of processes in the mammalian brain, but the mechanisms of neuronal NO signaling are poorly understood. In the periphery, many effects of NO are mediated via the generation of the second messenger cyclic guanosine-3',5'-monophosphate (cGMP) and activation of the cGMP-dependent protein kinase type I (cGKI). However, previous studies suggested that the expression of cGKI in the nervous system is rather restricted, thus, questioning the functional significance of the cGMP/cGKI pathway as a mediator of NO signaling in the brain. Here we have performed a detailed immunohistochemical study to elucidate the distribution of cGKI in the CNS and eye of the mouse. Expression of cGKI protein was detected not only in the previously described areas (cerebellum, hippocampus, dorsomedial hypothalamus) but also in a number of additional regions, such as medulla, subcommissural organ, cerebral cortex, amygdala, habenulae, various hypothalamic regions, olfactory bulb, pituitary gland, and retina. Immunoblotting with isoform-specific antibodies indicated that the cGKIalpha isoform is prominent in the cerebellum and medulla, whereas the cGKIbeta isoform is predominant in the cortex, hippocampus, hypothalamus, and olfactory bulb. Similar levels of the isoforms were detected in the pituitary gland and eye. Thus, it appears that distinct brain regions express distinct cGKI isoforms that signal via distinct pathways. Together, these results improve our understanding of the cellular and molecular mechanisms of NO/cGMP/cGKI signaling and indicate that the distribution and functional relevance of this pathway in the mammalian brain is broader than previously thought.

Distribution of IRAG and cGKI-isoforms in murine tissues.

cGMP kinase I (cGKI) signaling modulates multiple physiological processes including smooth muscle relaxation. The expression of cGKI and its substrate IRAG (Inositol 1,4,5-trisphosphate receptor associated cGMP kinase substrate) was studied. IRAG and cGKI were colocalized in the smooth muscle of aorta and colon. IRAG was present in the thalamus and in most of the myenteric plexus in the absence of cGKI. Coexpression of IRAG and cGKIbeta or cGKIalpha in COS-7 cells revealed that IRAG recruits cGKIbeta but not cGKIalpha to the endoplasmic reticulum. These results suggest that IRAG may be involved in cGKI-dependent and -independent pathways.

Function of cGMP-dependent protein kinases as revealed by gene deletion.

Over the past few years, a wealth of biochemical and functional data have been gathered on mammalian cGMP-dependent protein kinases (cGKs). In mammals, three different kinases are encoded by two genes. Mutant and chimeric cGK proteins generated by molecular biology techniques yielded important biochemical knowledge, such as the function of the NH(2)-terminal domains of cGKI and cGKII, the identity of the cGMP-binding sites of cGKI, and the substrate specificity of the enzymes. Genetic approaches have proven especially useful for the analysis of the biological functions of cGKs. Recently, some of the in vivo targets and mechanisms leading to changes in neuronal adaptation, smooth muscle relaxation and growth, intestinal water secretion, bone growth, renin secretion, and other important functions have been identified. These data show that cGKs are signaling molecules involved in many biological functions.

Assembly of the preprotein receptor MOM72/MAS70 into the protein import complex of the outer membrane of mitochondria.

Membrane integration and assembly of MOM72 from Neurospora crassa and its yeast homolog MAS70 was studied with isolated mitochondria. After synthesis in vitro, the precursors of MOM72/MAS70 are tightly folded and expose only their N-terminal amino acid residues comprising the targeting and the membrane anchor domain. Insertion of the protein into the mitochondrial outer membrane (MOM) occurs in a time- and temperature-dependent manner and is stimulated by ATP. MOM72/MAS70 is then assembled into the outer membrane MOM complex. Whereas membrane insertion occurred independently of the presence of protease-sensitive surface components, the assembly reaction depended on such components. In the MOM complex MOM72 and MAS70 were found in the neighborhood of different components in yeast and N. crassa mitochondria. MOM72 was found in association with MOM22 in N. crassa mitochondria, whereas MAS70 was in proximity to a 37-kDa component in yeast outer mitochondrial membrane. The interaction with the 37-kDa protein is important for integration of MAS70 into the MOM complex. Thus, the 37-kDa protein plays an important role in the biogenesis of MAS70.

Rising behind NO: cGMP-dependent protein kinases.

Over the past few years, a wealth of biochemical and functional data has been gathered on mammalian cGMP-dependent protein kinases (cGKs). In mammals, three different kinases are encoded by two genes. Mutant and chimeric cGMP kinase proteins generated by molecular biology techniques have yielded important biochemical knowledge, such as the function of the N-terminal domains of cGKI and cGKII, the identity of the cGMP-binding sites of cGKI, the substrate specificity of the enzymes and structural details of the catalytic center. Genetic approaches have proved to be especially useful for the analysis of the biological function of cGKs. Recently, some of the in vivo targets and mechanisms leading to smooth muscle relaxation have been identified. In vivo targets are the myosin-binding subunit of myosin phosphatase (PP1M), a member of the protein phosphatase 1, the calcium-activated maxi K(+) channel and a new protein named IRAG that forms a complex with the inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) receptor and cGKI. Phosphorylation of PP1M by cGKI(alpha) activates myosin phosphatase, whereas phosphorylation of IRAG by cGKI(beta) decreases Ins(1,4, 5)P(3)-induced calcium release. cGKII regulates in vivo intestinal fluid secretion by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), bone growth and renal renin secretion by phosphorylation of unknown proteins.

Specific recognition of mitochondrial preproteins by the cytosolic domain of the import receptor MOM72.

The import receptor MOM72 constitutes part of the protein translocation machinery of the outer mitochondrial membrane, the receptor-general insertion pore complex. The protein contains a membrane anchor at the NH2 terminus and a large cytosolic domain. In yeast and Neurospora crassa the cytosolic domain comprises about 570-580 amino acid residues. The cytosolic domain of yeast MOM72 was purified after expression in Escherichia coli as a homogeneous monomeric protein. It can recognize precursor proteins as demonstrated by its ability to compete for binding and import into the mitochondria and to physically interact with preproteins. A subset of preproteins including the ADP/ATP carrier and the phosphate carrier interact with very high affinity, precursors that are known to be targeted via MOM72. Thus, the cytosolic domain of MOM72 plays a critical function in the recognition of preproteins by directly binding to precursor proteins and thereby facilitating their targeting to mitochondria.

Molecular determinants of the interaction between the inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) and cGMP kinase Ibeta.

Cyclic GMP-dependent protein kinase I (cGKI) affects the inositol 1,4,5-trisphosphate (InsP(3))-dependent release of intracellular calcium by phosphorylation of IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate). IRAG is present in a macromolecular complex with the InsP(3) receptor type I (InsP(3)RI) and cGKIbeta. The specificity of the interaction between these three proteins was investigated by using the yeast two-hybrid system and by co-precipitation of expressed proteins. The amino-terminal region containing the leucine zipper (amino acids 1-53) of cGKIbeta but not that of cGKIalpha or cGKII interacted with the sequence between amino acids 152 and 184 of IRAG in vitro and in vivo most likely through electrostatic interaction. cGKIbeta did not interact with the InsP(3)RI, but co-precipitated the InsP(3)RI in the presence of IRAG indicating that IRAG bound to the InsP(3)RI and to cGKIbeta. cGKIbeta phosphorylated up to four serines in IRAG. Mutation of these four serines to alanine showed that cGKIbeta-dependent phosphorylation of Ser(696) is necessary to decrease calcium release from InsP(3)-sensitive stores. These results show that cGMP induced reduction of cytosolic calcium concentrations requires cGKIbeta and phosphorylation of Ser(696) of IRAG.

Tom71, a novel homologue of the mitochondrial preprotein receptor Tom70.

The protein Tom71 is encoded by the open reading frame YHR117w (yeast chromosome VIII) and shares 53% amino acid sequence identity with Tom70, a protein import receptor of the mitochondrial outer membrane. We investigated the cellular function of Tom71 and addressed the question of whether Tom71 and Tom70 fulfill similar functions. Like Tom70, Tom71 is anchored to the mitochondrial outer membrane via its N terminus, thereby exposing a large C-terminal domain to the cytosol. Tom71 is associated with the protein import complex of this membrane and can be cross-linked to a protein with a molecular mass of 30-35 kDa. Disruption of the TOM71 gene does not reduce cell growth, except on nonfermentable carbon sources at elevated temperatures. Deletion of both the TOM71 and TOM70 genes does not acerbate this growth defect. In vitro import studies demonstrated no functional requirement for Tom71 in the import of several preproteins destined for each of the mitochondrial subcompartments. In particular, the import of Tom70-dependent preproteins is minimally affected by the deletion of Tom71, irrespective of the presence or absence of the Tom70 receptor. Thus, despite their strikingly similar biochemical properties, Tom71 and Tom70 do not perform identical functions.

Mapping of the protein import machinery in the mitochondrial outer membrane by crosslinking of translocation intermediates.

Mitochondria contain a complex machinery for the import of nuclear-encoded proteins. Receptor proteins exposed on the outer membrane surface are required for the specific binding of precursor proteins to mitochondria, either by binding of cytosolic signal recognition factors or by direct recognition of the precursor polypeptides. Subsequently, the precursors are inserted into the outer membrane at the general insertion site GIP (general insertion protein). Here we report the analysis of receptors and GIP by crosslinking of translocation intermediates and by coimmunoprecipitation. Surface-accumulated precursors were crosslinked to the receptors MOM19 and MOM72, suggesting a direct interaction of preproteins with surface receptors. We identified three novel mitochondrial outer membrane proteins, MOM7, MOM8, and MOM30 that, together with the previously identified MOM38, seem to form the GIP site and are present in the mitochondrial receptor complex.

Regulation of stably expressed and native BK channels from human myometrium by cGMP- and cAMP-dependent protein kinase.

The cloned BK channel alpha subunit from human myometrium was stably expressed in Chinese hamster ovary cells, either alone (CHOalpha cells) or in combination with the auxiliary beta subunit (CHOalpha+beta cells). We studied basic channel properties and the effects of cGMP- and cAMP-dependent protein kinases on the BK channel activity. Coexpression of alpha and beta subunits enhanced the Ca2+ and voltage sensitivity of the BK channel, and decreased the inhibitory potency of iberiotoxin. Blocking and stimulating effects on BK channel activity by charybdotoxin and nitric oxide, respectively, were independent of the beta subunit. The cGMP kinase Ialpha and cAMP kinase failed to affect BK channel activity in CHOalpha and CHOalpha+beta cells at different [Ca2+]i and voltages. In contrast, BK channels in freshly isolated myometrial cells from postmenopausal women responded to cAMP kinase and cGMP kinase with a fourfold and twofold decrease in their open probability (NPo), respectively. These effects could be reversed by alkaline phosphatase and remained unaffected by the phosphatase inhibitor okadaic acid (100 nM). In 28% of myometrial cells, however, cAMP and cGMP kinases increased NPo 2-fold and 3.5-fold, respectively. This stimulation was enhanced rather than reversed by alkaline phosphatase and was abolished by 100 nM okadaic acid. The results suggest that in stably transfected CHO cells the expressed BK channel is not regulated by cAMP kinase and cGMP kinase. However, in native myometrial cells stimulatory and inhibitory regulation of BK channels by cAMP kinase and cGMP kinase was observed, suggesting that channel regulation by the protein kinases requires factors that are not provided by CHO cells. Alternatively, failure of regulation may have been due to the primary structure of the myometrial BK channel protein used in this study.

Protein phosphatase 2A is essential for the activation of Ca2+-activated K+ currents by cGMP-dependent protein kinase in tracheal smooth muscle and Chinese hamster ovary cells.

The regulation of Ca2+-activated K+ channels (KCa channels) by cGMP-dependent protein kinase (cGMP kinase) and its molecular mechanism were investigated in Chinese hamster ovary (CHO) and tracheal smooth muscle cells. In CHO wild-type cells (CHO-WT cells) and in CHO cells stably transfected with cGMP kinase Ialpha (CHO-cGK cells), KCa channels with intermediate conductance (approximately 50 picosiemens) were identified. Due to the basal activity of cGMP kinase, Ca2+-activated K+ currents had a higher sensitivity toward the cytosolic Ca2+ concentration in CHO-cGK cells than in CHO-WT cells. Dialysis of the active fragment of cGMP kinase (300 n) into CHO-WT cells or of cGMP into CHO-cGK cells increased the Ca2+-activated K+ current, while the catalytic subunit of cAMP-dependent protein kinase (cAMP kinase) was without effect. In cell-attached patches obtained from freshly isolated bovine tracheal smooth muscle cells, the open state probability (NPo) of maxi-KCa channels (conductance of approximately 260 picosiemens) was enhanced by 300 microM 8-(4-chlorophenylthio)-cGMP, a specific and potent activator of cGMP kinase. In contrast, 1 microM isoprenaline, 20 microM forskolin, and 3 mM 8-bromo-cAMP failed to enhance KCa channel activity. In excised inside-out patches, only the active fragment of cGMP kinase (but not that of cAMP kinase) increased NPo when applied to the cytosolic side of the patch. The enhancement of NPo by cGMP kinase was inhibited in CHO cells as well as in tracheal smooth muscle cells by the cGMP kinase inhibitor KT 5823 (1 microM) and the protein phosphatase (PP) inhibitors microcystin (5 microM) and okadaic acid (10 nM). The catalytic subunit of PP2A (but not that of PP1) mimicked the effect of cGMP kinase on NPo in excised inside-out patches. The results show that cGMP kinase regulates two different KCa channels in two unrelated cell types by the same indirect mechanism, which requires the activity of PP2A. The regulation of the KCa channel is specific for cGMP kinase and is not mimicked by cAMP kinase.

Targeting and translocation of the phosphate carrier/p32 to the inner membrane of yeast mitochondria.

We analyzed the submitochondrial location and biogenesis pathway of the phosphate carrier (PiC), also termed p32, of Saccharomyces cerevisiae mitochondria, PiC/p32 was found to behave as an integral membrane protein that cofractionated with the ADP/ATP carrier of the inner membrane. Import of the precursor of PiC/p32 required a membrane potential across the inner membrane, supporting its localization to the inner membrane. This makes it unlikely that the major function of PiC/p32 is that of an import receptor on the surface of the mitochondrial outer membrane. Furthermore, we found that both receptors MOM72 and MOM19 were involved in the import pathway of the precursor of PiC/p32 with MOM72 being responsible for the bulk of import. Yeast PiC/p32 is thus not only structurally homologous to the ADP/ATP carrier, but has a similar targeting mechanism and submitochondrial location, supporting its classification as a member of the inner membrane carrier family.

Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta.

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.