Calmodulin-dependent protein kinase IV during T-cell development.

In a primary cell culture system of fetal rat brain, the calmodulin-dependent protein-kinase IV (CaMKIV) could be induced by the thyroid hormone T3 in a time- and concentration-dependent manner, provided the tissue was excised not later than day 15 of gestation (E15) (Krebs et al., J. Biol. Chem. 271, 11055, 1996). We report here that in the fetal thymus CaMKIV could not be detected earlier than day 16 of gestation and that the expression of this enzyme was fully upregulated at day 18. In mouse fetal thymus organ culture (FTOC) of day 14 embryonic thymus, CaMKIV could not be detected, even after several days of culture if a minimal culture medium lacking fetal calf serum was used. However, after addition of fetal calf serum to the culture medium the expression of CaMKIV could be specifically induced. Furthermore, it could also be shown that during T-cell development in the adult murine thymus the expression of CaMKIV was tightly regulated. Taken together, these results demonstrate that the expression of CaMKIV, an enzyme involved in the regulation of Ca(2+)-dependent gene expression, is itself under stringent regulatory control during tissue development.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.

Induction of calmodulin kinase IV by the thyroid hormone during the development of rat brain.

This communication reports the specific induction of calmodulin kinase IV by the thyroid hormone 3,3',5-triiodo-L-thyronine (T3) in a time- and concentration-dependent manner at a very early stage of brain differentiation using a fetal rat telencephalon primary cell culture system, which can grow and differentiate under chemically defined conditions. The induction of the enzyme that can be observed both on the mRNA and on the protein level is T3-specific, i.e. it cannot be induced by retinoic acid or reverse T3, and can be inhibited on both the transcriptional and the translational level by adding to the culture medium actinomycin D or cycloheximide, respectively. The earliest detection of calmodulin kinase IV in the fetal brain tissue of the rat is at days E16/E17, both on the mRNA as well as on the protein level. This is the first report in which a second messenger-dependent kinase involved in the control of cell regulatory processes is itself controlled by a primary messenger, the thyroid hormone.

Calmodulin kinase IV: expression and function during rat brain development.

The expression of calmodulin kinase IV (CaMKIV) can be induced by the thyroid hormone T3 in a time- and concentration-dependent manner at a very early stage of brain differentiation using a fetal rat telencephalon primary cell culture system which can grow and differentiate under chemically defined conditions (Krebs et al. (1996) J. Biol. Chem. 271, 11055-11058). After the induction of CaMKIV by T3 we examined the influence of prolonged absence of T3 from the culture medium on the expression of CaMKIV. We could demonstrate that after the T3-dependent induction of CaMKIV, omission of the hormone, even for 8 days, from the medium did not downregulate the expression of CaMKIV indicating that different regulatory mechanisms became important for the expression of the enzyme. We further showed that CaMKIV could be involved in the Ca(2+) -dependent expression of the immediate early gene c-fos, probably via phosphorylation of the transcription factor CREB. Convergence of signal transduction pathways on this transcription factor by using different protein kinases may explain the importance of CREB for the regulation of different cellular processes.

MyD88, an adapter protein involved in interleukin-1 signaling.

MyD88 has a modular organization, an N-terminal death domain (DD) related to the cytoplasmic signaling domains found in many members of the tumor necrosis factor receptor (TNF-R) superfamily, and a C-terminal Toll domain similar to that found in the expanding family of Toll/interleukin-1-like receptors (IL-1R). This dual domain structure, together with the following observations, supports a role for MyD88 as an adapter in IL-1 signal transduction; MyD88 forms homodimers in vivo through DD-DD and Toll-Toll interactions. Overexpression of MyD88 induces activation of the c-Jun N-terminal kinase (JNK) and the transcription factor NF-kappaB through its DD. A point mutation in MyD88, MyD88-lpr (F56N), which prevents dimerization of the DD, also blocks induction of these activities. MyD88-induced NF-kappaB activation is inhibited by the dominant negative versions of TRAF6 and IRAK, which also inhibit IL-1-induced NF-kappaB activation. Overexpression of MyD88-lpr or MyD88-Toll (expressing only the Toll domain) acted to inhibit IL-1-induced NF-kappaB and JNK activation in a 293 cell line overexpressing the IL-1RI. MyD88 coimmunoprecipitates with the IL-1R signaling complex in an IL-1-dependent manner.

Identification of CARDIAK, a RIP-like kinase that associates with caspase-1.

Members of the tumor necrosis factor receptor (TNFR) superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation and death. TNFR-1 recruits and assembles a signaling complex containing a number of death domain (DD)-containing proteins, including the adaptor protein TRADD and the serine/threonine kinase RIP, which mediates TNF-induced NF-kappa B activation. RIP also recruits caspase-2 to the TNFR-1 signaling complex via the adaptor protein RAIDD, which contains a DD and a caspase-recruiting domain (CARD). Here, we have identified a RIP-like kinase, termed CARDIAK (for CARD-containing interleukin (IL)-1 beta converting enzyme (ICE) associated kinase), which contains a serine/threonine kinase domain and a carboxy-terminal CARD. Overexpression of CARDIAK induced the activation of both NF-kappa B and Jun N-terminal kinase (JNK). CARDIAK interacted with the TNFR-associated factors TRAF-1 and TRAF-2, and a dominant-negative form of TRAF-2 inhibited CARDIAK-induced NF-kappa B activation. Interestingly, CARDIAK specifically interacted with the CARD of caspase-1 (previously known as ICE), and this interaction correlated with the processing of pro-caspase-1 and the formation of the active p20 subunit of caspase-1. Together, these data suggest that CARDIAK may be involved in NF-kappa B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta through activation of caspase-1.

Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c-Jun N-terminal kinase.

We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.

Association of brain spectrin isoforms with microtubules.

The relationship of rat brain spectrin isoforms to microtubules of newborn and adult animals was studied. Spectrins were minor components in microtubule preparations. The microtubule-associated spectrin is a major calmodulin-binding protein. Radiolabelled brain spectrin(240/235) revealed specific microtubule binding activity in vitro, possibly via a tubulin.

An SH2 domain-containing 5' inositolphosphatase inhibits insulin-induced GLUT4 translocation and growth factor-induced actin filament rearrangement.

Tyrosine kinase receptors lead to rapid activation of phosphatidylinositol 3-kinase (PI3 kinase) and the subsequent formation of phosphatidylinositides (PtdIns) 3,4-P2 and PtdIns 3,4, 5-P3, which are thought to be involved in signaling for glucose transporter GLUT4 translocation, cytoskeletal rearrangement, and DNA synthesis. However, the specific role of each of these PtdIns in insulin and growth factor signaling is still mainly unknown. Therefore, we assessed, in the current study, the effect of SH2-containing inositol phosphatase (SHIP) expression on these biological effects. SHIP is a 5' phosphatase that decreases the intracellular levels of PtdIns 3,4,5-P3. Expression of SHIP after nuclear microinjection in 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation by 100 +/- 21% (mean +/- the standard error) at submaximal (3 ng/ml) and 64 +/- 5% at maximal (10 ng/ml) insulin concentrations (P < 0.05 and P < 0.001, respectively). A catalytically inactive mutant of SHIP had no effect on insulin-induced GLUT4 translocation. Furthermore, SHIP also abolished GLUT4 translocation induced by a membrane-targeted catalytic subunit of PI3 kinase. In addition, insulin-, insulin-like growth factor I (IGF-I)-, and platelet-derived growth factor-induced cytoskeletal rearrangement, i.e., membrane ruffling, was significantly inhibited (78 +/- 10, 64 +/- 3, and 62 +/- 5%, respectively; P < 0.05 for all) in 3T3-L1 adipocytes. In a rat fibroblast cell line overexpressing the human insulin receptor (HIRc-B), SHIP inhibited membrane ruffling induced by insulin and IGF-I by 76 +/- 3% (P < 0.001) and 68 +/- 5% (P < 0.005), respectively. However, growth factor-induced stress fiber breakdown was not affected by SHIP expression. Finally, SHIP decreased significantly growth factor-induced mitogen-activated protein kinase activation and DNA synthesis. Expression of the catalytically inactive mutant had no effect on these cellular responses. In summary, our results show that expression of SHIP inhibits insulin-induced GLUT4 translocation, growth factor-induced membrane ruffling, and DNA synthesis, indicating that PtdIns 3,4,5-P3 is the key phospholipid product mediating these biological actions.

Phosphorylation regulates the microtubule-destabilizing activity of stathmin and its interaction with tubulin.

Stathmin is a regulator of microtubule dynamics which undergoes extensive phosphorylation during the cell cycle as well as in response to various extracellular factors. Four serine residues are targets for protein kinases: Ser-25 and Ser-38 for proline-directed kinases such as mitogen-activated protein kinase and cyclin-dependent protein kinase, and Ser-16 and Ser-63 for cAMP-dependent protein kinase. We studied the effect of phosphorylation on the microtubule-destabilizing activity of stathmin and on its interaction with tubulin in vitro. We show that triple phosphorylation on Ser-16, Ser-25, and Ser-38 efficiently inhibits its activity and prevents its binding to tubulin.

Subcellular localization, distribution and expression of calmodulin in Zea mays roots.

The subcellular localization, distribution and the steady state level of calmodulin from maize roots (Zea mays L., cv. LG 11) were studied. To analyze the subcellular localization, 2-day old root membranes were fractionated by sucrose density gradient centrifugation and immunoblotting was done with an antibody raised against a vertebrate calmodulin (SWant) which recognized the plant calmodulin. Calmodulin was principally associated with high density fractions and particularly plasmalemma. For studying the distribution of calmodulin in various zones of Zea mays roots, a micro method of membrane preparation was developed. Most of the calmodulin was present in microsomes isolated from the root apex corresponding to the first 4 mm of a 15 +/- 2 mm root. An identical distribution was found by studying the steady state level of the protein by Northern blotting using a cDNA clone of Zea mays calmodulin.

Tonoplast localization of a calmodulin-stimulated Ca2+-pump from maize roots.

The subcellular localization of a calmodulin-stimulated calcium (Ca2+)-ATPase activity from maize roots (Zea mays L., cv LG 11) was studied. For this purpose, an efficient procedure was developed to prepare sealed plasma membrane vesicles allowing the measurement of proton and Ca2+ transport activities. Two-day-old root membranes were fractionated by sucrose and dextran density gradient centrifugation. Marker enzymes were used to study the distribution of the different membranes in the gradients and a filtration technique was developed to measure Ca-45(2+) transport in sealed vesicles. Most of the ATP-dependent Ca2+ transport activity was associated with the ER. However, a small part of this activity was associated with the tonoplast (corresponding to the activity of the H+/Ca2+ antiport) and the plasma membrane. When the Ca2+ transport was measured in the presence of exogenous calmodulin (1 muM), a 3-5-fold increase of uptake was measured. The calmodulin-stimulated activity was associated with the tonoplast vesicles only. This activity was insensitive to monensin, a proton ionophore, ruling out a direct effect of calmodulin on the H+/Ca2+ antiport. In conclusion, four different Ca2+ transporters are present in young maize root cells. A Ca2+/H+ antiport system is present on the tonoplast, whereas, the plasma membrane and the ER possess each a calmodulinin-sensitive Ca2+-ATPase. Finally, a calmodulin-stimulated Ca2+-ATPase is associated with the tonoplast.

Novel calmodulin mutations associated with congenital arrhythmia susceptibility.

BACKGROUND: Genetic predisposition to life-threatening cardiac arrhythmias such as congenital long-QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) represent treatable causes of sudden cardiac death in young adults and children. Recently, mutations in calmodulin (CALM1, CALM2) have been associated with severe forms of LQTS and CPVT, with life-threatening arrhythmias occurring very early in life. Additional mutation-positive cases are needed to discern genotype-phenotype correlations associated with calmodulin mutations. METHODS AND RESULTS: We used conventional and next-generation sequencing approaches, including exome analysis, in genotype-negative LQTS probands. We identified 5 novel de novo missense mutations in CALM2 in 3 subjects with LQTS (p.N98S, p.N98I, p.D134H) and 2 subjects with clinical features of both LQTS and CPVT (p.D132E, p.Q136P). Age of onset of major symptoms (syncope or cardiac arrest) ranged from 1 to 9 years. Three of 5 probands had cardiac arrest and 1 of these subjects did not survive. The clinical severity among subjects in this series was generally less than that originally reported for CALM1 and CALM2 associated with recurrent cardiac arrest during infancy. Four of 5 probands responded to β-blocker therapy, whereas 1 subject with mutation p.Q136P died suddenly during exertion despite this treatment. Mutations affect conserved residues located within Ca(2+)-binding loops III (p.N98S, p.N98I) or IV (p.D132E, p.D134H, p.Q136P) and caused reduced Ca(2+)-binding affinity. CONCLUSIONS: CALM2 mutations can be associated with LQTS and with overlapping features of LQTS and CPVT.

A Mutation in CALM1 Encoding Calmodulin in Familial Idiopathic Ventricular Fibrillation in Childhood and Adolescence.

OBJECTIVES: This study aimed to identify the genetic defect in a family with idiopathic ventricular fibrillation (IVF) manifesting in childhood and adolescence. BACKGROUND: Although sudden cardiac death in the young is rare, it frequently presents as the first clinical manifestation of an underlying inherited arrhythmia syndrome. Gene discovery for IVF is important as it enables the identification of individuals at risk, because except for arrhythmia, IVF does not manifest with identifiable clinical abnormalities. METHODS: Exome sequencing was carried out on 2 family members who were both successfully resuscitated from a cardiac arrest. RESULTS: We characterized a family presenting with a history of ventricular fibrillation (VF) and sudden death without electrocardiographic or echocardiographic abnormalities at rest. Two siblings died suddenly at the ages of 9 and 10 years, and another 2 were resuscitated from out-of-hospital cardiac arrest with documented VF at ages 10 and 16 years, respectively. Exome sequencing identified a missense mutation affecting a highly conserved residue (p.F90L) in the CALM1 gene encoding calmodulin. This mutation was also carried by 1 of the siblings who died suddenly, from whom DNA was available. The mutation was present in the mother and in another sibling, both asymptomatic but displaying a marginally prolonged QT interval during exercise. CONCLUSIONS: We identified a mutation in CALM1 underlying IVF manifesting in childhood and adolescence. The causality of the mutation is supported by previous studies demonstrating that F90 mediates the direct interaction of CaM with target peptides. Our approach highlights the utility of exome sequencing in uncovering the genetic defect even in families with a small number of affected individuals.

Calcineurin blockade prevents cardiac mitogen-activated protein kinase activation and hypertrophy in renovascular hypertension.

Chronic stimulation of the renin-angiotensin system induces an elevation of blood pressure and the development of cardiac hypertrophy via the actions of its effector, angiotensin II. In cardiomyocytes, mitogen-activated protein kinases as well as protein kinase C isoforms have been shown to be important in the transduction of trophic signals. The Ca(2+)/calmodulin-dependent phosphatase calcineurin has also been suggested to play a role in cardiac growth. In the present report, we investigate possible cross-talks between calcineurin, protein kinase C, and mitogen-activated protein kinase pathways in controlling angiotensin II-induced hypertrophy. Angiotensin II-stimulated cardiomyocytes and mice with angiotensin II-dependent renovascular hypertension were treated with the calcineurin inhibitor cyclosporin A. Calcineurin, protein kinase C, and mitogen-activated protein kinase activations were determined. We show that cyclosporin A blocks angiotensin II-induced mitogen-activated protein kinase activation in cultured primary cardiomyocytes and in the heart of hypertensive mice. Cyclosporin A also inhibits specific protein kinase C isoforms. In vivo, cyclosporin A prevents the development of cardiac hypertrophy, and this effect appears to be independent of hemodynamic changes. These data suggest cross-talks between the calcineurin pathway, the protein kinase C, and the mitogen-activated protein kinase signaling cascades in transducing angiotensin II-mediated stimuli in cardiomyocytes and could provide the basis for an integrated model of cardiac hypertrophy.