Ubiquitous Kinase Research Articles

42: Enhanced sensitivity of human colon cancer cells to 5-fluorouracil and trail by selective inhibition of the PI3K/Akt pathway

Introduction: Despite improvements in the survival of patients with colorectal cancers (CRC), systemic metastasis remains a significant cause of morbidity and mortality. 5-fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents for advanced CRC, yet overall response rates are as low as 10-20%. TNF-related apoptosis-inducing ligand (TRAIL) is a novel member of the TNF family which selectively induces apoptosis in many transformed cells; however, recent studies indicate that many cancers are resistant to TRAIL. We have shown that activation of phosphatidylinositol 3-kinase (PI3K), a ubiquitous lipid kinase composed of regulatory (p85) and catalytic (p110) subunits, and its downstream effector protein, Akt, is associated with growth and progression of CRC; inhibition of PI3K/Akt may sensitize resistant cancers to current therapies. The purpose of this study was to determine whether selective inhibition of PI3K/Akt can sensitize resistant CRC to either 5-FU or TRAIL. Methods: (1) In initial experiments, human colon cancer cells KM20, KM12C and KML4A were treated with increasing concentrations of 5-FU or TRAIL. Cytotoxicity assays were performed using the WST-1 and sulforhodamine B techniques representing the percentage of cell growth compared to a control after exposure to increasing drug concentrations. As an indicator of apoptosis, DNA fragmentation was measured by ELISA. (2) To determine whether direct inhibition of PI3K/Akt could sensitize resistant CRC, synthetic chemically-modified siRNA duplexes directed to p85α, p110α, Akt2 or non-targeting control (NTC) were transfected into KM20 cells via electroporation. Morphologic changes were assessed by phase-contrast microscopy; cytotoxicity was determined by sulforhodamine B assay. Results: (1) KM20 cells were resistant to TRAIL treatment, with IC50 > 1 mg · L-1 and dose-independent cytotoxicity. KM12C and KM12L4A cells were relatively sensitive to TRAIL, with IC50 > 0.05 mg · L-1. The partial response of KM12C and KM12L4A cells to TRAIL treatment was due to increased apoptosis detected by DNA fragmentation assay. KM20, KM12C and KM12L4A cells were not sensitive to 5-FU treatment; an increase in DNA fragmentation was observed only in KM12L4A cell line. (2) Combination treatment showed significant TRAIL sensitization of KM20 cells with IC50 > 0.0125 mg · L-1 with transfection of Akt2 siRNA and IC50 > 0.05 mg · L-1 with p110α siRNA. Combination treatment with 5-FU resulted in increased cytotoxicity in KM20 cells transfected with either p110α or Akt2 siRNA in a time-dependent fashion. Conclusions: Selective targeting of the PI3K pathway components enhances the effects of standard chemotherapeutic agents and TRAIL and provides a novel adjuvant treatment strategy for selected colon cancers.

Creatine promotes the GABAergic phenotype in human fetal spinal cord cultures

In the present study, we investigated the expression pattern of cytosolic brain specific-BB-CK and ubiquitous mitochondrial-creatine kinases (uMt-CK) in developing human spinal cord. Consequently, we studied the effects of creatine treatment on cultured fetal human spinal cord tissue. We found that both CK isoforms were expressed in fetal spinal cord at all time points investigated (5 to 11.5 weeks post conception) and correspondingly specific CK activity was detected. Chronic creatine exposure resulted in significantly higher densities of GABA-immunoreactive neurons in the cultures, while total neuronal cell density was not altered, suggesting a differentiation inducing mechanism of creatine supplementation. Taken together, our observations favour the view that the creatine phosphocreatine system plays an important role in the developing CNS.

Identifying interaction motifs in CK2β – a ubiquitous kinase regulatory subunit

Casein kinase 2 (CK2) is probably the most ubiquitous serine/threonine kinase found in eukaryotes: it phosphorylates >300 cellular proteins, ranging from transcription factors to proteins involved in chromatin structure and cell division. CK2 is a heterotetrameric enzyme that induces neoplastic growth when overexpressed. The beta subunit of CK2 (CK2beta) functions as the regulator of the catalytic CK2alpha and CK2alpha' subunits, enhancing their stability, activity and specificity. However, CK2beta also functions as a multisubstrate docking platform for several other binding partners. Here, we discuss the organization and roles of interaction motifs of CK2beta, postulate new protein-interaction sites and map these to the known interaction motifs, and show how the resulting complexity of interactions mediated by CK2 gives rise to the versatile functions of this pleiotropic protein kinase.

Characterization of protein kinase CK2 from Trypanosoma brucei

CK2 is a ubiquitous but enigmatic kinase. The difficulty in assigning a role to CK2 centers on the fact that, to date, no biologically relevant modulator of its function has been identified. One common theme revolves around a constellation of known substrates involved in growth control, compatible with its concentration in the nucleus and nucleolus. We had previously described the identification of two catalytic subunits of CK2 in Trypanosoma brucei and characterized one of them. Here we report the characterization of the second catalytic subunit, CK2α′, and the identification and characterization of the regulatory subunit CK2β. All three subunits are primarily localized to the nucleolus in T. brucei. We also show that CK2β interacts with the nucleolar protein NOG1, adding to the interaction map which previously linked CK2α to the nucleolar protein NOPP44/46, which in turn associates with the rRNA binding protein p37. CK2 activity has four distinctive features: near equal affinity for GTP and ATP, heparin sensitivity, and stimulation by polyamines and polybasic peptides. Sequence comparison shows that the parasite orthologues have mutations in residues previously mapped as important in specifying affinity for GTP and stimulation by both polyamines and polybasic peptides. Studies of the enzymatic activity of the T. brucei CK2s show that both the affinity for GTP and stimulation by polyamines have been lost and only the features of heparin inhibition and stimulation by polybasic peptides are conserved.

Multiple myeloma cell survival relies on high activity of protein kinase CK2

Casein kinase 2 (CK2) is a ubiquitous cellular serine-threonine kinase that regulates relevant biologic processes, many of which are dysregulated in malignant plasma cells. Here we investigated its role in multiple myeloma (MM). Analysis of MM cell lines and highly purified malignant plasma cells in patients with MM revealed higher protein and CK2 activity levels than in controls (normal in vitro-generated polyclonal plasma cells and B lymphocytes). The inhibition of CK2 with specific synthetic compounds or by means of RNA interference caused a cytotoxic effect on MM plasma cells that could not be overcome by IL-6 or IGF-I and that was associated with the activation of extrinsic and intrinsic caspase cascades. CK2 blockage lowered the sensitivity threshold of MM plasma cells to the cytotoxic effect of melphalan. CK2 inhibition also resulted in impaired IL-6-dependent STAT3 activation and in decreased basal and TNF-alpha-dependent I kappaB alpha degradation and NF-kappaB-driven transcription. Our data show that CK2 was involved in the pathophysiology of MM, suggesting that it might play a crucial role in controlling survival and sensitivity to chemotherapeutics of malignant plasma cells.

Recoverin Binds Exclusively to an Amphipathic Peptide at the N Terminus of Rhodopsin Kinase, Inhibiting Rhodopsin Phosphorylation without Affecting Catalytic Activity of the Kinase

Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate.

Tissue-specific expression and regulation of GSK-3 in human skeletal muscle and adipose tissue

Glycogen synthase kinase-3 (GSK-3) is a ubiquitous kinase implicated in both insulin action and adipogenesis. To determine how these multiple roles may relate to insulin resistance, we studied the regulation of GSK-3 protein expression and phosphorylation in skeletal muscle and isolated adipocytes from nonobese healthy control (HC), obese control (OC), and obese type 2 diabetic (OT2D) subjects. At baseline there were no differences in the GSK-3 protein expression in adipocytes. OC subjects underwent a 6-mo caloric restriction resulting in a 7% decrease in body mass index (BMI) and a 21% improvement in insulin-stimulated whole body glucose disposal rate (GDR). GSK-3alpha and GSK-3beta expression decreased in adipocytes (P < 0.05), whereas GSK-3alpha protein expression increased in skeletal muscle (P < 0.05). OT2D subjects were treated with troglitazone or metformin for 3-4 mo. After troglitazone treatment GDR improved (P < 0.05) despite an increase in BMI (P < 0.05), whereas metformin had no significant effect on GDR. There was no significant change in GSK-3 expression in adipocytes following troglitazone, whereas both GSK-3alpha and -beta were decreased in skeletal muscle (P < 0.05). Metformin treatment had no significant impact on GSK-3 protein expression in either adipocytes or skeletal muscle. Neither treatment influenced GSK-3 serine phosphorylation in skeletal muscle or adipocytes. These results suggest that there is tissue specificity for the regulation of GSK-3 in humans. In skeletal muscle GSK-3 plays a role in control of metabolism and insulin action, whereas the function in adipose tissue is less clear.

Efficient transduction of monocyte‐ and CD34+‐ derived Langerhans cells with lentiviral vectors in the absence of phenotypic and functional maturation

Gene delivery in dendritic cells (DC) has raised considerable interest to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific fashion. Among immature DC, Langerhans cells (LC) are attractive candidates for antigen delivery using lentiviral vectors (LV). LC derived from monocytes (Mo-LC), or derived from CD34+ cells (CD34-LC) in the presence of cytokine cocktail, were transduced with LV expressing enhanced green fluorescent protein (E-GFP) under the control of the ubiquitous phosphoglycerate kinase (PGK) promoter at a multiplicity of infection of 18, at days 0 to 3 for Mo-LC, or at days 0 to 12 for CD34-LC. We assessed gene transfer levels from the percentage of E-GFP+ cells in the final cultures, and examined the morphology, immunophenotype, state of differentiation and function of transduced LC. Day 0 transduction of monocytes or CD34+ progenitors before cytokine pre-activation and LC differentiation resulted in stable gene expression in 7.8% of Mo-LC and 24% of CD34-LC. Monocyte-derived DC (Mo-DC) differentiated in serum-free medium were also efficiently transduced up to 13.2%. Interestingly, Mo-LC cells committed towards LC phenotype were permissive for transduction up to day 3. Transduction levels of CD34-LC peaked at day 6 to 44% and decreased thereafter. LV transduction did not perturb viability, phenotype and function of E-GFP-expressing LC. LC generated ex vivo can serve as vaccine vehicles in humans through efficient transduction by LV. These LC will be helpful to assess in vitro the immunogenicity of gene therapy vectors, from the characterization of their phenotypic and functional maturation.

Kinase Suppressor of Ras1 Compartmentalizes Hippocampal Signal Transduction and Subserves Synaptic Plasticity and Memory Formation

The ERK/MAP kinase cascade is important for long-term memory formation and synaptic plasticity, with a myriad of upstream signals converging upon ERK activation. Despite this convergence of signaling, neurons routinely activate appropriate biological responses to different stimuli. Scaffolding proteins represent a mechanism to achieve compartmentalization of signaling and the appropriate targeting of ERK-dependent processes. We report that kinase suppressor of Ras (KSR1) functions biochemically in the hippocampus to scaffold the components of the ERK cascade, specifically regulating the cascade when a membrane fraction of ERK is activated via a PKC-dependent pathway but not via a cAMP/PKA-dependent pathway. Specificity of KSR1-dependent signaling also extends to specific downstream targets of ERK. Behaviorally and physiologically, we found that the absence of KSR1 leads to deficits in associative learning and theta burst stimulation-induced LTP. Our report provides novel insight into the endogenous scaffolding role of KSR1 in controlling kinase activation within the nervous system.

PKCδ is necessary for Smad3 expression and TGFβ‐induced fibronectin synthesis in vascular smooth muscle cells

Objective Production of extracellular matrix (ECM) by vascular smooth muscle cells (SMCs) is critical for the development of intimal hyperplasia (IH), a process that leads to failure of vascular reconstructions. TGFβ is a potent mediator of ECM synthesis including fibronectin (FN), a matrix glycoprotein abundant in IH. The purpose of these studies is to investigate the mechanism by which TGFβ regulates the synthesis of fibronectin. Methods and Results TGFβ induced FN protein and mRNA in A10 rat aortic SMCs. Overexpression of Smad3 in A10 SMCs via adenoviral transfection stimulated both basal and TGFβ induced FN protein and mRNA, while ectopic expression of Smad7 eliminated the TGFβ response. To understand the downstream signals from the TGFβ receptor, we manipulated PKCδ, a ubiquitous kinase and intermediate for various cytokine receptors. Inhibition of PKCδ using the chemical inhibitor rottlerin or dominant negative adenovirus (AdPKCδ DN) blocked TGFβ's induction of FN protein and mRNA. Moreover, aortic SMCs isolated from PKCδ −/ − mice exhibited diminished FN induction in response to TGFβ. Furthermore, we found Smad3 protein and mRNA levels markedly reduced in AdPKCδ DN-treated A10 cells and in PKCδ −/ − SMCs. Finally, restoring Smad3 in rottlerin-treated A10 SMCs rescued the ability of TGFβ to upregulate FN protein and mRNA expression. Conclusion Our data suggest that both PKCδ and Smad3 are required for induction of FN by TGFβ. Furthermore, PKCδ regulates FN expression, at least in part, through maintaining normal expression of Smad3. PKCδ may be a novel target for treating the fibroproliferative response after arterial injury.

Use of Chuk as an internal standard suitable for quantitative RT-PCR in mouse preimplantation embryos

Analysis of gene expression changes during preimplantation development by quantitative reverse transcription-polymerase chain reaction (Q-PCR) requires appropriate internal standards. Ideally, such a gene should show a constant level of transcripts per embryo across all preimplantation stages from unfertilized eggs to blastocysts. By analysing the microarray-based gene expression profiles of preimplantation embryos, it was found that a conserved helix–loop–helix ubiquitous kinase gene ( Chuk, also known as IκB kinase α, IKKα or IKK1) satisfied this criterion. To test the utility of this gene as an internal standard for Q-PCR, the expression levels of two known genes ( Nalp5/Mater, Pou5f1/Oct3/Oct4) were normalized by Chuk and other housekeeping genes ( Actb, Gapdh, Eef1a1, and H2afz) and demonstrated that the former was more consistent with the expression patterns obtained by a whole-mount in-situ hybridization than those reported previously with the latter. It is concluded that Chuk, unlike other commonly used normalization controls, is a reliable and suitable internal standard for measuring gene expression levels by Q-PCR in mouse oocytes and preimplantation embryos.

MAP kinases p38 and JNK are activated by the steroid hormone 1α,25(OH)2‐vitamin D3 in the C2C12 muscle cell line

In chick skeletal muscle cell primary cultures, we previously demonstrated that 1alpha,25(OH)2-vitamin D3 [1alpha,25(OH)2D3], the hormonally active form of vitamin D, increases the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2, their subsequent translocation to the nucleus and involvement in DNA synthesis stimulation. In this study, we show that other members of the MAP kinase superfamily are also activated by the hormone. Using the muscle cell line C2C12 we found that 1alpha,25(OH)2D3 within 1 min phosphorylates and increases the activity of p38 MAPK. The immediately upstream mitogen-activated protein kinase kinases 3/6 (MKK3/MKK6) were also phosphorylated by the hormone suggesting their participation in p38 activation. 1Alpha,25(OH)2D3 was able to dephosphorylate/activate the ubiquitous cytosolic tyrosine kinase c-Src in C2C12 cells and studies with specific inhibitors imply that Src participates in hormone induced-p38 activation. Of relevance, 1alpha,25(OH)2D3 induced in the C2C12 line the stimulation of mitogen-activated protein kinase activating protein kinase 2 (MAPKAP-kinase 2) and subsequent phosphorylation of heat shock protein 27 (HSP27) in a p38 kinase activation-dependent manner. Treatment with the p38 inhibitor, SB203580, blocked p38 phosphorylation caused by the hormone and inhibited the phosphorylation of its downstrean substrates. 1Alpha,25(OH)2D3 also promotes the phosphorylation of c-jun N-terminal protein kinases (JNK 1/2), the response is fast (0.5-1 min) and maximal phosphorylation of the enzyme is observed at physiological doses of 1alpha,25(OH)2D3 (1 nM). The relative contribution of ERK-1/2, p38, and JNK-1/2 and their interrelationships in hormonal regulation of muscle cell proliferation and differentiation remain to be established.

Interleukin-2 family cytokines: potential for therapeutic immmunoregulation

IL-2 is a four-helix bundle cytokine that was initially identified as a T cell growth factor more than two decades ago. Since then, extensive investigations have led to the discovery of additional cytokines, including IL-4, IL-7, IL-9, IL-15 and IL-21, which display many similarities with IL-2 and have been grouped, together with the latter, as a distinct IL-2 cytokine family. These cytokines are all globular proteins that contain two sets of short-chain α-helical domains. They all play major roles in promoting and maintaining T lymphocyte populations, albeit in non-redundant and often complementary manners, whilst acting on other cell lineages as well. Most remarkably, their cell surface receptor complexes, which consist of two or three subunits, including a cytokine-specific private subunit, all use a shared subunit called the common γ chain (γc). This common subunit was shown to be mutated in X-linked severe combined immunodeficiency and to uniquely recruit the Janus kinase, Jak3. Accordingly, IL-2 family cytokines all utilise Jak3 in their mechanisms of intracellular signalling, together with more ubiquitous kinases. Owing to their prominent roles in lymphocyte development and homeostasis, IL-2 family cytokines have been the focus of much interest for the purpose of therapeutic applications. They may provide tools to bolster immune responses for the treatment of neoplastic diseases, viral infections and immunodeficiencies. Conversely, their blockade may help achieve immunosuppression for the treatment of transplant rejection, as well as inflammatory, autoimmune and allergic disorders. Here, the patent literature of the past four years pertaining to these important cytokines will be reviewed in the context of the current understanding of their biological significance.

Active Kinase Proteome Screening Reveals Novel Signal Complexity in Cardiomyopathy

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions.

Src Kinase Activity Is Required for Integrin αVβ3-Mediated Activation of Nuclear Factor-κB

Src Kinase Activity Is Required for Integrin αVβ3-Mediated Activation of Nuclear Factor-κB

Surfactant phospholipid DPPC downregulates monocyte respiratory burst via modulation of PKC

Pulmonary surfactant phospholipids have been shown previously to regulate inflammatory functions of human monocytes. This study was undertaken to delineate the mechanisms by which pulmonary surfactant modulates the respiratory burst in a human monocytic cell line, MonoMac-6 (MM6). Preincubation of MM6 cells with the surfactant preparations Survanta, Curosurf, or Exosurf Neonatal inhibited the oxidative response to either lipopolysaccharide (LPS) and zymosan or phorbol 12-myristate 13-acetate (PMA) by up to 50% (P < 0.01). Preincubation of MM6 cells and human peripheral blood monocytes with dipalmitoyl phosphatidylcholine (DPPC), the major phospholipid component of surfactant, inhibited the oxidative response to zymosan. DPPC did not directly affect the activity of the NADPH oxidase in a MM6 reconstituted cell system, suggesting that DPPC does not affect the assembly of the individual components of this enzyme into a functional unit. The effects of DPPC were evaluated on both LPS/zymosan and PMA activation of protein kinase C (PKC), a ubiquitous intracellular kinase, in MM6 cells. We found that DPPC significantly inhibited the activity of PKC in stimulated cells by 70% (P < 0.01). Western blotting experiments demonstrated that DPPC was able to attenuate the activation of the PKCdelta isoform but not PKCalpha. These results suggest that DPPC, the major component of pulmonary surfactant, plays a role in modulating leukocyte inflammatory responses in the lung via downregulation of PKC, a mechanism that may involve the PKCdelta isoform.

Multiple Myeloma Cells Survival and Proliferation Rely on High Levels and Activity of the Serine-Threonine Kinase CK2.

Survival and proliferation of Multiple Myeloma plasma cells (MMPCs) depend on the activation of signaling pathways through the interaction with the surrounding bone marrow microenvironment. CK2 is a ubiquitous cellular serine-threonine kinase, whose involvement in oncogenic transformation, apoptosis and cell cycle progression has recently become matter of intense research. Due to its connection with signaling molecules pivotal for plasma cell (PCs) survival, such as those implicated in the TNF-α/NF-κB, IGF1/PI3K/AKT and Wnt/β-catenin pathways, CK2 is likely to play a central role in MM biology. We investigated CK2 function in MMPCs survival and cell cycle progression, in the modulation of the sensitivity to chemotherapeutics and in the regulation of the I-κB/NF-κB dependent pathway.We first analysed the CK2 protein levels and specific kinase activity in MMPCs. Different cell lines and highly purified CD138+ PCs from 5 patients were used. We observed higher protein levels of the CK2 catalytic subunit αin the neoplastic MMPCs than in controls (resting peripheral blood and splenic B lymphocytes). Moreover, also the total CK2-dependent kinase activity was found significantly increased in MMPCs. We also assessed the levels and pattern of total protein phosphorylation by radioactive phosphate incorporation assay. We found that MMPCs share a similar pattern of phoshorylated proteins. The degree of phosphorylation of some of these proteins was significantly reduced in the presence of specific CK2 inhibitors. Next, using a panel of highly specific CK2 inhibitors, we studied the effects of hampering CK2 function in MMPCs. A dose-dependent cytotoxic effect was observed after the treatment with such compounds that was associated with the activation of both the extrinsic and intrinsic caspase-dependent pathways, the release from mitochondria of cytochrome c and smac/diablo and cell cycle arrest in G2-M. A possible role for CK2 inhibition in sensitising MMPCs to melphalan-induced apoptosis was also investigated. Indeed, CK2 blockade lowered the threshold of sensitivity of MMPCs to the cytotoxic effect of melphalan. We then looked at the consequences of CK2 blockade on the NF-κB dependent signaling cascade. Basal and TNF-α-dependent I-κB-αdegradation, as well as NF-κB transcriptional activity upon TNF-αstimulation, were partially impaired by CK2 blockade in MMPCs. Finally, we detected association between the endogenous αcatalytic subunit of CK2 and the NF-κB p50/p105 member by confocal microscopy and co-immunoprecipitation.Altogether, our data suggest a pivotal role for CK2 in controlling survival, proliferation and sensitivity to chemotherapeutics of MMPCs and implicate this kinase in the regulation of the NF-κB pathway in MM through the modulation of I-κB protein levels and NF-κB transcriptional activity. This latter effect is possibly exerted through physical association of CK2 with NF-κB transcription factors. Our findings also suggest that CK2 inhibition could be exploited as a novel therapeutic approach for MM.

Structural and behavioural consequences of double deficiency for creatine kinases BCK and UbCKmit

The cytosolic brain-type creatine kinase (BCK) isoform and the mitochondrial ubiquitous creatine kinase (UbCKmit) isoform are both important for the maintenance and distribution of cellular energy in neurons and astrocytes. Previously, we reported that mice deficient for BCK or UbCKmit each showed a surprisingly mild phenotype, probably due to reciprocal functional compensation by the remaining creatine kinase. This study shows that adult male mice lacking both creatine kinase isoforms (CK--/-- double knockout mice) have a reduced body weight, and demonstrate a severely impaired spatial learning in both a dry and a wet maze, lower nestbuilding activity and diminished acoustic startle reflex responses when compared to age-matched male wildtype mice with the same genetic background. In contrast, their visual and motor functions, exploration behaviour, prepulse inhibition and anxiety-related responses were not changed, suggesting no global deficit in sensorimotor function, hearing or motivation. Morphological analysis of CK--/-- double knockout brains revealed a reduction of ∼7% in wet brain weight and hippocampal size, a ∼15% smaller regio-inferior and relatively larger supra-pyramidal, and intra-infra-pyramidal mossy fiber areas. These results suggest that lack of both brain specific creatine kinase isoforms renders the synaptic circuitry in adult brain less efficient in coping with sensory or cognitive activity related challenges.

Mammalian Target of Rapamycin Positively Regulates Collagen Type I Production via a Phosphatidylinositol 3-Kinase-independent Pathway

The mammalian target of rapamycin (mTOR) is a multifunctional protein involved in the regulation of cell growth, proliferation, and differentiation. The goal of this study was to determine the role of mTOR in type I collagen regulation. The pharmacological inhibitor of phosphatidylinositol (PI) 3-kinase, LY294002, significantly inhibited collagen type I protein and mRNA levels. The effects of LY294002 were more pronounced on the collagen alpha1(I) chain, which was inhibited at the transcriptional and mRNA stability levels versus collagen alpha2(I) chain, which was inhibited through a decrease in mRNA stability. In contrast, addition of the PI 3-kinase inhibitor, wortmannin, did not alter type I collagen steady-state mRNA levels. This observation and further experiments using an inactive LY294002 analogue suggested that collagen mRNA levels are inhibited independent of PI 3-kinase. Additional experiments have established that mTOR positively regulates collagen type I synthesis in human fibroblasts. These conclusions are based on results demonstrating that inhibition of mTOR activity using a specific inhibitor, rapamycin, reduced collagen mRNA levels. Furthermore, decreasing mTOR expression by about 50% by using small interfering RNA resulted in a significant decrease of collagen mRNA (75% COL1A1 decrease and 28% COL1A2 decrease) and protein levels. Thus, mTOR plays an essential role in regulating basal expression of collagen type I gene in dermal fibroblasts. Together, our data suggest that the classical PI 3-kinase pathway, which places mTOR downstream of PI 3-kinase, is not involved in mTOR-dependent regulation of type I collagen synthesis in dermal fibroblasts. Because collagen overproduction is a main feature of fibrosis, identification of mTOR as a critical mediator of its regulation may provide a suitable target for drug or gene therapy.

GSK3 beta-dependent phosphorylation of the alpha NAC coactivator regulates its nuclear translocation and proteasome-mediated degradation.

c-Jun is an immediate-early gene whose degradation by the proteasome pathway is required for an efficient transactivation. In this report, we demonstrated that the c-Jun coactivator, nascent polypeptide associated complex and coactivator alpha (alphaNAC) was also a target for degradation by the 26S proteasome. The proteasome inhibitor lactacystin increased the metabolic stability of alphaNAC in vivo, and lactacystin, MG-132, or epoxomicin treatment of cells induced nuclear translocation of alphaNAC. We have shown that the ubiquitous kinase glycogen synthase kinase 3beta (GSK3beta) directly phosphorylated alphaNAC in vitro and in vivo. Inhibition of the endogenous GSKappa3beta activity resulted in the stabilization of this coactivator in vivo. We identified the phosphoacceptor site in the C-terminal end of the coactivator, on position threonine 159. We demonstrated that the inhibition of GSK3beta activity by treatment of cells with the inhibitor 5-iodo-indirubin-3'-monoxime, as well as with a dominant-negative GSK3beta mutant, induced the accumulation of alphaNAC in the nuclei of cells. Mutation of the GSK3beta phosphoacceptor site on alphaNAC induced a significant increase of its coactivation potency. We conclude that GSK3beta-dependent phosphorylation of alphaNAC was the signal that directed the protein to the proteasome. The accumulation of alphaNAC caused by the inhibition of the proteasome pathway or the activity of GSK3beta contributes to its nuclear translocation and impacts on its coactivating function.