Protein kinase D is a downstream target of protein kinase Ctheta

Protein kinase D (PKD/PKCmu immunoprecipitated from either COS-7 cells or Jurkat T lymphocytes transiently transfected with a constitutively active mutant of PKCtheta AE (PKCthetaAE) exhibited a marked increase in basal activity. In contrast, coexpression of constitutively active mutant of PKCzeta does not induce PKD activation in both types of cells. PKCthetaAE does not induce kinase activity in immunocomplexes of PKD kinase-deficient mutants PKDK618N or PKDD733A. PKD activation in response to PKCthetaAE signaling was completely prevented by treatment with the protein kinase C (PKC) inhibitors, GF I or Ro 31-8220, or by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Our results show that PKD is a downstream target of the theta isoform of PKC in both COS-7 cells and lymphocytes. The regulation of PKD by PKCtheta reveals a new pathway in the signaling network existing between multiple members of the PKC superfamily and PKD.     

Cyclic phosphatidic acid influences the expression and regulation of cyclic nucleotide phosphodiesterase 3B and lipolysis in 3T3-L1 cells

Cyclic phosphatidic acid (cPA) is found in cells from slime mold to humans and has a largely unknown function. We previously reported that cPA significantly inhibited the lipid accumulation in 3T3-L1 adipocytes through inhibition of PPARγ activation. We find here that cPA reduced intracellular triglyceride levels and inhibited the phosphodiesterase 3B (PDE3B) expression in 3T3-L1 adipocytes. PPARγ activation in adipogenesis that can be blocked by treatment with cPA then participates in adipocyte function through inhibition of PDE3B expression. We also found the intracellular cAMP levels in 3T3-L1 adipocytes increased after exposure to cPA. These findings contribute to the participation of cPA on the lipolytic activity in 3T3-L1 adipocytes. Our studies imply that cPA might be a therapeutic compound in the treatment of obesity and obesity-related diseases.     

Cyclic phosphatidic acid influences the expression and regulation of cyclic nucleotide phosphodiesterase 3B and lipolysis in 3T3-L1 cells

The abundance of transforming growth factor-beta (TGF-β) in normal airway epithelium suggests its participation in physiological processes to maintain airway homeostasis. The current study was designed to address the hypothesis that TGF-β1 and TGF-β2 might contribute to normal reparative response of airway epithelial cells (AECs). Treatments with exogenous TGF-β1 or TGF-β2 significantly enhanced wound repair of confluent AEC monolayers. Mechanical injury of AEC monolayers induced production of both TGF-β1 and TGF-β2. Wound repair of AECs was significantly reduced by a specific inhibitor of TGF-β type I receptor kinase activity. We investigated whether the TGF-β-enhanced repair required epidermal growth factor receptor (EGFR) transactivation and secretion of EGFR ligands. Both TGF-β1 and TGF-β2 enhanced EGFR phosphorylation and induced production of heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-α) in AECs. Moreover, treatment with a broad-spectrum metalloproteinase inhibitor or anti-HB-EGF and anti-TGF-α antibodies inhibited the wound repair and the EGFR phosphorylation by TGF-β1 and TGF-β2, indicating that the TGF-β1 and TGF-β2 effects on wound repair required the release of HB-EGF and TGF-α. Our data, for the first time, have shown that both TGF-β1 and TGF-β2 play a stimulatory role in airway epithelial repair through EGFR phosphorylation following autocrine production of HB-EGF and TGF-α. These findings highlight an important collaborative mechanism between TGF-β and EGFR in maintaining airway epithelial homeostasis.     

B23 is a downstream target of polyamine- modulated CK2

Our previous studies have shown that the overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, increases the enzymatic activity of the polyamine-responsive enzyme casein kinase 2 (CK2). Because CK2 is known to preferentially associate with the nuclear matrix in response to other trophic stimuli, we investigated the effects of ODC overexpression on CK2 localisation and on the CK2-mediated phosphorylation of a known CK2 substrate, the nucleolar phosphoprotein B23. Immunofluorescence analysis of CK2 and B23 in primary keratinocytes revealed that ODC overexpression resulted in the colocalisation of CK2 with B23 at the nucleolar borders. ODC overexpression also increased CK2 kinase activity 2-fold at the nuclear matrix, a response which could be abrogated by treatment of K6/ODC transgenic keratinocytes with the ODC inhibitor α-difluoromethylornithine (DFMO). Levels of B23 protein were also elevated in ODC-overexpressing cells compared to normal cells or transgenic cells treated with DFMO. This increase in protein level was neither due to an increase in steady-state mRNA levels, nor was it due to increased stability of B23 protein. Phosphorylation of B23 was also increased in ODC-overexpressing cells, and this increased phosphorylation could be blocked by treatment of the cells with the CK2 kinase inhibitors apigenin or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). These data suggest that B23 may be a downstream effector of polyamines via phosphorylation by the protein kinase CK2.     

Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes

In adipocytes, protein kinase B (PKB) has been suggested to be the enzyme that phosphorylates phosphodiesterase 3B (PDE3B), a key enzyme in insulin's antilipolytic signalling pathway. In order to screen for PKB phosphatases, adipocyte homogenates were fractionated using ion-exchange chromatography and analysed for PKB phosphatase activities. PKB phosphatase activity eluted as one main peak, which coeluted with serine/threonine phosphatases (PP)2A. In addition, adipocytes were incubated with inhibitors of PP. Incubation of adipocytes with 1 μM okadaic acid inhibited PP2A by 75% and PP1 activity by only 17%, while 1 μM tautomycin inhibited PP1 activity by 54% and PP2A by only 7%. Okadaic acid, but not tautomycin, induced the activation of both PKBα and PKBβ. Finally, PP2A subunits were found in several subcellular compartments, including plasma membranes (PM) where the phosphorylation of PKB is thought to occur. In summary, our results suggest that PP2A is the principal phosphatase that dephosphorylates PKB in adipocytes.     

Long-term regulation of cyclic nucleotide phosphodiesterase type 3B and 4 in 3T3-L1 adipocytes

Phosphodiesterase 3B (PDE3B), is known to play an important role in acute insulin and cAMP-mediated regulation of lipid metabolism, and PDE4 are the main PDE types expressed in adipocytes. Here, we show that members of all PDE4 isoforms are expressed in 3T3-L1 and primary mouse adipocytes. Long-term treatment of 3T3-L1 adipocytes with insulin induced up-regulation of PDE3B and PDE4D in a phosphatidylinositol 3-kinase-dependent manner whereas long-term treatment with beta-adrenergic agonists induced down-regulation of PDE3B and up-regulation of PDE4D. Thus, PDE3B and PDE4D can be added to the list of genes regulated by insulin and cAMP-increasing hormones. Altered expression of PDE3B and PDE4D in response to long-term treatment with insulin and catecholamines may contribute to altered regulation of metabolism in diabetes.     

Tyrosine protein kinase is involved in anti-IgM-mediated signaling in BAL17 B lymphoma cells.

BAL17 B lymphoma cells, representing mature B lymphocytes, were used to analyze the role of tyrosine kinase in B cell activation. Anti-IgM-induced tyrosine phosphorylation was inhibited by preincubation of cells with tyrosine kinase inhibitor herbimycin A. Enzymatic activity of lyn protein was also inhibited by this drug, accompanied by down-regulation of p53lyn and p56lyn. However, a protein kinase C-mediated event was intact in the herbimycin A-pretreated cells, suggesting that the inhibitor acts selectively on tyrosine kinase. Anti-IgM failed to stimulate herbimycin A-pretreated cells to induce increases in inositol phospholipid metabolism or increased [Ca2+]i, whereas aluminum fluoride-induced metabolism was not altered. Moreover, membrane IgM density as revealed by flow cytometry was not changed by herbimycin A. These results indicate that tyrosine kinase(s) participates in the coupling of an Ag receptor cross-linkage to phospholipase C activation through a phosphorylation event in B lymphoma cells.     

Phosphorylation and activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase by a brain Ca2+, calmodulin-dependent protein kinase

A Ca2+, calmodulin-dependent protein kinase from rat brain with a MW of 640,000 phosphorylated calmodulin-sensitive phosphodiesterase from the brain cytosol. The Km of the enzyme for the phosphodiesterase was 5.0 microM and the Vmax was 212 nmol/mg/min. The amount of phosphate incorporated into the phosphodiesterase was 0.7 mol/mol subunit. Phosphorylation of the phosphodiesterase enhanced the enzyme activity by about 20% for hydrolysis of a higher concentration of cyclic AMP.     

Activation of a cyclic nucleotide phosphodiesterase and of a protein kinase by chemically modified calmodulin

1. Several calmodulin derivatives prepared by chemical modification of lysine residues were tested using bovine heart cyclic nucleotide phosphodiesterase and wheat germ calmodulin-dependent protein kinase. 2. The effect of chemical modification on the activation capacity of calmodulin for the two studied enzymes was different. 3. This was particularly noticeable in the case of alkylated derivatives which exhibited a higher affinity than native calmodulin towards phosphodiesterase but a lower affinity towards protein kinase. 4. The efficiency of these derivatives (maximal activation) was higher than that of native calmodulin in relation with the protein kinase.     

High-mobility group protein 2 may be involved in the locus control region regulation of the beta-globin gene cluster.

Expression regulation of the beta-globin gene cluster is a result of synergistic interactions between cis-elements and trans-acting factors. Previous studies usually concentrated on the core sequence of each hypersensitive site in the locus control region of the beta-globin gene cluster. But more and more evidence illustrates that the flanking regions are indispensable also. Using electrophoretic mobility shift assay and solid-phase DNase I footprinting methods, we identified a small nuclear protein from K562 cells that binds specifically to the first AT-rich region flanking the hypersensitive site 2 core sequence of the human beta-globin gene locus control region. N-terminal sequencing of the enriched protein proved that it is a member of the high-mobility group protein 2 family. This indicates that the AT-rich region in human hypersensitive site 2 may take part in the regulation of the beta-globin gene cluster by facilitating DNA bending, which is a prerequisite for the looping mechanism in this region.     

Mitogen-activated protein kinase phosphatase-3 is a tumor promoter target in initiated cells that express oncogenic Ras

We have capitalized on the unique properties of the skin tumor promoter palytoxin, which does not activate protein kinase C, to investigate alternative mechanisms by which major signaling molecules can be modulated during carcinogenesis. We report here that palytoxin activates extracellular signal-regulated kinase (ERK) through a novel mechanism that involves inactivation of an ERK phosphatase in keratinocytes derived from initiated mouse skin (308 cells). Use of U0126 revealed that palytoxin requires the ERK kinase MEK to stimulate ERK activity, although palytoxin did not activate MEK. We found that 308 keratinocytes highly express mitogen-activated protein kinase phosphatase-3 (MKP-3), which selectively inactivates ERK. Palytoxin induced the loss of MKP-3 in a manner that corresponded to increased ERK phosphorylation. Complementary studies showed that sustained expression of exogenous MKP-3 inhibited palytoxin-stimulated ERK activation. As is characteristic of initiated keratinocytes, 308 cells express activated H-Ras. To investigate whether expression of oncogenic Ras is key to palytoxin-stimulated ERK activation, we determined how palytoxin affected ERK and MKP-3 in MCF10A human breast epithelial cells and in H-ras MCF10A cells, which stably express activated H-Ras. Palytoxin did not affect ERK activity in MCF10A cells, which had no detectable MKP-3. Like 308 cells, H-ras MCF10A cells highly express MKP-3. Strikingly, palytoxin stimulated ERK activity and induced a corresponding loss of MKP-3 in H-ras MCF10A cells. These studies indicate that in initiated cells palytoxin unleashes ERK activity by down-regulating MKP-3, an ERK inhibitor, and further suggest that MKP-3 may be a vulnerable target in cells that express oncogenic Ras.     

A diacylglycerol kinase is involved in the regulation of interleukin-2 synthesis in Jurkat T cells.

Diacylglycerol (DAG) plays a prominent role in several activation processes because of its ability, in the presence of calcium ions and phosphatidylserine, to activate C kinase. In T lymphocytes, however, DAG, produced in response to activating mAb or lectins, is rapidly metabolized into phosphatidic acid (PA) which may participate in further steps of activation. We thus investigated the involvement of a DAG kinase in several events subsequent to the activation of Jurkat T cells by CD3 mAb or phytohemagglutinin (PHA). We showed that three inhibitors of DAG kinase abrogated PA production and impaired calcium release from intracellular compartment, restored phosphatidylserine synthesis, and finally blocked IL-2 production in CD3- and PHA-stimulated cells. Postactivation DAG levels were not modified and DAG kinase inhibitors lowered IL-2 secretion even in the presence of phorbol ester that directly activates the C kinase. These results clearly demonstrate that, beside the effect of DAG on C kinase, DAG kinase dependent production of PA is crucial for further steps of T cell activation.     

Tip60 and HDAC7 interact with the endothelin receptor a and may be involved in downstream signaling

Endothelins exert their biological effects through G protein-coupled receptors. However, the precise mechanism of downstream signaling and trafficking of the receptors is largely unknown. Here we report that the histone acetyltransferase Tip60 and the histone deacetylase HDAC7 interact with one of the ET receptors, ETA, as determined by yeast two-hybrid analysis, glutathione S-transferase pull-down assays, and co-immunoprecipitation from transfected COS-7 cells. In the absence of ET-1, Tip60 and HDAC7 were localized mainly in the cell nucleus while ETA was predominantly confined to the plasma membrane. Stimulation with ET-1 resulted in the internalization of ETA to the perinuclear compartment and simultaneously in the efflux of Tip60 and HDAC7 from the nucleus to the same perinuclear compartment where each protein co-localized with the receptor. Upon co-transfection with ETA into COS-7 cells, Tip60 strongly increased ET-1-induced ERK1/2 phosphorylation, whereas HDAC7 had no significant effect. We thus suggest that protein acetylase and deacetylase interact with ETA in a ligand-dependent fashion and may participate in ET signal transduction.     

Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca(2+) sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin, a Ca(2+) ionophore that bypasses K(+) and Ca(2+) channels. Overexpression of NCS-1 caused a shift in the dose-response curve of inhibition of ATP-evoked secretion using phenylarsine oxide, an inhibitor of phosphatidylinositol 4-OH kinase (PI4K). Plasma membrane phosphatidylinositol 4,5-bisphosphate pools were increased upon NCS-1 transfection as visualized using a phospholipase C-delta pleckstrin homology domain-green fluorescent protein construct. NCS-1-transfected cell extracts displayed increased phosphatidylinositol-4-phosphate biosynthesis, indicating an increase in PI4K activity. Mutations in NCS-1 equivalent to those that abolish the interaction of recoverin, another EF-hand-containing Ca(2+) sensor, with its downstream target rhodopsin kinase, lost their ability to enhance exocytosis. Taken together, the present data indicate that NCS-1 modulates the activity of PI4K, leading to increased levels of phosphoinositides and concomitant enhancement of exocytosis.