Neu differentiation factor stimulates phosphorylation and activation of the Sp1 transcription factor.
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Neu differentiation factors (NDFs), or neuregulins, are epidermal growth factor-like growth factors which bind to two tyrosine kinase receptors, ErbB-3 and ErbB-4. The transcription of several genes is regulated by neuregulins, including genes encoding specific subunits of the acetylcholine receptor at the neuromuscular junction. Here, we have examined the promoter of the acetylcholine receptor epsilon subunit and delineated a minimal CA-rich sequence which mediates transcriptional activation by NDF (NDF-response element [NRE]). Using gel mobility shift analysis with an NRE oligonucleotide, we detected two complexes that are induced by treatment with neuregulin and other growth factors and identified Sp1, a constitutively expressed zinc finger phosphoprotein, as a component of one of these complexes. Phosphatase treatment, two-dimensional gel electrophoresis, and an in-gel kinase assay indicated that Sp1 is phosphorylated by a 60-kDa kinase in response to NDF-induced signals. Moreover, Sp1 seems to act downstream of all members of the ErbB family and thus may funnel the signaling of the ErbB network into the nucleus. (+info)
Karyotyping of human oocytes by chromosomal analysis of the second polar bodies.
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This paper describes a method for obtaining metaphase chromosomes from human second polar bodies. The second polar body nucleus was injected into the cytoplasm of an enucleated oocyte, which is activated shortly after injection. When the polar body nucleus is transformed into a haploid pronucleus, treatment with okadaic acid was used to induce premature chromosome condensation. A total of 25 analysable chromosome plates were obtained from 38 polar bodies karyotyped using this technique. Whole chromosome painting was used to detect second polar bodies (and respectively, oocytes) with unbalanced translocations. In combination with the first polar body analysis, this technique may be useful in preimplantation genetic diagnosis for patients carrying maternal translocations. (+info)
A unique Na+/H+ exchanger, analogous to NHE1, in the chicken embryonic fibroblast.
(3/1378)
We report the characterization of an Na+/H+ exchanger (NHE) in embryonic fibroblasts (SL-29 cells) of the chicken, a terrestrial vertebrate, where Na+ conservation is important. This exchanger is electroneutral, has a single Na+ binding site, and is highly sensitive to amiloride (IC50 2 microM), dimethyl amiloride (350 nM), and ethyl-isopropyl amiloride (25 nM). It is stimulated by serum, transforming growth factor-alpha, hypertonicity, and okadaic acid. Although these features make it resemble mammalian NHE1, other characteristics suggest distinct differences. First, in contrast to mammalian NHE1 it is inhibited by cAMP and shows a biphasic response to phorbol esters and a highly variable response to increased intracellular Ca2+ concentration. Second, whereas full-length human and rat NHE1 cDNA probes recognize a 4.8-kb transcript in rat tissues, they recognize only a 3.9-kb transcript in chicken tissues. An antibody against amino acids 631-746 of human NHE1 sequence fails to recognize a protein in SL-29 cells. Rat NHE2 and NHE3 probes do not recognize any transcript in chicken fibroblasts. The SL-29 exchanger differs markedly from the previously characterized chicken intestinal apical exchanger in its amiloride sensitivity and regulation by phorbol esters. These results suggest that a modified version of mammalian NHE1 is present in chicken tissues and imply that another functionally distinct Na+/H+ exchanger is expressed in aves. (+info)
Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells.
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The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c). (+info)
The expression of casein kinase 2alpha' and phosphatase 2A activity.
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Protein phosphatase 2A (PP2A) activity may be differentially regulated by the expression of proteins containing a related amino acid sequence motif such as the casein kinase 2alpha (CK2alpha) subunit or SV40 small t antigen (SVt). Expression of CK2alpha increases PP2A activity whereas SVt decreases its activity. In this work we have tested for the effect of the expression of a third protein containing a similar motif that could be involved in PP2A regulation, the catalytic casein kinase 2alpha' subunit. Our results show that despite the structural similarity of this protein with the other CK2 catalytic (alpha) subunit, the function of the two subunits with respect to the modulation of PP2A activity is quite different: CK2alpha increases whereas CK2alpha' slightly decreases PP2A activity. (+info)
Differential effects of a calcineurin inhibitor on glutamate-induced phosphorylation of Ca2+/calmodulin-dependent protein kinases in cultured rat hippocampal neurons.
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Calcium/calmodulin-dependent protein kinases (CaM kinases) are major multifunctional enzymes that play important roles in calcium-mediated signal transduction. To characterize their regulatory mechanisms in neurons, we compared glutamate-induced phosphorylation of CaM kinase IV and CaM kinase II in cultured rat hippocampal neurons. We observed that dephosphorylation of these kinases followed different time courses, suggesting different regulatory mechanisms for each kinase. Okadaic acid, an inhibitor of protein phosphatase (PP) 1 and PP2A, increased the phosphorylation of both kinases. In contrast, cyclosporin A, an inhibitor of calcineurin, showed different effects: the phosphorylation and activity of CaM kinase IV were significantly increased with this inhibitor, but those of CaM kinase II were not significantly increased. Cyclosporin A treatment of neurons increased phosphorylation of Thr196 of CaM kinase IV, the activated form with CaM kinase kinase, which was recognized with an anti-phospho-Thr196 antibody. Moreover, recombinant CaM kinase IV was dephosphorylated and inactivated with calcineurin as well as with PP1, PP2A, and PP2C in vitro. These results suggest that CaM kinase IV, but not CaM kinase II, is directly regulated with calcineurin. (+info)
NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation.
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The cloned epithelial cell-specific Na+/H+ exchanger (NHE) isoform NHE2 is stimulated by fibroblast growth factor (FGF), phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), and fetal bovine serum (FBS) through a change in maximal velocity of the transporter. In the present study, we used COOH-terminal truncation mutants to delineate specific domains in the COOH terminus of NHE2 that are responsible for growth factor and/or protein kinase regulation. Five truncation mutants (designated by the amino acid number at the truncation site) were stably expressed in NHE-deficient PS120 fibroblasts. The effects of PMA, FGF, OA, FBS, and W-13 [a Ca2+/calmodulin (CaM) inhibitor] were studied. Truncation mutant E2/660, but not E2/573, was stimulated by PMA. OA stimulated E2/573 but not E2/540. FGF stimulated E2/540 but not E2/499. The most truncated mutant, E2/499, was stimulated by FBS. W-13 stimulated the basal activity of the wild-type NHE2. However, W-13 had no effect on E2/755. By monitoring the emission spectra of dansylated CaM fluorescence, we showed that dansylated CaM bound directly to a purified fusion protein of glutathione S-transferase and the last 87 amino acids of NHE2 in a Ca2+-dependent manner, with a stoichiometry of 1:1 and a dissociation constant of 300 nM. Our results showed that the COOH terminus of NHE2 is organized into separate stimulatory and inhibitory growth factor/protein kinase regulatory subdomains. This organization of growth factor/protein kinase regulatory subdomains is very similar to that of NHE3, suggesting that the tertiary structures of the putative COOH termini of NHE2 and NHE3 are very similar despite the minimal amino acid identity in this part of the two proteins. (+info)
Extracellular-regulated kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-kappa B-dependent IL-6 expression in human monocytes.
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In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-kappa B activity. Freshly isolated monocytes treated with the protein phosphatase inhibitor okadaic acid secreted high levels of IL-6 protein, which coincided with enhanced binding activity of NF-kappa B as well as with phosphorylation and activation of the ERK1/2 and JNK proteins. The ERK pathway-specific inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient overexpression of inactive mutants of either Raf-1 or JNK1 showed that both pathways were involved in kappa B-dependent IL-6 promoter activity. By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did not affect the DNA binding of NF-kappa B but, rather, acted at the level of transcriptional activity of NF-kappa B. Interestingly, it was shown that NF-kappa B-mediated gene transcription, both in the context of the IL-6 promoter as well as on its own, was dependent on both serine kinase activity and interaction with c-Jun protein. We conclude that okadaic acid-induced IL-6 gene expression is at least partly mediated through the ERK1/2 and JNK pathway-dependent activation of NF-kappa B transcriptional capacity. Our results suggest that the JNK pathway may regulate NF-kappa B-mediated gene transcription through its phosphorylation and activation of c-Jun. (+info)