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- Wolf-Watz, M, et al.
(författare)
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Solution properties of the free and DNA-bound Runt domain of AML1.
- 1999
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 261:1, s. 251-60
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Tidskriftsartikel (refereegranskat)abstract
- The Runt domain is responsible for specific DNA and protein-protein interactions in a family of transcription factors which includes human AML1. Structural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here we describe the optimization and characterization of a 140-residue fragment, containing the Runt domain of AML1, which is suitable for structural studies. The fragment of AML1 including amino acids 46-185 [AML1 Dm(46-185)] contains a double cysteine-->serine mutation which does not affect Runt domain structure or DNA-binding affinity. Purified AML1 Dm(46-185) is soluble and optimally stable in a buffer containing 200 mm MgSO4 and 20 mm sodium phosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spectroscopy indicate that the Runt domain contains beta-sheet, but little or no alpha-helical secondary structure elements. The 45 N-terminal residues of AML1 are unstructured and removal of the N-terminal enhances sequence-specific DNA binding. The NMR spectrum of AML1 Dm(46-185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily identified, suggesting that a structure determination of this Runt domain fragment is feasible. A titration of 15N-labelled AML1 Dm(46-185) with a 14-bp cognate DNA duplex results in changes in the 15N NMR heteronuclear single quantum coherence spectrum which indicate the formation of a specific complex and structural changes in the Runt domain upon DNA binding.
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- Grundström, Gunilla, et al.
(författare)
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Integrin αvβ3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin α2β1
- 2003
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 291:2, s. 463-473
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Tidskriftsartikel (refereegranskat)abstract
- The interplay between the collagen-binding integrin, α2β1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type α2β1 (α2β1A) or α2β1 with a Y783/795F mutation in the cytoplasmic tail of the β1 subunit (α2β1Amut) in the β1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the α1 and α2 integrin subunits and do not adhere to collagen even after transfection with β1A. Cells expressing α2β1Amut contracted three-dimensional collagen lattices less efficiently than those expressing α2β1A. PDGF-BB significantly stimulated lattice contraction by GD25-α2β1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130Cas were phosphorylated when GD25-α2β1A cells, but not GD25-α2β1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130Cas only in GD25-α2β1A cells. However, when cultured within collagen lattices, FAK and p130Cas phosphorylation were stimulated in both α2β1A- and α2β1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-α2β1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-α2β1Amut cells were mediated by the αvβ3 integrin. Phosphorylation of p130Cas, but not FAK, in GD25-α2β1Amut cells seeded in collagen lattices also depended on αvβ3. Our results show that PDGF-BB stimulation of fibroblast–collagen interactions is mediated by the αvβ3 integrin when β1 integrin function is impaired.
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- Hughes, K, et al.
(författare)
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Calmodulin-dependent kinase II mediates T cell receptor/CD3- and phorbol ester-induced activation of IkappaB kinase.
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:38, s. 36008-36013
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Tidskriftsartikel (refereegranskat)abstract
- Numerous fundamental biological processes involve the NFkappaB family of transcription factors. The mechanisms by which this family of proteins is regulated are therefore of widespread importance. In most cells, NFkappaB is bound to inhibitory IkappaB proteins and sequestered in the cytoplasm. NFkappaB-inducing signals result in activation of a large multisubunit kinase complex, IKK, which phosphorylates IkappaB. IkappaB is subsequently degraded, releasing NFkappaB, which translocates to the nucleus. We previously reported that inhibitors of the calcium-binding protein calmodulin (CaM) prevent phorbol ester-induced phosphorylation of IkappaB. Here we show that KN93, an inhibitor of CaM-dependent kinases (CaMKs), also inhibits the phosphorylation of IkappaB. The effect of both CaM and CaMK inhibitors on IkappaB phosphorylation is due to the inhibition of the activity of CaMK II because neither drug has any effect when a derivative of CaMK II that is insensitive to these inhibitors is expressed. When CaMK II is inhibited, phorbol ester is no longer able to activate IKK, placing CaMK II in the signaling pathway that leads to IKK activation. CaM and CaMK inhibitors also block T cell receptor/CD3-induced activation but have no effect on the ability of the cytokine tumor necrosis factor alpha or the phosphatase inhibitor calyculin A to induce degradation of IkappaB. Finally we show that expression of a constitutively active CaMK II results in the activation of NFkappaB. The results identify CaMK II as a mediator of IKK activation specifically in response to T cell receptor/CD3 and phorbol ester stimulation.
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- Lawrence, D, et al.
(författare)
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Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells.
- 1989
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 186:3, s. 523-33
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Tidskriftsartikel (refereegranskat)abstract
- Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1. In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E. coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAI-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin).
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- Liljeholm, S, et al.
(författare)
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NF-kappaB only partially mediates Epstein-Barr virus latent membrane protein 1 activation of B cells.
- 1998
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Ingår i: Journal of General Virology. - 0022-1317 .- 1465-2099. ; 79 ( Pt 9), s. 2117-25
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Tidskriftsartikel (refereegranskat)abstract
- The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is required for EBV-induced immortalization of human B cells and causes tumorigenic transformation of cell lines. LMP1 expression induces phenotypic changes resembling B cell activation, such as cell size increase and up-regulation of cell surface activation markers. LMP1 contains two domains that activate the transcription factor NF-kappaB, one through interactions with TRAF proteins and the other with the TRADD protein. The purpose of the present study was to investigate the importance of NF-kappaB induction in the up-regulation of the B cell activation markers ICAM-1 and CD71 by LMP1. This study shows that expression of LMP1 activates transcription from p50/p65- and c-Rel-responsive promoters, and that this activity can be completely inhibited by expression of a dominant inhibitory IkappaB mutant. ICAM-1 and CD71 are nevertheless up-regulated by LMP1 in primary B cells and cell lines expressing the dominant IkappaB. Furthermore, LMP1-induced cell size increase of primary B cells was unaffected by IkappaB expression. It was concluded that even when LMP1 is unable to activate NF-kappaB, it is still capable of inducing certain characteristics of activated B cells, strongly suggesting that LMP1 can also activate cells independently of NF-kappaB.
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