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Biomedical subjects

John E Eriksson

Publications and source records attributed to John E Eriksson.

At least 19 recordsLinked to original sources
PubMed2007

Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography.

While immobilized metal affinity chromatography (IMAC) has been widely used for affinity purification of phosphopeptides, the technique suffers from insufficient specificity. Therefore, there is an urgent need for IMAC optimization to yield the selectivity and sensitivity that is required for more challenging analyses. Recently, 2,5-dihydroxybenzoic acid (DHB) and phosphoric acid mixture has been reported as an efficient IMAC eluant. The disadvantage of DHB is that is not suitable for electrospray ionization-mass spectrometry. While further developing the IMAC elution protocol to overcome this problem, we noticed that DHB is not necessary and found a novel combination of phosphoric acid and acetonitrile to be more efficient. The purification efficacy of the novel protocol is superior to all previously described methods, while still being compatible with the most commonly used mass-spectrometric techniques in phosphoproteomics.

Chromatography, Affinity↗
PubMed2006

Intermediate filaments as signaling platforms.

Intermediate filaments (IFs) are cytoskeletal structures that are crucial for maintaining the structural and mechanical integrity of cells and tissues. Intriguingly, a wide range of previously unknown nonmechanical roles for the IF cytoskeleton are emerging: Recent studies have linked IFs to the integration of signals related to the determination of cell size, the regulation of cell migration and cell survival, and the buffering of the effects of stress-activated kinases. The characteristic structural features and expression patterns of the different members of this diverse family of highly abundant proteins make them well suited to act as cell- and tissue-specific modifiers and organizers of signaling.

Animals↗
PubMed2006

A nestin scaffold links Cdk5/p35 signaling to oxidant-induced cell death.

The intermediate filament protein, nestin, has been implicated as an organizer of survival-determining signaling molecules. When nestin expression was related to the sensitivity of neural progenitor cells to oxidant-induced apoptosis, nestin displayed a distinct cytoprotective effect. Oxidative stress in neuronal precursor cells led to downregulation of nestin with subsequent activation of cyclin-dependent kinase 5 (Cdk5), a crucial kinase in the nervous system. Nestin downregulation was a prerequisite for the Cdk5-dependent apoptosis, as overexpression of nestin efficiently inhibited induction of apoptosis, whereas depletion of nestin by RNA interference had a sensitizing effect. When the underlying link between nestin and Cdk5 was analyzed, we observed that nestin serves as a scaffold for Cdk5, with binding restricted to a specific region following the alpha-helical domain of nestin, and that the presence and organization of nestin regulated the sequestration and activity of Cdk5, as well as the ubiquitylation and turnover of its regulator, p35. Our data imply that nestin is a survival determinant whose action is based upon a novel mode of Cdk5 regulation, affecting the targeting, activity, and turnover of the Cdk5/p35 signaling complex.

Apoptosis↗
PubMed2006

Vimentin function in lymphocyte adhesion and transcellular migration.

Although the adhesive interactions of leukocytes with endothelial cells are well understood, little is known about the detailed mechanisms underlying the actual migration of leukocytes across the endothelium (diapedesis). Leukocytes have been shown to use both paracellular and transcellular routes for transendothelial migration. Here we show that peripheral blood mononuclear cells (PBMCs; T- and B-lymphocytes) preferentially use the transcellular route. The intermediate filaments of both endothelial cells and lymphocytes formed a highly dynamic anchoring structure at the site of contact between these two cell types. The initiation of this process was markedly reduced in vimentin-deficient (vim(-/-)) PBMCs and endothelial cells. When compared with wild-type PBMCs, vim(-/-) PBMCs showed a markedly reduced capacity to home to mesenteric lymph nodes and spleen. Furthermore, endothelial integrity was compromised in vim(-/-) mice, demonstrating that intermediate filaments also regulate the barrier that governs leukocyte extravasation. Absence of vimentin resulted in highly aberrant expression and distribution of surface molecules critical for homing (ICAM-1 and VCAM-1 on endothelial cells and integrin-beta1 on PBMCs). These data show that intermediate filaments are active in lymphocyte adhesion and transmigration.

Animals↗
PubMed2006

Fast track to a phosphoprotein sketch - MALDI-TOF characterization of TLC-based tryptic phosphopeptide maps at femtomolar detection sensitivity.

Tryptic phosphopeptide mapping by TLC on microcrystalline cellulose has been a convenient method to get a fast and highly reproducible overview of the number of phosphopeptides present in any given (32)P-labeled phosphoprotein. This method also provides an immediate presentation of the relative phosphorylation stoichiometry between individual phosphopeptides. However, so far, traditional tryptic phosphopeptide maps have not been useful for phosphoproteomics applications, as the S/N has been very poor, due to the large number of quenching substances and contaminants present on cellulose plates. In this study, we present a rapid and easy method for phosphopeptides identification from 2-D phosphopeptide maps (2-D-PPMs). We obtain improved sensitivity (femtomole levels) upon MALDI-TOF MS analysis of phosphopeptides extracted from 2-D-PPMs. Using this approach we could confidently characterize the major phosphorylation sites of in vivo and in vitro (32)P-labeled proteins.

Chromatography, Thin Layer↗
PubMed2005

Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct.

A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. We conclude that CD95-induced caspase-3 activation in HT-22 cells was readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells.

Amino Acid Sequence↗
PubMed2005

Fas costimulation of naive CD4 T cells is controlled by NF-kappaB signaling and caspase activity.

Fas ligation induces apoptosis of activated T cells via the caspase cascade but can also mediate costimulatory signals to naïve T cells at the time of activation. We have previously shown that Fas ligation of naïve CD4 T cells activated by dendritic cells induces death or accelerates their proliferation and increases interferon-gamma (IFN-gamma) production. To understand this costimulation, we investigated the roles of caspases and nuclear factor (NF)-kappaB in survival and proliferation of responding T cells. Fas ligation increased caspase-3 and -8 activities during T cell activation, irrespective of cell fate. The accelerated proliferation induced by Fas ligation could be reduced by selective inhibition of both caspases. Inhibition of NF-kappaB simultaneously with Fas ligation inhibited the increased IFN-gamma production and caused uniform death of all responding T cells. Thus, Fas-mediated costimulation of naïve CD4 T cells is driven by active caspases, and NF-kappaB acts as a dominant survival-supporting factor of Fas-costimulated cells containing high levels of activated caspase-8 and -3.

Animals↗
PubMed2005

Binding and phosphorylation of par-4 by akt is essential for cancer cell survival.

Activation of the PI3K-Akt pathway by loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) function, increased growth factor signaling, or oncogene expression renders cancer cells resistant to apoptotic signals and promotes tumor growth. Although Akt acts as a global survival signal, the molecular circuits of this pathway have not been completely established. We report that Akt physically binds to the pro-apoptotic protein Par-4 via the Par-4 leucine zipper domain and phosphorylates Par-4 to inhibit apoptosis. Suppression of Akt activation by the PI3K-inhibitor PTEN or LY294002, Akt expression by RNA-interference, or Akt function by dominant-negative Akt caused apoptosis in cancer cells. Apoptosis induced by inhibiting Akt was blocked by inhibition of Par-4 expression, but not by inhibition of other apoptosis agonists that are Akt substrates, suggesting that inhibition of the PI3K-Akt pathway leads to Par-4-dependent apoptosis. Thus, Par-4 is essential for PTEN-inducible apoptosis, and inactivation of Par-4 by Akt promotes cancer cell survival.

Apoptosis↗
PubMed2005

Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail.

The caspase-8 inhibitor c-FLIP exists as two splice variants, c-FLIP(L) and c-FLIP(S), with distinct roles in death receptor signaling. The mechanisms determining their turnover have not been established. We found that in differentiating K562 erythroleukemia cells both c-FLIP isoforms were inducibly degraded by the proteasome, but c-FLIP(S) was more prone to ubiquitylation and had a considerably shorter half-life. Analysis of the c-FLIP(S)-specific ubiquitylation revealed two lysines, 192 and 195, C-terminal to the death effector domains, as principal ubiquitin acceptors in c-FLIP(S) but not in c-FLIP(L). Furthermore the c-FLIP(S)-specific tail of 19 amino acids, adjacent to the two target lysines, was demonstrated to be the key element determining the isoform-specific instability of c-FLIP(S). Molecular modeling in combination with site-directed mutagenesis demonstrated that the C-terminal tail is required for correct positioning and subsequent ubiquitylation of the target lysines. Because the antiapoptotic operation of c-FLIP(S) was not affected by the tail deletion, the antiapoptotic activity and ubiquitin-mediated degradation of c-FLIP(S) are functionally and structurally independent processes. The presence of a small destabilizing sequence in c-FLIP(S) constitutes an important determinant of c-FLIP(S)/c-FLIP(L) ratios by allowing differential degradation of c-FLIP isoforms. The conformation-based predisposition of c-FLIP(S) to ubiquitin-mediated degradation introduces a novel concept to the regulation of the death-inducing signaling complex.

Amino Acid Sequence↗
PubMed2005

CD95 capping is ROCK-dependent and dispensable for apoptosis.

Upon engagement, the CD95 receptor is rapidly clustered into cellular 'caps'. This receptor capping is one of the first events to take place following activation and it has been proposed to be important for the initiation of apoptotic signaling. As the biological roles of CD95 capping are still elusive, we explored in detail the role of capping in induction of apoptosis in lymphocytes. CD95 capping was shown to be uncoupled from apoptosis, as apoptosis could occur in the absence of CD95 capping and, vice versa, capping could occur without inducing apoptosis. CD95 capping occurred concomitantly with reorganization of the actin cytoskeleton and aggregation of lipid rafts. While inhibition of actin polymerization and caspase-8 activity had cell type-specific effects on capping in type I and type II cells, the rapid CD95-mediated cellular polarization, as visualized by the orchestrated reorganization of CD95, F-actin and lipid rafts, was shown to be dependent on signaling by Rho kinase (ROCK) in both cell types, however, by distinct activation mechanisms in the respective cell type. CD95 activated RhoA exclusively in the type II cell, whereas ROCK activation was caspase-dependent in the type I cell. Taken together, our results imply that CD95 capping and the subsequent cellular polarization is a ROCK signaling-regulated process that does not correlate with the induction of apoptosis, but is more likely to be involved in the emerging non-apoptotic functions of CD95.

Actins↗
PubMed2004

Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments.

Intermediate filaments (IFs) continuously exchange between a small, depolymerized fraction of IF protein and fully polymerized IFs. To elucidate the possible role of phosphorylation in regulating this equilibrium, we disrupted the exchange of phosphate groups by specific inhibition of dephosphorylation and by specific phosphorylation and site-directed mutagenesis of two of the major in vivo phosphorylation sites determined in this study. Inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases in BHK-21 fibroblasts with calyculin-A, induced rapid vimentin phosphorylation in concert with disassembly of the IF polymers into soluble tetrameric vimentin oligomers. This oligomeric composition corresponded to the oligopeptides released by cAMP-dependent kinase (PKA) following in vitro phosphorylation. Characterization of the (32)P-labeled vimentin phosphopeptides, demonstrated Ser-4, Ser-6, Ser-7, Ser-8, Ser-9, Ser-38, Ser-41, Ser-71, Ser-72, Ser-418, Ser-429, Thr-456, and Ser-457 as significant in vivo phosphorylation sites. A number of the interphase-specific high turnover sites were shown to be in vitro phosphorylation sites for PKA and protein kinase C (PKC). The effect of presence or absence of phosphate groups on individual subunits was followed in vivo by microinjecting PKA-phosphorylated (primarily S38 and S72) and mutant vimentin (S38:A, S72:A), respectively. The PKA-phosphorylated vimentin showed a clearly decelerated filament formation in vivo, whereas obstruction of phosphorylation at these sites by site-directed mutagenesis had no significant effect on the incorporation rates of subunits into assembled polymers. Taken together, our results suggest that elevated phosphorylation regulates IF assembly in vivo by changing the equilibrium constant of subunit exchange towards a higher off-rate.

Amino Acid Sequence↗
PubMed2004

Instant decisions: transcription-independent control of death-receptor-mediated apoptosis.

Transcription-independent modulation of signaling mediated by death receptors (DRs) has emerged as an important determinant of cell survival during both development and cellular homeostasis. Frequently, a given DR signal must be redirected rapidly either to inhibit or to potentiate the apoptotic response. This process requires immediate, protein-synthesis-independent modifications of the regulatory molecules involved. Numerous mechanisms have been shown to regulate DR responses without engaging the apoptosis-directing transcription machinery. These mechanisms involve key posttranslational modifications such as phosphorylation, ubiquitination and proteolytic degradation, all of which affect the activities of proteins at different levels in the DR signaling pathways. Changes in the organization of regulatory molecules and in their interactions with other factors also affect the DR signaling pathways. The balance between these modulatory signals rapidly decides the fate of a cell.

Animals↗
PubMed2003

Type-2A protein phosphatase activity is required to maintain death receptor responsiveness.

Type-2A protein phosphatase (PP2A) is a key regulator in many different cell signaling pathways and an important determinant in tumorigenesis. One of the signaling targets of PP2A is the mitogen-activated protein kinase (MAPK/ERK) cascade. In this study, we wanted to determine whether PP2A could be involved in regulation of death receptor activity through its capacity to regulate MAPK/ERK. To this end, we studied the effects of two different routes of protein phosphatase inhibition on death receptor-mediated apoptosis. We demonstrated that the apoptosis mediated by Fas, TNF-alpha, and TRAIL in U937 cells is suppressed by calyculin A, an inhibitor of type-1 and type-2A protein phosphatases. The inhibition of the protein phosphatase activity was shown to subsequently increase the MAPK activity in these cells, and the level of activation corresponded to the degree of suppression of cytokine-mediated apoptosis. A more physiological inhibitor, the intracellular PP2A inhibitor protein I2(PP2A), protected transfected HeLa cells in a similar way from Fas-mediated apoptosis and induced activation of MAPK in I2(PP2A) transfected cells. A corresponding inhibition could also be obtained by stable transfection with a constitutively active form of the MAPK kinase, MKK1 (also referred to as MEK1). The inhibitor-mediated protection was highly efficient in preventing early stages of apoptosis, as no caspase-8 cleavage occurred in these cells. The observed apoptosis suppression is likely to facilitate the tumor-promoting effect of a range of different type-2A protein phosphatase inhibitors, and could explain the reported tumor association of I2(PP2A).

3T3 Cells↗
PubMed2003

Tissue inhibitor of metalloproteinases-3 induces apoptosis in melanoma cells by stabilization of death receptors.

Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin (ADAM) activity. We have previously shown that adenovirally expressed tissue inhibitor of metalloproteinases-3 (TIMP-3) induces apoptosis in melanoma cells and inhibits growth of human melanoma xenografts. Here, we have studied the role of death receptors in apoptosis of melanoma cells induced by TIMP-3. Our results show, that the exposure of three metastatic melanoma cell lines (A2058, SK-Mel-5, and WM-266-4) to recombinant TIMP-3, N-terminal MMP inhibitory domain of TIMP-3, as well as to adenovirally expressed TIMP-3 results in stabilization of tumor necrosis factor receptor-1 (TNF-RI), FAS, and TNF-related apoptosis inducing ligand receptor-1 (TRAIL-RI) on melanoma cell surface and sensitizes these cells to apoptosis induced by TNF-alpha, anti-Fas-antibody and TRAIL. Stabilization of death receptors by TIMP-3 results in activation of caspase-8 and caspase-3, and subsequent apoptosis is blocked by specific caspase-8 inhibitor (Z-IETD-FMK) and by pan-caspase inhibitor (Z-DEVD-FMK). Adenovirus-mediated expression of TIMP-3 in human melanoma xenografts in vivo resulted in increased immunostaining for TNF-RI, FAS, and cleaved caspase-3, and in apoptosis of melanoma cells. Taken together, these results show that TIMP-3 promotes apoptosis in melanoma cells through stabilization of three distinct death receptors and activation of their apoptotic signaling cascade through caspase-8.

Animals↗
PubMed2003

Identification of ATP-synthase as a novel intracellular target for microcystin-LR.

Microcystins (MCs) are a group of closely related cyclic heptapeptides produced by a variety of common cyanobacteria. These are potent and highly specific hepatotoxins, the toxicity of which is based upon their inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases. Apart from protein phosphatases, it is not known whether these phosphatase-inhibiting peptides could bind any other cellular proteins. We wanted to determine whether any possible unknown MC-adducts could explain the apoptotic effects observed at high concentrations of MCs. The question of other possible cellular proteins binding to MCs is also relevant when these compounds are employed for affinity purification of protein phosphatases. In MC-treated cell lysates, antibodies to MC recognized three protein adducts of 35-37 and 55 kD. By immunochemical and proteomics approaches, these proteins were identified as the catalytic subunits of type-1 and type-2A protein phosphatases and the ATP-synthase beta-subunit. The latter target could be associated with the suggested apoptosis-inducing potential of MCs.

Amino Acid Sequence↗
PubMed2003

Synthesis and organotropism of 3H-dihydro derivatives of the cyanobacterial peptide hepatotoxin nodularin.

Tritium-labelled dihydro derivatives of the cyanobacterial peptide hepatotoxin nodularin were prepared by reduction with sodium boro[3H]hydride. The optimised reaction gave two dihydronodularin stereoisomers which were purified by high-performance liquid chromatography with a mobile phase of methanol-0.7% sodium sulfate (6:4) and a C(18) stationary phase. The specific activities of the stereoisomers were 1780-1807 dis min(-1) ng(-1). The radiolabelled dihydronodularins were tested for stability and used for toxicokinetic studies in mice. Liver was the main site of toxin accumulation.

Animals↗
PubMed2003

Cdk5 regulates the organization of Nestin and its association with p35.

The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity-either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitor-blocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. Interestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.

Amino Acid Sequence↗