Identification of 14-3-3ζ as an EGF receptor interacting protein

AbstractThe 14-3-3 proteins are known to interact with a number of proteins involved in the regulation of cell signaling. Here, we describe an association of 14-3-3ζ with the epidermal growth factor receptor (EGFR) that is rapidly induced by EGF. The 1028-EGFR truncated mutant which lacks the cytoplasmic tail from amino acids 1029–1186 identified the binding site for 14-3-3 to be between amino acid 1028 and the receptor carboxyl terminus. Mutational deletion of serine residues 1046, 1047, 1057 and 1142 did not inhibit EGF-induced 14-3-3 association with the receptor. Immunofluorescence microscopy indicated an EGF-induced co-localization of EGFR and HA-14-3-3ζ along the plasma membrane. Our finding adds to the growing complexity of EGF receptor signaling and indicates a role for 14-3-3 proteins in EGF receptor signaling or regulation

Hva betyr naturen for deg?: En kvalitativ studie om naturens betydning for barnehagelærerstudenter med fordypning i natur- og friluftsliv

Er det slik at natur og friluftsliv har en så stor effekt på oss som mennesker? I Stortingsmelding nr. 18 (Det kongelige klima- og miljødepartement (2015-2016)) beskrives friluftslivet som en del av den norske identiteten og er godt forankret i vår kulturarv. Begrepet friluftsliv ble først brukt i skriftlig form av Henrik Ibsen i diktet På Vidderne, der han beskriver hvordan mennesket opplever sine individuelle styrker og særegenheter via innsikten man får ute i naturen. I 1960-årene bodde 70% av befolkningen her i landet på bygda, med umiddelbar nærhet til naturen som en naturlig følge av gårdsdriften. Barndommen var preget av frihet, dyreliv og natur. I dag bor åtte av ti nordmenn i større samfunn, gjerne byer, og tilhørigheten til naturen er en helt annen enn før (Dragland, 2018, s. 12). Dette danner et bakteppe for dagens barnehagekultur, og er noe som påvirker både barnas og barnehagelærerens forhold til natur og friluftsliv. I Rammeplanen for barnehagen presiseres det under fagområdet Natur, miljø og teknologi, at barnehagen skal bidra til at naturen skal være en arena for lek, læring og utvikling, og at det skal legges til rette for mangfoldige naturopplevelser for barna (Kunnskapsdepartementet, 2017, s. 53). Under Nasjonale retningslinjer for barnehagelærerutdanningen pekes det på at en del av profesjonskompetansen til barnehagelæreren etter endt utdanning, nettopp fordrer trygghet i naturen og å se mulighetene for utforskning, undring og utvikling som er til stede. Dette er også en del av det å være en god rollemodell og støtte for barna i uterommet (Kunnskapsdepartementet, 2018, s. 17). Jeg er nysgjerrig på hvilken utvikling studentene selv føler de har hatt etter tre år på barnehagelærerutdanningen, med vekt på fordypningen i natur og friluftsliv. Jeg er også spent på å høre om deres syn på barn og natur nå som de snart er ferdigutdannet, og om dette gjenspeiler noen av deres egne erfaringer med natur og friluftsliv gjort underveis i studiet. På bakgrunn av dette har jeg valgt følgende problemstilling for min oppgave: Med vekt på fordypning i natur og friluftsliv; på hvilken måte preger studentenes egen utvikling under barnehagelærerstudiet deres syn på barn og natur?publishedVersionBNBAC390

TGF-β-induced growth inhibition in B-cell lymphoma correlates with Smad1/5 signalling and constitutively active p38 MAPK

<p>Abstract</p> <p>Background</p> <p>Cytokines of the transforming growth factor β (TGF-β) superfamily exert effects on proliferation, apoptosis and differentiation in various cell types. Cancer cells frequently acquire resistance to the anti-proliferative signals of TGF-β, which can be due to mutations in proteins of the signalling cascade. We compared the TGF-β-related signalling properties in B-cell lymphoma cell lines that were sensitive or resistant to TGF-β-induced anti-proliferative effects.</p> <p>Results</p> <p>TGF-β sensitive cell lines expressed higher cell surface levels of the activin receptor-like kinase 5 (Alk-5), a TGF-β receptor type 1. The expression levels of the other TGF-β and bone morphogenetic protein receptors were comparable in the different cell lines. TGF-β-induced phosphorylation of Smad2 was similar in TGF-β sensitive and resistant cell lines. In contrast, activation of Smad1/5 was restricted to cells that were sensitive to growth inhibition by TGF-β. Moreover, with activin A we detected limited anti-proliferative effects, strong phosphorylation of Smad2, but no Smad1/5 phosphorylation. Up-regulation of the TGF-β target genes Id1 and Pai-1 was identified in the TGF-β sensitive cell lines. Constitutive phosphorylation of MAPK p38 was restricted to the TGF-β sensitive cell lines. Inhibition of p38 MAPK led to reduced sensitivity to TGF-β.</p> <p>Conclusions</p> <p>We suggest that phosphorylation of Smad1/5 is important for the anti-proliferative effects of TGF-β in B-cell lymphoma. Alk-5 was highly expressed in the sensitive cell lines, and might be important for signalling through Smad1/5. Our results indicate a role for p38 MAPK in the regulation of TGF-β-induced anti-proliferative effects.</p

Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit

<p>Abstract</p> <p>Background</p> <p>Epidermal Growth Factor Receptor (EGFR) is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA) induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits.</p> <p>Results</p> <p>We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Cα and Cβ in two different models. The first model used targeted disruption of either Cα or Cβ in mice whereas the second model used Cα and Cβ RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level.</p> <p>Conclusion</p> <p>Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.</p

The neuropeptide landscape of human prefrontal cortex

Human prefrontal cortex (hPFC) is a complex brain region involved in cognitive and emotional processes and several psychiatric disorders. Here, we present an overview of the distribution of the peptidergic systems in 17 subregions of hPFC and three reference cortices obtained by microdissection and based on RNA sequencing and RNAscope methods integrated with published single-cell transcriptomics data. We detected expression of 60 neuropeptides and 60 neuropeptide receptors in at least one of the hPFC subregions. The results reveal that the peptidergic landscape in PFC consists of closely located and functionally different subregions with unique peptide/transmitter–related profiles. Neuropeptide-rich PFC subregions were identified, encompassing regions from anterior cingulate cortex/orbitofrontal gyrus. Furthermore, marked differences in gene expression exist between different PFC regions (>5-fold; cocaine and amphetamine–regulated transcript peptide) as well as between PFC regions and reference regions, for example, for somatostatin and several receptors. We suggest that the present approach allows definition of, still hypothetical, microcircuits exemplified by glutamatergic neurons expressing a peptide cotransmitter either as an agonist (hypocretin/orexin) or antagonist (galanin). Specific neuropeptide receptors have been identified as possible targets for neuronal afferents and, interestingly, peripheral blood-borne peptide hormones (leptin, adiponectin, gastric inhibitory peptide, glucagon-like peptides, and peptide YY). Together with other recent publications, our results support the view that neuropeptide systems may play an important role in hPFC and underpin the concept that neuropeptide signaling helps stabilize circuit connectivity and fine-tune/modulate PFC functions executed during health and disease

Activation of p38 Mitogen-Activated Protein Kinase Promotes Epidermal Growth Factor Receptor Internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions

Additional Serine/Threonine Phosphorylation Reduces Binding Affinity but Preserves Interface Topography of Substrate Proteins to the c-Cbl TKB Domain

The E3-ubiquitin ligase, c-Cbl, is a multi-functional scaffolding protein that plays a pivotal role in controlling cell phenotype. As part of the ubiquitination and downregulation process, c-Cbl recognizes targets, such as tyrosine kinases and the Sprouty proteins, by binding to a conserved (NX/R)pY(S/T)XXP motif via its uniquely embedded SH2 domain (TKB domain). We previously outlined the mode of binding between the TKB domain and various substrate peptide motifs, including epidermal growth factor receptor (EGFR) and Sprouty2 (Spry2), and demonstrated that an intrapetidyl hydrogen bond forms between the (pY-1) arginine or (pY-2) asparagine and the phosphorylated tyrosine, which is crucial for binding. Recent reports demonstrated that, under certain types of stimulation, the serine/threonine residues at the pY+1 and/or pY+2 positions within this recognition motif of EGFR and Sprouty2 may be endogenously phosphorylated. Using structural and binding studies, we sought to determine whether this additional phosphorylation could affect the binding of the TKB domain to these peptides and consequently, whether the type of stimulation can dictate the degree to which substrates bind to c-Cbl. Here, we show that additional phosphorylation significantly reduces the binding affinity between the TKB domain and its target proteins, EGFR and Sprouty2, as compared to peptides bearing a single tyrosine phosphorylation. The crystal structure indicates that this is accomplished with minimal changes to the essential intrapeptidyl bond and that the reduced strength of the interaction is due to the charge repulsion between c-Cbl and the additional phosphate group. This obvious reduction in binding affinity, however, indicates that Cbl's interactions with its TKB-centered binding partners may be more favorable in the absence of Ser/Thr phosphorylation, which is stimulation and context specific in vivo. These results demonstrate the importance of understanding the environment in which certain residues are phosphorylated, and the necessity of including this in structural investigations

Differential Effects of EGFR Ligands on Endocytic Sorting of the Receptor

Endocytic downregulation is a pivotal mechanism turning off signalling from the EGF receptor (EGFR). It is well established that whereas EGF binding leads to lysosomal degradation of EGFR, transforming growth factor (TGF)-α causes receptor recycling. TGF-α therefore leads to continuous signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-α, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking

A global view of protein expression in human cells, tissues, and organs

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry-based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced

Exosome enrichment of human serum using multiple cycles of centrifugation

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113694/1/elps5528.pd