Structural and biochemical characterisation of the KLHL3-WNK kinase interaction important in blood pressure regulation
This study provides further insights into how kelch-like adaptor proteins recognise their substrates and provides a structural basis for how mutations in WNK4 and KLHL3 lead to hypertension. (Source: BJ Signal)
Source: BJ Signal - March 18, 2014 Category: Biochemistry Authors: F Schumacher, F J. Sorrell, D R. Alessi, A N. Bullock, T Kurz Tags: BJ Signal Source Type: research
GDF-15 regulates Kv2.1-mediated outward K{+} current through Akt/mTOR signaling pathway in rat cerebellar granule cells
VGDF-15, a novel member of the transforming growth factor-β (TGF-β) superfamily, plays critical roles in the central and peripheral nervous systems, but the signal transduction pathways and receptor subtypes involved are not well understood. Here, we report that GDF-15 specifically increases the delayed rectifier outward K+ currents (IK) in rat cerebellar granule neurons (CGNs) in time- and concentration-dependent manners. The GDF-15-induced amplification of IK is mediated by the increased expression and reduced lysosome-dependent degradation of the Kv2.1 protein, the main α-subunit of IK. Expos...
Source: BJ Signal - March 6, 2014 Category: Biochemistry Authors: C Wang, A Huang, M Zhou, Y Mei Tags: BJ Signal Source Type: research
Glce regulates PC12 cell neuritogenesis induced by nerve growth factor through activating Smad/Id3 signaling
Neurodevelopment is orchestrated by a series of growth factor-heparan sulfate (HS) interactions which were involved in neuritogenesis. Glucuronic acid epimerase (Glce) is a critical enzyme involving in HS synthesis, which converts D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). However, function of Glce in neuritogenesis is largely unknown. Here we showed that Glce depletion caused PC12 cell growth arrested and promoted the cell neuritogenesis and differentiation induced by nerve growth factor (NGF). PC12 cell growth was boosted by overexpression of Glce, and the neuritogenesis was impaired when Glce-depletion was resc...
Source: BJ Signal - February 5, 2014 Category: Biochemistry Authors: J Li, J Fang, Y Qin, W Liao, H Liu, Y Zhou, K Ding Tags: BJ Biomolecules Source Type: research
Phosphorylation by Akt within the ST loop of AMPK-{alpha}1 down-regulates its activation in tumour cells
The insulin/IGF1-activated protein kinase Akt phosphorylates Ser487 in the “ST loop” within the C-terminal domain of AMPK-α1, leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr172. Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. Prior stimulation of HEK-293 cells with IGF1 caused reduced subsequent Thr172 phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca2+ ionophore A23187, effects we show to be dependent on Akt activation and Ser4...
Source: BJ Signal - January 27, 2014 Category: Biochemistry Authors: S A. Hawley, F A. Ross, G J. Gowans, P Tibarewal, N R. Leslie, D Grahame Hardie Tags: BJ Signal Source Type: research
Stability of the human pregnane X receptor is regulated by E3 ligase UBR5 and serine/threonine kinase DYRK2
The human pregnane X receptor (hPXR), a major chemical toxin sensor, is a ligand-induced transcription factor activated by various xenobiotics and toxins, resulting in the transcriptional upregulation of detoxifying enzymes. To date, little is known about the upstream regulation of hPXR. Using mass spectrometry analysis and kinome-wide siRNA screen, we report that the E3 ligase UBR5 and DYRK2 regulates hPXR stability. UBR5 knockdown resulted in accumulation of cellular hPXR and a concomitant increase in hPXR activity, whereas the rescue of UBR5 knockdown decreased the cellular hPXR level and activity. Importantly, UBR5 exe...
Source: BJ Signal - January 20, 2014 Category: Biochemistry Authors: S Ong, A N. Goktug, A Elias, J Wu, D Saunders, T Chen Tags: BJ Signal Source Type: research
Structure of Cyclin G associated kinase (GAK) Trapped in different Conformations using Nanobodies
Cyclin G associated kinase (GAK) is a key regulator of clathrin-coated vesicles trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain it is a frequent “off-target” of clinical kinase inhibitors associated with respiratory side effects of these drugs. Here, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo-structure revealed a dimeric inactive state of the catalytic domain ...
Source: BJ Signal - January 17, 2014 Category: Biochemistry Authors: A Chaikuad, T Keates, C Vincke, M Kaufholz, M Zenn, B Zimmermann, C Gutiérrez, R Zhang, C Hatzos-Skintges, A Joachimiak, S Muyldermans, F W. Herberg, S Knapp, S Müller Tags: BJ Biomolecules Source Type: research
K11- and K48-Linked Ubiquitin Chains Interact with p97 during Endoplasmic Reticulum-Associated Degradation
The AAA+ ATPase p97 has a critical function in the cytoplasmic degradation of proteins misfolded in the endoplasmic reticulum through a mechanism known as ER-associated degradation (ERAD). During this process, p97 binds polyubiquitinated ERAD substrates and couples ATP hydrolysis to their dislocation from the ER as a prerequisite to destruction by the proteasome. The ubiquitin signals important for this process are not fully understood. Here we report that p97 interacts with lysine 11 (K11) and K48-linked ubiquitin polymers, but not those containing K63 linkages. Disruption of p97 through siRNA-mediated depletion, d...
Source: BJ Signal - January 14, 2014 Category: Biochemistry Authors: M Locke, J Irene Toth, M David Petroski Tags: BJ Signal Source Type: research
Signal transduction mechanism of biased ligands at histamine H2 receptors.
7TMR exists as conformational collections in which different conformations would lead to differential downstream behaviors such as receptor phosphorylation, G-protein activation and receptor internalization among others. In this context, a ligand may cause differential activation of some, but not all, of the signaling events, which are associated to a particular receptor, and it would lead to biased agonism. The aim of the present work is to study if histamine H2 receptor (H2R) ligands -described as inverse agonists because of their negative efficacy at modulating adenylyl cyclase- could display some positive efficacy conc...
Source: BJ Signal - January 14, 2014 Category: Biochemistry Authors: N Alonso, F Monczor, E Echeverría, C Davio, C Shayo, N Fernandez Tags: BJ Signal Source Type: research
The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K{+}-Cl- co-transporters
Precise homeostasis of intracellular Cl- is achieved via the coordinated activities of the Cl- influx and efflux. We demonstrate that the WNK activated SPAK/OSR1 kinases known to directly phosphorylate and stimulate the sodium-potassium co-transporters (N[K]CC), also promote inhibition of the potassium-chloride co-transporters (KCCs) by directly phosphorylating a recently-described C-terminal threonine conserved in all KCC isoforms (Site-2 [Thr1048]). First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Second, STOCK1S-50699, ...
Source: BJ Signal - January 7, 2014 Category: Biochemistry Authors: P de los Heros, D R. Alessi, R Gourlay, D G. Campbell, M Deak, T J. Macartney, K T. Kahle, J Zhang Tags: BJ Signal Source Type: research
The ability of TRIM3 to induce growth arrest depends on RING-dependent E3 ligase activity
Mutation of the TRIM-NHL family members brat and mei-P26 in Drosophilia perturb the differentiation of transit amplifying progenitor cells resulting intumor-like phenotypes. The NHL domain is essential for their growth suppressive activity, and they can induce cell cycle exit in a RING-independentmanner. TRIM3 is the only bona fide tumor suppressor in the mammalian TRIM-NHL subfamily and like the other members of this family its ability to inhibit cell proliferation depends on the NHL domain. However, whether the RINGdomain was required for TRIM3 dependent cell cycle exit had not been investigated. Here we establish that t...
Source: BJ Signal - January 6, 2014 Category: Biochemistry Authors: R Raheja, Y Liu, E Hukkelhoven, N Yeh, A Koff Tags: BJ Signal Source Type: research
Mechanistic insights into activation and SOCS3-mediated inhibition of myeloproliferative neoplasm-associated JAK2 mutants from biochemical and structural analyses
JAK2 (Janus kinase 2) initiates the intracellular signalling cascade downstream of cell surface receptor activation by cognate haematopoietic cytokines, including erythropoietin and thrombopoietin. The pseudokinase (JH2) domain of JAK2 negatively regulates the catalytic activity of the adjacent tyrosine kinase (JH1) domain and mutations within the pseudokinase domain underlie human myeloproliferative neoplasms, including polycythaemia vera and essential thrombocytosis. To date, the mechanism of JH2-mediated inhibition of JH1 kinase activation as well as the susceptibility of pathological mutant JAK2 to inhibition by the ph...
Source: BJ Signal - December 19, 2013 Category: Biochemistry Authors: L N Varghese, D Ungureanu, N P.D. Liau, S N Young, A Laktyushin, H Hammarén, I S. Lucet, N A Nicola, O Silvennoinen, J J Babon, J M Murphy Tags: BJ Signal Source Type: research
The inhibition of functional expression of calcium channels by prion protein demonstrates competition with {alpha}2{delta} for GPI-anchoring pathways
We examined whether there was an effect of PrP on calcium currents. We show that when PrP is co-expressed with calcium channels formed from CaV2.1/β and α2δ-1 or α2δ-2, this results in a consistent decrease in calcium current density. This reduction was absent when PrP lacked its glycosyl-phosphatidylinositol (GPI) anchor. We have found that α2δ subunits are able to form GPI-anchored proteins [2] and present further evidence here. We have recently characterised a C-terminally truncated α2δ-1 construct, α2δ-1ΔC, and found that, despite loss ...
Source: BJ Signal - December 12, 2013 Category: Biochemistry Authors: A Alvarez-Laviada, I Kadurin, A Senatore, R Chiesa, A C Dolphin Tags: BJ Signal Source Type: research
Ca2{+}/S100 proteins inhibit the interactions of FKBP38 with Bcl-2 and Hsp90
FKBP38 (FK506-binding protein 38), a membrane-anchored, tetratricopeptide repeat (TPR)-containing immunophilin, regulates signaling pathways such as cell survival, apoptosis, proliferation, and metastasis. However, the mechanisms that regulate the activity of FKBP38 are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR-proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin-light chain, Tom70, FKBP52, CyP40, C-terminus of Hsc70-interacting protein (CHIP) and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR-pro...
Source: BJ Signal - December 3, 2013 Category: Biochemistry Authors: S Shimamoto, M Tsuchiya, F Yamaguchi, Y Kubota, H Tokumitsu, R Kobayashi Tags: BJ Signal Source Type: research
Investigation of role that LKB1 Ser431 phosphorylation and Cys433 farnesylation play by mouse knock-in analysis reveals an unexpected role of prenylation in regulating AMPK activity
The LKB1 tumour suppressor protein kinase functions to activate two isoforms of AMP-activated protein kinase (AMPK) and 12 members of the AMPK-related family of protein kinase. The highly conserved C-terminal residues of LKB1 are phosphorylated (Ser431) by PKA and RSK and farnesylated (Cys433) within a CAAX motif. To better define the role that these posttranslational modifications play, we created homozygous LKB1S431A/S431A and LKB1C433S/C433S knock-in mice. These animals were viable, fertile and displayed no overt phenotypes. Employing a farnesylation-specific monoclonal antibody that we generated, we establish by immuno...
Source: BJ Signal - December 3, 2013 Category: Biochemistry Authors: V P. Houde, M Ritorto, R Gourlay, J Varghese, P Davies, N Shpiro, K Sakamoto, D R. Alessi Tags: BJ Signal Source Type: research
c-Src-induced activation of ceramide metabolism impairs membrane microdomains and promotes malignant progression by facilitating the translocation of c-Src to focal adhesions
The proto-oncogenic tyrosine kinase c-Src is upregulated in various human cancers, implicating its role in tumor progression. Upon activation, c-Src translocates to focal adhesions and initiates intracellular signalling cascades that promote malignant transformation, but the underlying mechanisms for c-Src translocation remain unclear. Here we show that c-Src upregulation perturbs sphingolipid/cholesterol enriched membrane microdomains by activating ceramide synthesis, resulting in promotion of c-Src translocation. Using an inducible c-Src expression system in Csk-deficient fibroblasts, we found that translocation of c-Src...
Source: BJ Signal - November 22, 2013 Category: Biochemistry Authors: K Kajiwara, T Yamada, T Bamba, E Fukusaki, F Imamoto, M Okada, C Oneyama Tags: BJ Signal Source Type: research
