The Role of the N-Terminal Domain in the Regulation of the “Constitutively Active” Conformation of Protein Kinase CK2α: Insight from a Molecular Dynamics Investigation

ChemMedChem ◽  
2011 ◽  
Vol 6 (7) ◽  
pp. 1207-1216 ◽  
Author(s):  
Andrea Cristiani ◽  
Giorgio Costa ◽  
Giorgio Cozza ◽  
Flavio Meggio ◽  
Leonardo Scapozza ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Protein Kinase ◽  
Active Conformation ◽  
Terminal Domain ◽  
Constitutively Active

scholarly journals Characterization of the Role of AMP-Activated Protein Kinase in the Regulation of Glucose-Activated Gene Expression Using Constitutively Active and Dominant Negative Forms of the Kinase

2000 ◽  
Vol 20 (18) ◽  
pp. 6704-6711 ◽  
Author(s):  
Angela Woods ◽  
Dalila Azzout-Marniche ◽  
Marc Foretz ◽  
Silvie C. Stein ◽  
Patricia Lemarchand ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Fatty Acid Synthase ◽  
Physiological Role ◽  
Rat Hepatocytes ◽  
Dominant Negative ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Constitutively Active

ABSTRACT In the liver, glucose induces the expression of a number of genes involved in glucose and lipid metabolism, e.g., those encoding L-type pyruvate kinase and fatty acid synthase. Recent evidence has indicated a role for the AMP-activated protein kinase (AMPK) in the inhibition of glucose-activated gene expression in hepatocytes. It remains unclear, however, whether AMPK is involved in the glucose induction of these genes. In order to study further the role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (α1312) acts as a constitutively active kinase, while the other (α1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to express these mutants in primary rat hepatocytes in culture in order to determine their effect on AMPK activity and the transcription of glucose-activated genes. Expression of α1312 increased AMPK activity in hepatocytes and blocked completely the induction of a number of glucose-activated genes in response to 25 mM glucose. This effect is similar to that observed following activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of α1DN markedly inhibited both basal and stimulated activity of endogenous AMPK but had no effect on the transcription of glucose-activated genes. Our results suggest that AMPK is involved in the inhibition of glucose-activated gene expression but not in the induction pathway. This study demonstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.

scholarly journals Continuous Activation of Gαq in Osteoblasts Results in Osteopenia through Impaired Osteoblast Differentiation

2007 ◽  
Vol 282 (49) ◽  
pp. 35757-35764 ◽  
Author(s):  
Naoshi Ogata ◽  
Hiroshi Kawaguchi ◽  
Ung-il Chung ◽  
Sanford I. Roth ◽  
Gino V. Segre
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Transgenic Mice ◽  
Trabecular Thickness ◽  
Kinase C ◽  
Protein Kinase C Inhibitor ◽  
Skeletal Homeostasis ◽  
Trabecular Bones ◽  
Constitutively Active

We explored the role of Gαq-mediated signaling on skeletal homeostasis by selectively expressing a constitutively active Gαq (mutation of Q209L) in osteoblasts. Continuous signaling via Gαq in mouse osteoblastic MC3T3-E1 cells impaired differentiation. Mice that expressed the constitutively active Gαq transgene in cells of the osteoblast lineage exhibited severe osteopenia in cortical and trabecular bones. Osteoblast number, bone volume, and trabecular thickness were reduced in transgenic mice, but the osteoclasts were unaffected. Osteoblasts from transgenic mice showed impaired differentiation and matrix formation. In the presence of a protein kinase C inhibitor GF109203X, this impairment was not seen, indicating mediation by the protein kinase C pathway. We propose that continuous activation of the Gαq signal in osteoblasts plays a crucial, previously unrecognized role in bone formation.

scholarly journals Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

2011 ◽  
Vol 437 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Suneet Jain ◽  
Takuya Suzuki ◽  
Ankur Seth ◽  
Geetha Samak ◽  
Radhakrishna Rao
Keyword(s):  
Protein Kinase ◽  
Tight Junction ◽  
Tight Junctions ◽  
Intercellular Junctions ◽  
Cell Monolayers ◽  
Terminal Domain ◽  
Tight Junction Regulation ◽  
Epithelial Tight Junctions ◽  
Zona Occludens

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin–Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.

scholarly journals The N-terminal domain of RfaH plays an active role in protein fold-switching

2021 ◽  
Vol 17 (9) ◽  
pp. e1008882
Author(s):  
Pablo Galaz-Davison ◽  
Ernesto A. Román ◽  
César A. Ramírez-Sarmiento
Keyword(s):  
Molecular Dynamics ◽  
Virulence Factors ◽  
Elongation Factor ◽  
Active Role ◽  
Structural Rearrangement ◽  
Umbrella Sampling ◽  
Protein Fold ◽  
Terminal Domain ◽  
Rnap Binding

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.

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