scholarly journals Potential Role for Protein Kinases in Regulation of Bidirectional Endoplasmic Reticulum-to-Golgi Transport Revealed by Protein Kinase Inhibitor H89

2000 ◽  
Vol 11 (8) ◽  
pp. 2577-2590 ◽  
Author(s):  
Tina H. Lee ◽  
Adam D. Linstedt
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Kinases ◽  
Potential Role ◽  
Kinase Inhibitor ◽  
Transport Processes ◽  
Protein Kinase Inhibitors ◽  
Er Export ◽  
Kinase A

Recent evidence suggests a regulatory connection between cell volume, endoplasmic reticulum (ER) export, and stimulated Golgi-to-ER transport. To investigate the potential role of protein kinases we tested a panel of protein kinase inhibitors for their effect on these steps. One inhibitor, H89, an isoquinolinesulfonamide that is commonly used as a selective protein kinase A inhibitor, blocked both ER export and hypo-osmotic-, brefeldin A-, or nocodazole-induced Golgi-to-ER transport. In contrast, H89 did not block the constitutive ER Golgi-intermediate compartment (ERGIC)-to-ER and Golgi-to-ER traffic that underlies redistribution of ERGIC and Golgi proteins into the ER after ER export arrest. Surprisingly, other protein kinase A inhibitors, KT5720 and H8, as well as a set of protein kinase C inhibitors, had no effect on these transport processes. To test whether H89 might act at the level of either the coatomer protein (COP)I or the COPII coat protein complex we examined the localization of βCOP and Sec13 in H89-treated cells. H89 treatment led to a rapid loss of Sec13-labeled ER export sites but βCOP localization to the Golgi was unaffected. To further investigate the effect of H89 on COPII we developed a COPII recruitment assay with permeabilized cells and found that H89 potently inhibited binding of exogenous Sec13 to ER export sites. This block occurred in the presence of guanosine-5′-O-(3-thio)triphosphate, suggesting that Sec13 recruitment is inhibited at a step independent of the activation of the GTPase Sar1. These results identify a requirement for an H89-sensitive factor(s), potentially a novel protein kinase, in recruitment of COPII to ER export sites, as well as in stimulated but not constitutive Golgi-to-ER transport.

Comparative Study of Active and Allosteric Interaction in Protein Kinases

2021 ◽  
Vol 8 (1) ◽  
pp. 23-31
Author(s):  
Jefrin Ahmed ◽  
Judith Mary Lamo ◽  
Baphilinia Jones Mylliemngap
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Kinases ◽  
Active Site ◽  
Cell Function ◽  
Binding Capacity ◽  
Second Messengers ◽  
Gene Families ◽  
Allosteric Site ◽  
Kinase A

Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. By adding phosphate groups to substrate proteins, they direct the activity, localization and overall function of many proteins, and serve to orchestrate the activity of almost all cellular processes. The main protein kinases consist of protein kinase A (PKA), protein kinase B (PKB), and protein kinase C (PKC) and are distinguished from each other by the different intracellular second messengers involved in their regulation and by the selective substrates they use. They all have a binding site for Mg2+-ATP (phosphate donor) and for substrate protein as well as various regulatory sites. We formulated to compare the binding capacity of protein kinases at the active site to allosteric sites. By comparing the active site and allosteric site of the protein kinases – A, B and C, using molecular docking it was found that in most of the cases the binding energy is high when an inhibitor binds to an active site as compared to the allosteric site. This comparison gave us an understanding of the interaction and inhibition of compounds to protein kinases in order to inhibit the activity of protein kinase A, B and C. It was concluded that for inhibiting the protein kinase function such as cell division and proliferation, binding of inhibitor to the allosteric site will be more effective.

scholarly journals A Stapled Peptide Mimic of the Pseudosubstrate Inhibitor PKI Inhibits Protein Kinase A

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1567 ◽  
Author(s):  
Jascha T. Manschwetus ◽  
George N. Bendzunas ◽  
Ameya J. Limaye ◽  
Matthias J. Knape ◽  
Friedrich W. Herberg ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Kinase Inhibitor ◽  
Stapled Peptide ◽  
Peptide Mimic ◽  
Cellular Environment ◽  
Ic50 Values ◽  
Dependent Protein ◽  
Peptide Targeting ◽  
Kinase A

Kinases regulate multiple and diverse signaling pathways and misregulation is implicated in a multitude of diseases. Although significant efforts have been put forth to develop kinase-specific inhibitors, specificity remains a challenge. As an alternative to catalytic inhibition, allosteric inhibitors can target areas on the surface of an enzyme, thereby providing additional target diversity. Using cAMP-dependent protein kinase A (PKA) as a model system, we sought to develop a hydrocarbon-stapled peptide targeting the pseudosubstrate domain of the kinase. A library of peptides was designed from a Protein Kinase Inhibitor (PKI), a naturally encoded protein that serves as a pseudosubstrate inhibitor for PKA. The binding properties of these peptide analogs were characterized by fluorescence polarization and surface plasmon resonance, and two compounds were identified with KD values in the 500–600 pM range. In kinase activity assays, both compounds demonstrated inhibition with 25–35 nM IC50 values. They were also found to permeate cells and localize within the cytoplasm and inhibited PKA activity within the cellular environment. To the best of our knowledge, these stapled peptide inhibitors represent some of the highest affinity binders reported to date for hydrocarbon stapled peptides.

scholarly journals Analysis of the Protein Kinase A-Regulated Proteome ofCryptococcus neoformansIdentifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
J. M. H. Geddes ◽  
M. Caza ◽  
D. Croll ◽  
N. Stoynov ◽  
L. J. Foster ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Camp Signaling ◽  
Ubiquitin Proteasome Pathway ◽  
Capsule Production ◽  
Life Threatening ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Kinase A

ABSTRACTThe opportunistic fungal pathogenCryptococcus neoformanscauses life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA) signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway inC. neoformansshowed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis.IMPORTANCEFungi cause life-threatening diseases, but very few drugs are available to effectively treat fungal infections. The pathogenic fungusCryptococcus neoformanscauses a substantial global burden of life-threatening meningitis in patients suffering from HIV/AIDS. An understanding of the mechanisms by which fungi deploy virulence factors to cause disease is critical for developing new therapeutic approaches. We employed a quantitative proteomic approach to define the changes in the protein complement that occur upon modulating the cAMP signaling pathway that regulates virulence inC. neoformans. This approach identified a conserved role for cAMP signaling in the regulation of the ubiquitin-proteasome pathway and revealed a link between this pathway and elaboration of a major virulence determinant, the polysaccharide capsule. Targeting the ubiquitin-proteasome pathway opens new therapeutic options for the treatment of cryptococcosis.

A-kinase-anchoring proteins and cytoskeletal signalling events

2003 ◽  
Vol 31 (1) ◽  
pp. 87-89 ◽  
Author(s):  
J.D. Scott
Keyword(s):  
Protein Kinase ◽  
Molecular Mass ◽  
Protein Kinase A ◽  
Protein Kinases ◽  
Recent Progress ◽  
Membrane Receptors ◽  
Anchoring Proteins ◽  
Syndrome Protein ◽  
Kinase A

Targeting of protein kinases and phosphatases to the cytoskeleton enhances the regulation of many signalling events. Cytoskeletal signalling complexes facilitate this process by optimizing the relay of messages from membrane receptors to specific sites on the actin cytoskeleton. These signals influence fundamental cell properties such as shape, movement and division. Targeting of the cAMP-dependent kinase (protein kinase A) and other enzymes to this compartment is achieved through interaction with A-kinase-anchoring proteins (AKAPs). The present paper discusses recent progress on dissecting the biological role of WAVE1 (Wiskott–Alrich syndrome protein family verprolin homology protein 1), an AKAP that assembles a cytoskeletal transduction complex in response to signals that emanate from the low-molecular-mass GTPase, Rac.

scholarly journals Adrenergic Regulation and Diurnal Rhythm of p38 Mitogen-Activated Protein Kinase Phosphorylation in the Rat Pineal Gland

Endocrinology ◽  
2004 ◽  
Vol 145 (11) ◽  
pp. 5194-5201 ◽  
Author(s):  
C. L. Chik ◽  
M. Mackova ◽  
D. Price ◽  
A. K. Ho
Keyword(s):  
Protein Kinase ◽  
Pineal Gland ◽  
Protein Kinase A ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Adrenergic Blocker ◽  
Rat Pineal Gland ◽  
Kinase A

Abstract In this study, we investigated adrenergic and photoneural regulation of p38MAPK phosphorylation in the rat pineal gland. Norepinephrine (NE), the endogenous neurotransmitter, dose-dependently increased the levels of phosphorylated MAPK kinase 3/6 (MKK3/6) and p38MAPK in rat pinealocytes. Time-course studies showed a gradual increase in MKK3/6 and p38MAPK phosphorylation that peaked between 1 and 2 h and persisted for 4 h post NE stimulation. In cells treated with NE for 2 and 4 h, the inclusion of prazosin or propranolol reduced NE-induced MKK3/6 and p38MAPK phosphorylation, indicating involvement of both α- and β-adrenergic receptors for the sustained response. Whereas treatment with dibutyryl cAMP or ionomycin mimicked the NE-induced MKK3/6 and p38MAPK phosphorylation, neither dibutyryl cGMP nor 4β-phorbol 12-myristate 13-acetate had an effect. The NE-induced increase in MKK3/6 and p38MAPK phosphorylation was blocked by KT5720 (a protein kinase A inhibitor) and KN93 (a Ca2+/calmodulin-dependent kinase inhibitor), but not by KT5823 (a protein kinase G inhibitor) or calphostin C (a protein kinase C inhibitor). In animals housed under a lighting regimen with 12 h of light, MKK3/6 and p38MAPK phosphorylation increased in the rat pineal gland at zeitgeber time 18. The nocturnal increase in p38MAPK phosphorylation was blocked by exposing the animal to constant light and reduced by treatment with propranolol, a β-adrenergic blocker. Together, our results indicate that activation of p38MAPK is under photoneural control in the rat pineal gland and that protein kinase A and intracellular Ca2+ signaling pathways are involved in NE regulation of p38MAPK.

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