scholarly journals PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization

2003 ◽  
Vol 370 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Miles D. HOUSLAY ◽  
David R. ADAMS
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Specific Interaction ◽  
Cellular Functions ◽  
Intracellular Targeting ◽  
Anchoring Proteins ◽  
Extracellular Signal ◽  
Catalytic Unit ◽  
Dependent Protein ◽  
Regulatory Functions

cAMP is a second messenger that controls many key cellular functions. The only way to inactivate cAMP is to degrade it through the action of cAMP phosphodiesterases (PDEs). PDEs are thus poised to play a key regulatory role. PDE4 cAMP-specific phosphodiesterases appear to have specific functions with selective inhibitors serving as potent anti-inflammatory agents. The recent elucidation of the structure of the PDE4 catalytic unit allows for molecular insight into the mode of catalysis as well as substrate and inhibitor selectivity. The four PDE4 genes encode over 16 isoforms, each of which is characterized by a unique N-terminal region. PDE4 isoforms play a pivotal role in controlling functionally and spatially distinct pools of cAMP by virtue of their unique intracellular targeting. Targeting occurs by association with proteins, such as arrestins, SRC family tyrosyl kinases, A-kinase anchoring proteins ('AKAPs') and receptor for activated C kinase 1 ('RACK1'), and, in the case of isoform PDE4A1, by a specific interaction (TAPAS-1) with phosphatidic acid. PDE4 isoforms are ‘designed’ to be regulated by extracellular-signal-related protein kinase (ERK), which binds to anchor sites on the PDE4 catalytic domain that it phosphorylates. The upstream conserved region 1 (UCR1) and 2 (UCR2) modules that abut the PDE4 catalytic unit confer regulatory functions by orchestrating the functional outcome of phosphorylation by cAMP-dependent protein kinase ('PKA') and ERK. PDE4 enzymes stand at a crossroads that allows them to integrate various signalling pathways with that of cAMP in spatially distinct compartments.

scholarly journals Activation State-Dependent Substrate Gating in Ca2+/Calmodulin-Dependent Protein Kinase II

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
D. E. Johnson ◽  
A. Hudmon
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Intrinsic Property ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
State Dependent ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Substrate Phosphorylation ◽  
The Brain

Calcium/calmodulin-dependent protein kinase II (CaMKII) is highly concentrated in the brain where its activation by the Ca2+sensor CaM, multivalent structure, and complex autoregulatory features make it an ideal translator of Ca2+signals created by different patterns of neuronal activity. We provide direct evidence that graded levels of kinase activity and extent of T287(T286αisoform) autophosphorylation drive changes in catalytic output and substrate selectivity. The catalytic domains of CaMKII phosphorylate purified PSDs much more effectively when tethered together in the holoenzyme versus individual subunits. Using multisubstrate SPOT arrays, high-affinity substrates are preferentially phosphorylated with limited subunit activity per holoenzyme, whereas multiple subunits or maximal subunit activation is required for intermediate- and low-affinity, weak substrates, respectively. Using a monomeric form of CaMKII to control T287autophosphorylation, we demonstrate that increased Ca2+/CaM-dependent activity for all substrates tested, with the extent of weak, low-affinity substrate phosphorylation governed by the extent of T287autophosphorylation. Our data suggest T287autophosphorylation regulates substrate gating, an intrinsic property of the catalytic domain, which is amplified within the multivalent architecture of the CaMKII holoenzyme.

scholarly journals Characterization of Transgenic Mice with Targeted Disruption of the Catalytic Domain of the Double-stranded RNA-dependent Protein Kinase, PKR

1999 ◽  
Vol 274 (9) ◽  
pp. 5953-5962 ◽  
Author(s):  
Ninan Abraham ◽  
David F. Stojdl ◽  
Peter I. Duncan ◽  
Nathalie Méthot ◽  
Tetsu Ishii ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Transgenic Mice ◽  
Catalytic Domain ◽  
Targeted Disruption ◽  
Dependent Protein Kinase ◽  
Double Stranded Rna ◽  
Dependent Protein

Cyclic GMP-dependent protein kinase is required for thrombospondin and tenascin mediated focal adhesion disassembly

1996 ◽  
Vol 109 (10) ◽  
pp. 2499-2508 ◽  
Author(s):  
J.E. Murphy-Ullrich ◽  
M.A. Pallero ◽  
N. Boerth ◽  
J.A. Greenwood ◽  
T.M. Lincoln ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Focal Adhesions ◽  
Matrix Proteins ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.

scholarly journals Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities

2015 ◽  
Vol 89 (19) ◽  
pp. 9709-9718 ◽  
Author(s):  
Hiroki Sato ◽  
Misako Yoneda ◽  
Reiko Honma ◽  
Fusako Ikeda ◽  
Shinya Watanabe ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Protein Phosphatase ◽  
Measles Virus ◽  
Housekeeping Genes ◽  
Cellular Protein ◽  
Cellular Functions ◽  
Dependent Protein Kinase ◽  
Protein Phosphatase 5 ◽  
Dependent Protein

ABSTRACTMeasles virus (MeV) causes several unique syndromes, including transient immunosuppression. To clarify the cellular responses to MeV infection, we previously analyzed a MeV-infected epithelial cell line and a lymphoid cell line by microarray and showed that the expression of numerous genes was up- or downregulated in the epithelial cells. In particular, there was a characteristic comprehensive downregulation of housekeeping genes during late stage infection. To identify the mechanism underlying this phenomenon, we examined the phosphorylation status of transcription factors and kinase/phosphatase activities in epithelial cells after infection. MeV infection inactivated cellular protein phosphatase 5 (PP5) that consequently inactivated DNA-dependent protein kinase, which reduced Sp1 phosphorylation levels, and c-Myc degradation, both of which downregulated the expression of many housekeeping genes. In addition, intracellular accumulation of viral nucleocapsid inactivated PP5 and subsequent downstream responses. These findings demonstrate a novel strategy of MeV during infection, which causes the collapse of host cellular functions.IMPORTANCEMeasles virus (MeV) is one of the most important pathogens in humans. We previously showed that MeV infection induces the comprehensive downregulation of housekeeping genes in epithelial cells. By examining this phenomenon, we clarified the molecular mechanism underlying the constitutive expression of housekeeping genes in cells, which is maintained by cellular protein phosphatase 5 (PP5) and DNA-dependent protein kinase. We also demonstrated that MeV targets PP5 for downregulation in epithelial cells. This is the first report to show how MeV infection triggers a reduction in overall cellular functions of infected host cells. Our findings will help uncover unique pathogenicities caused by MeV.

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A

2001 ◽  
Vol 359 (3) ◽  
pp. 497-505 ◽  
Author(s):  
Sunke HIMPEL ◽  
Pascal PANZER ◽  
Klaus EIRMBTER ◽  
Hanna CZAJKOWSKA ◽  
Muhammed SAYED ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Molecular Model ◽  
Enzymic Activity ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Family ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

scholarly journals cGMP-dependent protein kinase protects cGMP from hydrolysis by phosphodiesterase-5

2003 ◽  
Vol 372 (2) ◽  
pp. 419-426 ◽  
Author(s):  
Jun KOTERA ◽  
Kennard A. GRIMES ◽  
Jackie D. CORBIN ◽  
Sharron H. FRANCIS
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Fold Increase ◽  
Binding Affinities ◽  
Dependent Protein Kinase ◽  
Physiological Concentration ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Pig Coronary Artery ◽  
Hydrolysis Of

The physiological effects of cGMP are largely determined by the activities of intracellular receptors, including cGMP-dependent protein kinase (PKG) and cGMP-binding cyclic nucleotide phosphodiesterases (PDEs), and the distribution of cGMP among these receptors dictates activity of the signalling pathway. In the present study, the effects of PKG-Iα or PKG-Iβ on the rate of cGMP hydrolysis by the isolated PDE5 catalytic domain were examined. PKG-Iα strongly inhibited cGMP hydrolysis with an IC50 value of 217 nM, which is similar to the physiological concentration of PKG in pig coronary artery reported previously. By contrast, PKG-Iβ, which has lower affinity for cGMP than does PKG-Iα, inhibited cGMP hydrolysis with an IC50 of approx. 1 μM. Inhibition by PKG-Iα was more effective than that by PKG-Iβ, consistent with their relative affinities for cGMP. Autophosphorylation of PKGs increased their cGMP-binding affinities and their inhibitory effects on PDE5 hydrolysis of cGMP. Autophosphorylation of PKG-Iβ increased its inhibitory potency on PDE5 hydrolysis of cGMP by 10-fold compared with a 2-fold increase upon autophosphorylation of PKG-Iα. The results indicate that cGMP bound to allosteric cGMP-binding sites of PKG is protected from hydrolysis by PDE5 and that persistent protection of cGMP by either non-phosphorylated or autophosphorylated PKGs may be a positive-feedback control to sustain cGMP signalling.

Evidence for the involvement of CaMKII and AMPK in Ca2+-dependent signaling pathways regulating FA uptake and oxidation in contracting rodent muscle

2008 ◽  
Vol 104 (5) ◽  
pp. 1366-1373 ◽  
Author(s):  
Marcella A. Raney ◽  
Lorraine P. Turcotte
Keyword(s):  
Electrical Stimulation ◽  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Calcium Calmodulin Dependent ◽  
Extracellular Signal ◽  
Regulation Of Contraction ◽  
Dependent Protein ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

Calcium-calmodulin/dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK1/2) have each been implicated in the regulation of substrate metabolism during exercise. The purpose of this study was to determine whether CaMKII is involved in the regulation of FA uptake and oxidation and, if it is involved, whether it does so independently of AMPK and ERK1/2. Rat hindquarters were perfused at rest with ( n = 16) or without ( n = 10) 3 mM caffeine, or during electrical stimulation ( n = 14). For each condition, rats were subdivided and treated with 10 μM of either KN92 or KN93, inactive and active CaMKII inhibitors, respectively. Both caffeine treatment and electrical stimulation significantly increased FA uptake and oxidation. KN93 abolished caffeine-induced FA uptake, decreased contraction-induced FA uptake by 33%, and abolished both caffeine- and contraction-induced FA oxidation ( P < 0.05). Caffeine had no effect on ERK1/2 phosphorylation ( P > 0.05) and increased α2-AMPK activity by 68% ( P < 0.05). Electrical stimulation increased ERK1/2 phosphorylation and α2-AMPK activity by 51% and 3.4-fold, respectively ( P < 0.05). KN93 had no effect on caffeine-induced α2-AMPK activity, ERK1/2 phosphorylation, or contraction-induced ERK1/2 phosphorylation ( P > 0.05). Alternatively, it decreased contraction-induced α2-AMPK activity by 51% ( P < 0.05), suggesting that CaMKII lies upstream of AMPK. These results demonstrate that regulation of contraction-induced FA uptake and oxidation occurs in part via Ca2+-independent activation of ERK1/2 as well as Ca2+-dependent activation of CaMKII and AMPK.

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