Quantum Chemical Study on the Catalytic Mechanism of the C-Subunit of cAMP-Dependent Protein Kinase

2002 ◽  
Vol 106 (22) ◽  
pp. 5788-5792 ◽  
Author(s):  
Yoshinori Hirano ◽  
Masayuki Hata ◽  
Tyuji Hoshino ◽  
Minoru Tsuda
Keyword(s):  
Protein Kinase ◽  
Quantum Chemical ◽  
Catalytic Mechanism ◽  
Quantum Chemical Study ◽  
Chemical Study ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
C Subunit ◽  
Dependent Protein

scholarly journals Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion.

1993 ◽  
Vol 4 (10) ◽  
pp. 993-1002 ◽  
Author(s):  
A T Harootunian ◽  
S R Adams ◽  
W Wen ◽  
J L Meinkoth ◽  
S S Taylor ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Intracellular Camp ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
Camp Dependent Protein Kinase ◽  
Block Movement ◽  
C Subunit ◽  
Dependent Protein

The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either as free C subunit or in the form of holoenzyme (PKA) in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine, respectively. Quantification of nuclear and cytoplasmic fluorescence intensities revealed that free C subunit nuclear accumulation was most similar to that of macromolecules that diffuse into the nucleus. A glutathione S-transferase-C subunit fusion protein did not enter the nucleus following cytoplasmic microinjection. Puncturing the nuclear membrane did not decrease the nuclear concentration of C subunit, and C subunit entry into the nucleus did not appear to be saturable. Cooling or depleting cells of energy failed to block movement of C subunit into the nucleus. Photobleaching experiments showed that even after reaching equilibrium at high [cAMP], individual molecules of C subunit continued to leave the nucleus at approximately the same rate that they had originally entered. These results indicate that diffusion is sufficient to explain most aspects of C subunit subcellular localization.

scholarly journals Identification of cAMP-dependent protein kinase holoenzymes in preantral- and preovulatory-follicle-enriched ovaries, and their association with A-kinase-anchoring proteins

1999 ◽  
Vol 344 (2) ◽  
pp. 613-623 ◽  
Author(s):  
Daniel W. CARR ◽  
Richard E. CUTLER ◽  
Joshua E. COTTOM ◽  
Lisa M. SALVADOR ◽  
Iain D. C. FRASER ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Sucrose Density Gradient Centrifugation ◽  
Camp Dependent Protein Kinase ◽  
Anchoring Proteins ◽  
C Subunit ◽  
Dependent Protein

Undifferentiated cells from preantral (PA) follicles respond to high levels of cAMP in a different manner than do differentiated cells from preovulatory (PO) follicles. We hypothesized that this differential response of PA and PO cells to cAMP could be due, in part, to either a difference in the profile of isoforms that comprise the cAMP-dependent protein kinase (PKA) holoenzymes and/or a difference in the interaction of PKA with A-kinase-anchoring proteins (AKAPs). To test these hypotheses, PKA activity, PKA holoenzymes, PKA subunits and AKAPs from PA and PO ovaries were compared. Soluble PKA holoenzymes and regulatory (R) subunits were separated by DEAE-cellulose chromatography and sucrose-density-gradient centrifugation. PKA R subunits were distinguished by photoaffinity labelling, autophosphorylation, size, isoelectric point and immunoreactivity. AKAPs were identified by RII subunit overlay assays and immunoreactivity. The results showed that extracts from PA and PO ovaries exhibited equivalent PKA holoenzyme profiles and activities, characterized by low levels of PKA type I (PKAI) holoenzyme and two distinct PKAII holoenzyme peaks, one containing only RIIβ subunits (PKAIIβ) and one containing both PKAIIβ and PKAIIα holoenzymes. Both PA and PO ovarian extracts also contained PKA catalytic (C)-subunit-free RIα, while only PO ovaries exhibited C-subunit-free RIIβ. Consistent with the elevated levels of C-subunit-free RIIβ in PO cells, PKA activation in PO cells required higher concentrations of forskolin than that in PA cells. While extracts of PA and PO ovaries exhibited a number of similar AKAPs, including four prominent ones reactive with anti-AKAP-KL antisera (where AKAP-KL is an AKAP especially abundant in kidney and liver), cAMP-agarose affinity chromatography revealed two major differences in AKAP binding to purified R subunits. PO ovaries contained increased levels of AKAP80 (AKAP of 80 kDa) bound selectively to R subunits in DEAE-cellulose peak 2 (comprising PKAIIβ and RIα), but not to R subunits in DEAE-cellulose peak 3 (comprising PKAIIα, PKAIIβ and RIIβ). PO ovaries also showed increased binding of R subunits to AKAPs reactive with anti-AKAP-KL antisera at 210, 175, 150 and 115 kDa. Thus in PO ovaries, unlike in PA ovaries, the majority of AKAPs are bound to R subunits. These results suggest that altered PKA-AKAP interactions may contribute to the distinct responses of PA and PO follicles to high levels of cAMP, and that higher cAMP levels are required to activate PKA in PO ovaries.

scholarly journals The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis

2001 ◽  
Vol 82 (7) ◽  
pp. 1637-1646 ◽  
Author(s):  
Mustapha Aoubala ◽  
John Holt ◽  
Roger A. Clegg ◽  
David J. Rowlands ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Atp Hydrolysis ◽  
Full Length ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
C Subunit ◽  
Dependent Protein ◽  
Arginine Rich Motif

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system – under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

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