scholarly journals Bacterial expression of Chinese hamster regulatory type-I and catalytic subunits of cyclic AMP-dependent protein kinase and mutational analysis of the type-I regulatory subunit

1994 ◽  
Vol 297 (1) ◽  
pp. 79-85 ◽  
Author(s):  
M E Gosse ◽  
R Fleischmann ◽  
M Marshall ◽  
N Wang ◽  
S Garges ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Mutational Analysis ◽  
Novel Mutation ◽  
Chinese Hamster ◽  
Alpha Subunit ◽  
Regulatory Subunit ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The type-I regulatory subunit (RI) of the cyclic AMP-dependent protein kinase (PKA) from Chinese hamster ovary (CHO) cells has been cloned and expressed in a strain of BL21(DE3) Escherichia coli lacking adenylate cyclase [BL21(DE3)/delta cya]. RI expressed in this bacterial system free of cyclic AMP is soluble and can reconstitute functional PKA. Recombinant CHO C alpha is predominantly insoluble with some active soluble protein. C beta is entirely insoluble and inactive. Soluble recombinant RI and soluble recombinant C alpha can associate in vitro and be activated by cyclic AMP. Six site-directed mutations of RI were generated to study the interaction of cyclic AMP with RI and RI-C alpha subunit interactions. Four cyclic AMP-binding-site point mutants were generated [W261R (tryptophan to arginine at position 261), a novel mutation in site A; V376G, a novel mutation in site B; G200E (site A), and Y370F (site B), previously described in bovine RI were introduced into the CHO RI for comparison purposes]. Mutants W261R, Y370F, and G200E demonstrated decreased 8-N3-[3H]cyclic AMP binding as well as 5-fold reduced affinity for [3H]cyclic AMP, with threefold increased EC50 values for cyclic AMP activation of kinase activity from reconstituted mutant holoenzymes. The mutation at V376G did not alter cyclic AMP binding or activation by cyclic AMP of mutant holoenzyme. A truncation mutant, G200Stop, which lacks both cyclic AMP-binding sites, did not bind cyclic AMP but can inhibit C alpha subunit activity. A novel mutation outside the cyclic AMP-binding regions of RI (V89A) weakened the interaction with C alpha indicated by a 7-fold lower EC50 for mutant holoenzyme activation by cyclic AMP.

scholarly journals Subcellular Localization and Biological Actions of Activated RSK1 Are Determined by Its Interactions with Subunits of Cyclic AMP-Dependent Protein Kinase

2006 ◽  
Vol 26 (12) ◽  
pp. 4586-4600 ◽  
Author(s):  
Deepti Chaturvedi ◽  
Helen M. Poppleton ◽  
Teresa Stringfield ◽  
Ann Barbier ◽  
Tarun B. Patel
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Nuclear Accumulation ◽  
Regulatory Subunit ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Anchoring Proteins ◽  
Dependent Protein ◽  
Ribosomal S6 Kinase ◽  
Biological Actions

ABSTRACT Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.

scholarly journals Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase.

1984 ◽  
Vol 4 (6) ◽  
pp. 1086-1095 ◽  
Author(s):  
R A Steinberg
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Regulatory Subunit ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Mapping Procedure ◽  
Dependent Protein ◽  
Structural Mutations ◽  
Limited Digestion ◽  
Charge Shift

A variety of structural mutations that alter functional properties of regulatory subunit (R) of type I cyclic AMP-dependent protein kinase are available in the cultured S49 mouse lymphoma cell system. Many of these mutations also alter the electrostatic charge of R by about 1 or 2 units. By a novel peptide mapping procedure, a number of these "charge-shift" structural mutations were localized to small regions within the R polypeptide. The procedure employed two-dimensional polyacrylamide gel electrophoresis to separate large overlapping fragments generated from denatured, affinity-purified R by limited digestion with papain. Mutations were mapped to intervals between the endpoints of these fragments. The position of one mutation was confirmed by mapping a new site for cleavage by Staphylococcus aureus V8 protease. Six different Ka mutations, which increase the concentrations of cyclic AMP required for kinase activation, mapped to three clusters in the carboxy-terminal half of R. Second-site mutations that cause phenotypic reversion of a single Ka mutant strain mapped to either side of the original mutation. By using charge-shift mutations for calibration, a map of charge density distribution was constructed for the R polypeptide. This map allowed tentative assignment of mutational lesions to portions of the R amino acid sequence implicated in cyclic AMP binding.

scholarly journals Regulation of cyclic AMP-dependent protein kinase levels during skeletal myogenesis

1989 ◽  
Vol 264 (1) ◽  
pp. 305-308 ◽  
Author(s):  
I A J Lorimer ◽  
B D Sanwal
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Type I ◽  
Skeletal Myogenesis ◽  
Dependent Protein Kinase ◽  
Skeletal Myoblasts ◽  
Dependent Protein ◽  
Degradation Studies

We showed previously that the levels of type I regulatory subunit of cyclic AMP-dependent protein kinase increase during differentiation of L6 skeletal myoblasts as a result of a specific decrease in its rate of degradation. Studies on the rates of degradation of the catalytic subunit show that unlike the type I regulatory subunit, catalytic subunit is degraded very slowly in myoblasts (t1/2 = 29 h) and more rapidly in myotubes (t1/2 = 14 h). As with the regulatory subunit, the degradation of catalytic subunit is increased by treatment of myoblasts with cyclic AMP analogues. These results suggest that the overall increase in the amount of type I cyclic AMP-dependent protein kinase holoenzyme during myogenesis is due to the increase in levels of mRNA for the catalytic subunit. This probably leads to an increase in the amount of catalytic subunit, which then stabilizes the regulatory subunit, thereby causing an increase in the levels of this protein also.

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