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Functional changes in the regulatory subunit of the type II cyclic adenosine 3':5'-monophosphate-dependent protein
Abstract:
The abilities of cyclic adenosine 3':5'-monophosphate (cAMP) and cyclic 8-azidoadenosine 3':5'-[32P]monophosphate (8-N3-[32P]cAMP) to bind to the regulatory subunit (RII) of the type II cAMP-dependent protein kinase isozyme and to cause subsequent dissociation of the holoenzyme were compared in extracts from adult and neonatal mouse lung and lung adenoma. RII in extracts from adult lung exhibits equal numbers of high- (Kd 15 nM) and low- (Kd 230 nM) affinity 8-N3-[32P]cAMP binding sites. In the neonate, the proportion of high-affinity sites is reduced to 20% while, in lung adenoma, only low-affinity RII binding is observed. Low-affinity RII binding is correlated with an inability of cAMP to dissociate the type II holoenzyme completely. Sucrose gradient sedimentation of adult lung cytosol in the presence of cAMP shows complete dissociation of the type I isozyme, while only some of the type II holoenzyme is dissociated. This is in contrast to the case with lung tumor cytosol, in which only low-affinity binding is observed and no apparent dissociation of the type II isozyme occurs. cAMP does promote RII dephosphorylation within the holoenzyme, however, suggesting that cAMP can bind to RII without dissociating the tetramer. Consistent with this interpretation, photoincorporation of 8-N3-[32P]cAMP prior to sucrose gradient sedimentation results in the formation of a photolabeled RII complex which sediments at the same rate as does the holoenzyme. Two-dimensional gel electrophoresis of RII photolabeled at low and high concentrations of 8-N3-[32P]cAMP suggests that these altered binding and dissociation characteristics of the type II isozyme are not due to the presence of a structurally altered RII molecule. After DEAE-cellulose chromatography of lung cytosol, only high-affinity RII binding is observed, and all of the RII can now be dissociated with cAMP. Low-affinity binding may thus reflect either an altered conformational state of RII or the interaction of the type II kinase with other cytosolic molecules which can affect RII binding and dissociation without altering the functional properties of the type I isozyme.
Insights
Cyclic adenosine monophosphate (cAMP) binding to the regulatory subunit (RII) of type II protein kinase differs between adult and neonatal mouse lung and lung adenoma. Altered RII binding in lung adenoma affects holoenzyme dissociation.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Cyclic adenosine monophosphate (cAMP) is a crucial second messenger regulating various cellular processes.
- cAMP-dependent protein kinase (PKA) isozymes, including type I and type II, play vital roles in signal transduction.
- The regulatory subunit (RII) of type II PKA is critical for holoenzyme assembly and cAMP binding.
Purpose of the Study:
- To compare the cAMP and 8-azidoadenosine 3':5'-[32P]monophosphate (8-N3-[32P]cAMP) binding affinities to RII in adult, neonatal mouse lung, and lung adenoma.
- To investigate the effect of altered RII binding on the dissociation of the type II PKA holoenzyme.
- To explore potential mechanisms underlying changes in RII binding and holoenzyme dissociation in lung adenoma.
Main Methods:
- Radioligand binding assays using 8-N3-[32P]cAMP to determine RII binding affinities (Kd).
- Sucrose gradient sedimentation to assess holoenzyme dissociation in response to cAMP.
- Photoaffinity labeling with 8-N3-[32P]cAMP followed by gel electrophoresis to analyze RII structure and interactions.
- DEAE-cellulose chromatography to investigate the influence of cytosolic factors on RII binding.
Main Results:
- Adult lung RII exhibits both high- (15 nM) and low-affinity (230 nM) 8-N3-[32P]cAMP binding sites.
- Neonatal lung shows reduced high-affinity RII binding (20%), while lung adenoma displays only low-affinity binding.
- Low-affinity RII binding in lung adenoma correlates with incomplete dissociation of the type II holoenzyme by cAMP.
- cAMP promotes RII dephosphorylation within the holoenzyme, indicating binding without complete dissociation.
- Altered binding and dissociation characteristics are not due to structural changes in RII.
- DEAE-cellulose chromatography reveals that high-affinity RII binding and cAMP-induced dissociation are restored after removing interacting cytosolic molecules.
Conclusions:
- Altered RII binding affinity and impaired holoenzyme dissociation in lung adenoma suggest a dysregulation of type II PKA signaling.
- Low-affinity RII binding may result from conformational changes in RII or interactions with other cytosolic proteins.
- These alterations in RII binding and dissociation could contribute to the development or progression of lung adenoma.