Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein: Cloning and characterization of the RII-binding domain

Daniel Carr, Zachary E. Hausken, Iain D C Fraser, Renata E. Stofko-Hahn, John D. Scott

Research output: Contribution to journalArticle

271 Citations (Scopus)

Abstract

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys- Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an α-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RIIα or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RIIα and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 μM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RIIα at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type IIα PKA holoenzyme may be specifically targeted to different locations in each type of cell.

Original languageEnglish (US)
Pages (from-to)13376-13382
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number19
StatePublished - Jul 5 1992
Externally publishedYes

Fingerprint

Cloning
Cyclic AMP-Dependent Protein Kinases
Organism Cloning
Thyroid Gland
Peptides
Protein Kinases
Holoenzymes
Proteins
Tissue
Dialysis
Circular Dichroism
Structural analysis
Conformations
human AKAP5 protein
Catalytic Domain
Carrier Proteins
Complementary DNA
Chemical activation
Amino Acids
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein : Cloning and characterization of the RII-binding domain. / Carr, Daniel; Hausken, Zachary E.; Fraser, Iain D C; Stofko-Hahn, Renata E.; Scott, John D.

In: Journal of Biological Chemistry, Vol. 267, No. 19, 05.07.1992, p. 13376-13382.

Research output: Contribution to journalArticle

Carr, Daniel ; Hausken, Zachary E. ; Fraser, Iain D C ; Stofko-Hahn, Renata E. ; Scott, John D. / Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein : Cloning and characterization of the RII-binding domain. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 19. pp. 13376-13382.
@article{68928c83544942c7b64414edbdd78166,
title = "Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein: Cloning and characterization of the RII-binding domain",
abstract = "The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys- Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an α-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RIIα or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RIIα and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 μM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RIIα at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type IIα PKA holoenzyme may be specifically targeted to different locations in each type of cell.",
author = "Daniel Carr and Hausken, {Zachary E.} and Fraser, {Iain D C} and Stofko-Hahn, {Renata E.} and Scott, {John D.}",
year = "1992",
month = "7",
day = "5",
language = "English (US)",
volume = "267",
pages = "13376--13382",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "19",

}

TY - JOUR

T1 - Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein

T2 - Cloning and characterization of the RII-binding domain

AU - Carr, Daniel

AU - Hausken, Zachary E.

AU - Fraser, Iain D C

AU - Stofko-Hahn, Renata E.

AU - Scott, John D.

PY - 1992/7/5

Y1 - 1992/7/5

N2 - The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys- Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an α-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RIIα or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RIIα and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 μM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RIIα at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type IIα PKA holoenzyme may be specifically targeted to different locations in each type of cell.

AB - The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys- Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an α-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RIIα or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RIIα and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 μM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RIIα at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type IIα PKA holoenzyme may be specifically targeted to different locations in each type of cell.

UR - http://www.scopus.com/inward/record.url?scp=0026680801&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026680801&partnerID=8YFLogxK

M3 - Article

C2 - 1618839

AN - SCOPUS:0026680801

VL - 267

SP - 13376

EP - 13382

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 19

ER -