Characterization of structural features that mediate the tethering of Caenorhabditis elegans protein kinase A to a novel A kinase anchor protein. Insights into the anchoring of PKAI isoforms

Robert G. Angelo, Charles S. Rubin

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30 Citations (Scopus)

Abstract

Caenorhabditis elegans protein kinase A (PKAI(CE)) is tethered to organelles in vivo. A unique A kinase anchor protein (AKAP(CE)) avidly binds the RI-like regulatory subunits (R(CE)) of PKAI(CE) and stringently discriminates against RIIα and RIIβ subunits, the preferred ligands for classical AKAPs. We elucidated structural features that stabilize AKAP(CE)·R(CE) complexes and confer atypical R isoform specificity on the anchor protein. Three large aliphatic amino acids (Leu236, Ile248, and Leu252) in the tethering domain of AKAP(CE) (residues 236-255) are crucial for ligation of R(CE). Their side chains apparently generate a precisely configured hydrophobic binding pocket that accommodates an apolar surface on R(CE) dimers. Basic residues (His254-Arg255-Lys256) at the C terminus of the tethering site set an upper limit on affinity for R(CE). A central dipeptide (Phe243-Ser244) contributes critical and distinctive properties of the tethering site. Ser244 is essential for selective binding of R(CE) and exclusion of RII isoforms. The aromatic hydrophobic character of Phe243 ensures maximal R(CE) binding activity, thereby supporting a 'gatekeeper' function of Ser244. Substitution of Phe243- Ser244 with Leu-Val generated an RII-specific AKAP·R(CE) and RII subunits contain similar dimerization domains. AKAP-binding domains of R(CE) (residues 23-47) and RII differ markedly in size, amino acid sequence, and docking specificity. Four hydrophobic residues (Cys23, Val27, Ile32, and Cys44) in R(CE) are crucial for avid binding with AKAP(CE), whereas side chains from Leu20, Leu35, Val36, Ile40, and Ile41 have little impact on complex formation. Tyr26 is embedded in the docking domain, but its aromatic ring is required for R(CE)-R(CE) dimerization. Residues 236-255 in AKAP(CE) also constitute a binding site for mammalian RIα. RIα (PKAIα) is tightly sequestered by AKAP(CE) in vitro (K(D) = ~10 nM) and in the environment of intact cells. The tethering domain of AKAP(CE) provides a molecular module for manipulating intracellular localization of RI and elucidating functions of anchored PKAI in eukaryotes.

Original languageEnglish (US)
Pages (from-to)4351-4362
Number of pages12
JournalJournal of Biological Chemistry
Volume275
Issue number6
DOIs
StatePublished - Feb 11 2000

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A Kinase Anchor Proteins
Caenorhabditis elegans Proteins
Dimerization
Cyclic AMP-Dependent Protein Kinases
Protein Isoforms
Amino Acids
Dipeptides
Eukaryota
Anchors
Organelles
Dimers
Ligation
Amino Acid Sequence
Substitution reactions
Fatty Acids
Binding Sites
Ligands
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{c2ffeefb6602460a91a1f94bff9e04a0,
title = "Characterization of structural features that mediate the tethering of Caenorhabditis elegans protein kinase A to a novel A kinase anchor protein. Insights into the anchoring of PKAI isoforms",
abstract = "Caenorhabditis elegans protein kinase A (PKAI(CE)) is tethered to organelles in vivo. A unique A kinase anchor protein (AKAP(CE)) avidly binds the RI-like regulatory subunits (R(CE)) of PKAI(CE) and stringently discriminates against RIIα and RIIβ subunits, the preferred ligands for classical AKAPs. We elucidated structural features that stabilize AKAP(CE)·R(CE) complexes and confer atypical R isoform specificity on the anchor protein. Three large aliphatic amino acids (Leu236, Ile248, and Leu252) in the tethering domain of AKAP(CE) (residues 236-255) are crucial for ligation of R(CE). Their side chains apparently generate a precisely configured hydrophobic binding pocket that accommodates an apolar surface on R(CE) dimers. Basic residues (His254-Arg255-Lys256) at the C terminus of the tethering site set an upper limit on affinity for R(CE). A central dipeptide (Phe243-Ser244) contributes critical and distinctive properties of the tethering site. Ser244 is essential for selective binding of R(CE) and exclusion of RII isoforms. The aromatic hydrophobic character of Phe243 ensures maximal R(CE) binding activity, thereby supporting a 'gatekeeper' function of Ser244. Substitution of Phe243- Ser244 with Leu-Val generated an RII-specific AKAP·R(CE) and RII subunits contain similar dimerization domains. AKAP-binding domains of R(CE) (residues 23-47) and RII differ markedly in size, amino acid sequence, and docking specificity. Four hydrophobic residues (Cys23, Val27, Ile32, and Cys44) in R(CE) are crucial for avid binding with AKAP(CE), whereas side chains from Leu20, Leu35, Val36, Ile40, and Ile41 have little impact on complex formation. Tyr26 is embedded in the docking domain, but its aromatic ring is required for R(CE)-R(CE) dimerization. Residues 236-255 in AKAP(CE) also constitute a binding site for mammalian RIα. RIα (PKAIα) is tightly sequestered by AKAP(CE) in vitro (K(D) = ~10 nM) and in the environment of intact cells. The tethering domain of AKAP(CE) provides a molecular module for manipulating intracellular localization of RI and elucidating functions of anchored PKAI in eukaryotes.",
author = "Angelo, {Robert G.} and Rubin, {Charles S.}",
year = "2000",
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T1 - Characterization of structural features that mediate the tethering of Caenorhabditis elegans protein kinase A to a novel A kinase anchor protein. Insights into the anchoring of PKAI isoforms

AU - Angelo, Robert G.

AU - Rubin, Charles S.

PY - 2000/2/11

Y1 - 2000/2/11

N2 - Caenorhabditis elegans protein kinase A (PKAI(CE)) is tethered to organelles in vivo. A unique A kinase anchor protein (AKAP(CE)) avidly binds the RI-like regulatory subunits (R(CE)) of PKAI(CE) and stringently discriminates against RIIα and RIIβ subunits, the preferred ligands for classical AKAPs. We elucidated structural features that stabilize AKAP(CE)·R(CE) complexes and confer atypical R isoform specificity on the anchor protein. Three large aliphatic amino acids (Leu236, Ile248, and Leu252) in the tethering domain of AKAP(CE) (residues 236-255) are crucial for ligation of R(CE). Their side chains apparently generate a precisely configured hydrophobic binding pocket that accommodates an apolar surface on R(CE) dimers. Basic residues (His254-Arg255-Lys256) at the C terminus of the tethering site set an upper limit on affinity for R(CE). A central dipeptide (Phe243-Ser244) contributes critical and distinctive properties of the tethering site. Ser244 is essential for selective binding of R(CE) and exclusion of RII isoforms. The aromatic hydrophobic character of Phe243 ensures maximal R(CE) binding activity, thereby supporting a 'gatekeeper' function of Ser244. Substitution of Phe243- Ser244 with Leu-Val generated an RII-specific AKAP·R(CE) and RII subunits contain similar dimerization domains. AKAP-binding domains of R(CE) (residues 23-47) and RII differ markedly in size, amino acid sequence, and docking specificity. Four hydrophobic residues (Cys23, Val27, Ile32, and Cys44) in R(CE) are crucial for avid binding with AKAP(CE), whereas side chains from Leu20, Leu35, Val36, Ile40, and Ile41 have little impact on complex formation. Tyr26 is embedded in the docking domain, but its aromatic ring is required for R(CE)-R(CE) dimerization. Residues 236-255 in AKAP(CE) also constitute a binding site for mammalian RIα. RIα (PKAIα) is tightly sequestered by AKAP(CE) in vitro (K(D) = ~10 nM) and in the environment of intact cells. The tethering domain of AKAP(CE) provides a molecular module for manipulating intracellular localization of RI and elucidating functions of anchored PKAI in eukaryotes.

AB - Caenorhabditis elegans protein kinase A (PKAI(CE)) is tethered to organelles in vivo. A unique A kinase anchor protein (AKAP(CE)) avidly binds the RI-like regulatory subunits (R(CE)) of PKAI(CE) and stringently discriminates against RIIα and RIIβ subunits, the preferred ligands for classical AKAPs. We elucidated structural features that stabilize AKAP(CE)·R(CE) complexes and confer atypical R isoform specificity on the anchor protein. Three large aliphatic amino acids (Leu236, Ile248, and Leu252) in the tethering domain of AKAP(CE) (residues 236-255) are crucial for ligation of R(CE). Their side chains apparently generate a precisely configured hydrophobic binding pocket that accommodates an apolar surface on R(CE) dimers. Basic residues (His254-Arg255-Lys256) at the C terminus of the tethering site set an upper limit on affinity for R(CE). A central dipeptide (Phe243-Ser244) contributes critical and distinctive properties of the tethering site. Ser244 is essential for selective binding of R(CE) and exclusion of RII isoforms. The aromatic hydrophobic character of Phe243 ensures maximal R(CE) binding activity, thereby supporting a 'gatekeeper' function of Ser244. Substitution of Phe243- Ser244 with Leu-Val generated an RII-specific AKAP·R(CE) and RII subunits contain similar dimerization domains. AKAP-binding domains of R(CE) (residues 23-47) and RII differ markedly in size, amino acid sequence, and docking specificity. Four hydrophobic residues (Cys23, Val27, Ile32, and Cys44) in R(CE) are crucial for avid binding with AKAP(CE), whereas side chains from Leu20, Leu35, Val36, Ile40, and Ile41 have little impact on complex formation. Tyr26 is embedded in the docking domain, but its aromatic ring is required for R(CE)-R(CE) dimerization. Residues 236-255 in AKAP(CE) also constitute a binding site for mammalian RIα. RIα (PKAIα) is tightly sequestered by AKAP(CE) in vitro (K(D) = ~10 nM) and in the environment of intact cells. The tethering domain of AKAP(CE) provides a molecular module for manipulating intracellular localization of RI and elucidating functions of anchored PKAI in eukaryotes.

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