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An Isoform-Specific Myristylation Switch Targets Type II PKA Holoenzymes to Membranes.

ABSTRACT: Cyclic AMP-dependent protein kinase (PKA) is regulated in part by N-terminal myristylation of its catalytic (C) subunit. Structural information about the role of myristylation in membrane targeting of PKA has been limited. In mammalian cells there are four functionally non-redundant PKA regulatory subunits (RI?, RI?, RII?, and RII?). PKA is assembled as an inactive R2C2 holoenzyme in cells. To explore the role of N-myristylation in membrane targeting of PKA holoenzymes, we solved crystal structures of RI?:myrC and RII?2:myrC2, and showed that the N-terminal myristylation site in the myrC serves as a flexible "switch" that can potentially be mobilized for membrane anchoring of RII, but not RI, holoenzymes. Furthermore, we synthesized nanodiscs and showed by electron microscopy that membrane targeting through the myristic acid is specific for the RII holoenzyme. This membrane-anchoring myristylation switch is independent of A Kinase Anchoring Proteins (AKAPs) that target PKA to membranes by other mechanisms.

SUBMITTER: Zhang P

PROVIDER: S-EPMC4558360 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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An Isoform-Specific Myristylation Switch Targets Type II PKA Holoenzymes to Membranes.

Zhang Ping P Ye Feng F Bastidas Adam C AC Kornev Alexandr P AP Wu Jian J Ginsberg Mark H MH Taylor Susan S SS

Structure (London, England : 1993) 20150813 9

Cyclic AMP-dependent protein kinase (PKA) is regulated in part by N-terminal myristylation of its catalytic (C) subunit. Structural information about the role of myristylation in membrane targeting of PKA has been limited. In mammalian cells there are four functionally non-redundant PKA regulatory subunits (RIα, RIβ, RIIα, and RIIβ). PKA is assembled as an inactive R2C2 holoenzyme in cells. To explore the role of N-myristylation in membrane targeting of PKA holoenzymes, we solved crystal structu ...[more]

PMID: 26278174

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Project description:The cAMP-dependent protein kinase catalytic (C) subunit is inhibited by two classes of functionally nonredundant regulatory (R) subunits, RI and RII. Unlike RI subunits, RII subunits are both substrates and inhibitors. Because RIIbeta knockout mice have important disease phenotypes, the RIIbeta holoenzyme is a target for developing isoform-specific agonists and/or antagonists. We also know little about the linker region that connects the inhibitor site to the N-terminal dimerization domain, although this linker determines the unique globular architecture of the RIIbeta holoenzyme. To understand how RIIbeta functions as both an inhibitor and a substrate and to elucidate the structural role of the linker, we engineered different RIIbeta constructs. In the absence of nucleotide, RIIbeta(108-268), which contains a single cyclic nucleotide binding domain, bound C subunit poorly, whereas with AMP-PNP, a non-hydrolyzable ATP analog, the affinity was 11 nM. The RIIbeta(108-268) holoenzyme structure (1.62 A) with AMP-PNP/Mn(2+) showed that we trapped the RIIbeta subunit in an enzyme:substrate complex with the C subunit in a closed conformation. The enhanced affinity afforded by AMP-PNP/Mn(2+) may be a useful strategy for increasing affinity and trapping other protein substrates with their cognate protein kinase. Because mutagenesis predicted that the region N-terminal to the inhibitor site might dock differently to RI and RII, we also engineered RIIbeta(102-265), which contained six additional linker residues. The additional linker residues in RIIbeta(102-265) increased the affinity to 1.6 nM, suggesting that docking to this surface may also enhance catalytic efficiency. In the corresponding holoenzyme structure, this linker docks as an extended strand onto the surface of the large lobe. This hydrophobic pocket, formed by the alphaF-alphaG loop and conserved in many protein kinases, also provides a docking site for the amphipathic helix of PKI. This novel orientation of the linker peptide provides the first clues as to how this region contributes to the unique organization of the RIIbeta holoenzyme.

Dataset Information

An Isoform-Specific Myristylation Switch Targets Type II PKA Holoenzymes to Membranes.

Publications

An Isoform-Specific Myristylation Switch Targets Type II PKA Holoenzymes to Membranes.

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