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Expression and mutagenesis of the regulatory subunit of cAMP-dependent protein kinase in Escherichia coli.

Methods in Enzymology (Impact Factor: 2). 02/1988; 159:325-36.
Source: PubMed
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    ABSTRACT: 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.
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    ABSTRACT: PKA holoenzymes containing two catalytic (C) subunits and a regulatory (R) subunit dimer are activated cooperatively by cAMP. While cooperativity involves the two tandem cAMP binding domains in each R-subunit, additional cooperativity is associated with the tetramer. Of critical importance is the flexible linker in R that contains an inhibitor site (IS). While the IS becomes ordered in the R:C heterodimer, the overall conformation of the tetramer is mediated largely by the N-Linker that connects the D/D domain to the IS. To understand how the N-Linker contributes to assembly of tetrameric holoenzymes, we engineered a monomeric RIα that contains most of the N-Linker, RIα(73-244), and crystallized a holoenzyme complex. Part of the N-linker is now ordered by interactions with a symmetry-related dimer. This complex of two symmetry-related dimers forms a tetramer that reveals novel mechanisms for allosteric regulation and has many features associated with full-length holoenzyme. A model of the tetrameric holoenzyme, based on this structure, is consistent with previous small angle X-ray and neutron scattering data, and is validated with new SAXS data and with an RIα mutation localized to a novel interface unique to the tetramer.
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    ABSTRACT: Previously we showed that the inactive form of p90 ribosomal S6 kinase 1 (RSK1) interacts with the regulatory subunit, PKARIalpha, of protein kinase A (PKA), whereas the active RSK1 interacts with the catalytic subunit (PKAc) of PKA. Herein, we demonstrate that the N-terminal kinase domain (NTK) of RSK1 is necessary for interactions with PKARIalpha. Substitution of the activation loop phosphorylation site (Ser-221) in the NTK with the negatively charged Asp residue abrogated the association between RSK1 and PKARIalpha. This explains the lack of an interaction between active RSK1 and PKARIalpha. Full-length RSK1 bound to PKARIalpha with an affinity of 0.8 nm. The NTK domain of RSK1 competed with PKAc for binding to the pseudosubstrate region (amino acids 93-99) of PKARIalpha. Overexpressed RSK1 dissociated PKAc from PKARIalpha, increasing PKAc activity, whereas silencing of RSK1 increased PKAc/PKARIalpha interactions and decreased PKAc activity. Unlike PKAc, which requires Arg-95 and -96 in the pseudosubstrate region of PKARIalpha for their interactions, RSK1/PKARIalpha association requires all four Arg residues (Arg-93-96) in the pseudosubstrate site of PKARIalpha. A peptide (Wt-PS) corresponding to residues 91-99 of PKARIalpha competed for binding of RSK1 with PKARIalpha both in vitro and in intact cells. Furthermore, peptide Wt-PS (but not control peptide Mut-PS), by dissociating RSK1 from PKARIalpha, activated RSK1 in the absence of any growth factors and protected cells from apoptosis. Thus, by competing for binding to the pseudosubstrate region of PKARIalpha, RSK1 regulates PKAc activity in a cAMP-independent manner, and PKARIalpha by associating with RSK1 regulates its activation and its biological functions.
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