A-kinase anchoring proteins (AKAPs) control the localization and substrate specificity of cAMP-dependent protein kinase (PKA), tetramers of regulatory (PKA-R) and catalytic (PKA-C) subunits, by ...binding to PKA-R subunits. Most mammalian AKAPs bind Type II PKA through PKA-RII (ref. 2), whereas dual specificity AKAPs bind both PKA-RI and PKA-RII (ref. 3). Inhibition of PKA-AKAP interactions modulates PKA signalling. Localized PKA activation in pseudopodia of migrating cells phosphorylates alpha4 integrins to provide spatial cues governing cell motility. Here, we report that the alpha4 cytoplasmic domain is a Type I PKA-specific AKAP that is distinct from canonical AKAPs in two ways: the alpha4 interaction requires the PKA holoenzyme, and is insensitive to amphipathic peptides that disrupt most PKA-AKAP interactions. We exploited type-specific PKA anchoring peptides to create genetically encoded baits that sequester specific PKA isoforms to the mitochondria and found that mislocalization of Type I, but not Type II, PKA disrupts alpha4 phosphorylation and markedly inhibits the velocity and directional persistence of cell migration.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that ...are either in or in close proximity to the cAMP binding sites. The photoaffinity analogue 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound per mole of type I regulatory subunit monomer (RI), the apparent Kd's observed were approximately 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and (4) the Hill coefficients were approximately 1.5-1.6. After photolysis and addition of an excess of cAMP, approximately 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that two unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the two residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue Kerlavage, A. R., & Taylor, S. S. (1980) J. Biol. Chem. 255, 8483-8488. RI contains two adjacent regions of sequence homology in the COOH-terminal fragment that binds two molecules of cAMP.
A template for the protein kinase family Taylor, S S; Knighton, D R; Zheng, J ...
Trends in biochemical sciences (Amsterdam. Regular ed.),
03/1993, Letnik:
18, Številka:
3
Journal Article
Recenzirano
The crystal structure of the catalytic subunit of cAMP-dependent protein kinase, complexed with ATP and a 20-residue inhibitor peptide, is reviewed and correlated with chemical and genetic data. The ...striking convergence of the structure with the biochemistry and genetics provides for the first time a molecular basis for understanding how this enzyme functions, as well as an explanation for the highly conserved residues that are scattered throughout the molecule. Because these residues probably serve a common role in all eukaryotic protein kinases, this first protein kinase structure serves as a general template for the entire family of enzymes.
A Kinase Anchoring Proteins (AKAPs) target PKA to specific microdomains using an amphipathic helix that docks to N-terminal Dimerization/Docking (D/D) domains of PKA regulatory (R) subunits. To ...understand specificity, we solved the crystal structure of the helical motif from d-AKAP2, a dual-specific AKAP, bound to the RIIα D/D domain. The 1.6 Å structure reveals for the first time how this dynamic, hydrophobic docking site is assembled. A stable, hydrophobic docking groove is formed by the helical interface of two RIIα protomers. The flexible N-terminus of one protomer is then recruited to the site, anchored to the peptide through two essential isoleucines. The other N-terminus is disordered. This asymmetry provides greater possibilities for AKAP docking. While there is strong discrimination against RIα in the N-terminus of the AKAP helix, the hydrophobic groove discriminates against RIIα. RIα, with a cavity in the groove, can accept a bulky tryptophan, while RIIα requires valine.
When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is expressed in Escherichia coli, it is autophosphorylated at four sites, Ser10, Ser139, Ser338 and Thr197 (49). Three of these ...sites, Ser10, Ser338 and Thr197, are also found in the mammalian enzyme. To understand the functional importance of these phosphorylation sites, each was replaced with Ala, Glu or Asp. The expression, solubility and phosphorylation state of each mutant protein was characterized by immunoprecipitation following in vivo labeling with 32Pi. When possible, isoforms were resolved and kinetic properties were measured. The two stable phosphorylation sites in the mammalian enzyme, Ser338 and Thr197, were shown to play different roles. Ser338, which stabilizes a turn near the C-terminus, is important for stability. Both rC(S338A) and rC(S338E) were very labile; however, the kinetic properties of rC(S338E) were similar to the wild-type catalytic subunit (C-subunit). Ser338 most likely helps to anchor the C-terminus to the surface of the small lobe. Thr197 is in the activation loop near the cleft interface. Mutagenesis of T197 caused a significant loss of catalytic activity with increases in Kms for both peptide and MgATP, as well as a small decrease in k(cat) indicating that this phosphate is important for the correct orientation of catalytic residues at the active site. Replacement of Ser139, positioned at the beginning of the E-helix, with Ala had no effect on the kinetic parameters, stability or phosphorylation at the remaining sites. In contrast, mutation of Ser10, located at the beginning of the A-helix, produced mostly insoluble, inactive, unphosphorylated protein, suggesting that this region, though far removed from the active site, is structurally important at least for the expression of soluble phosphoprotein in E.coli. Since the mutation of active site residues as well as deletion mutants generate underphosphorylated proteins, these phosphorylations in E.coli all result from autophosphorylation.
Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzyme results in a proteolytic-resistant Î(1â72) regulatory
subunit core, indicating that interaction between the regulatory ...and catalytic subunits extends beyond the autoinhibitory
site in the R subunit at the NH 2 terminus. Sequence alignment of the two R subunit isoforms, RI and RII, reveals a significantly sequence diversity at this
specific region. To determine whether this sequence diversity is functionally important for interaction with the catalytic
subunit, specific mutations, R133A and D328A, are introduced into sites adjacent to the active site cleft in the catalytic
subunit. While replacing Arg 133 with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast,
mutant C(D328A) showed a decrease in affinity for binding RI while maintaining similar affinities for RII as compared with
the wild-type catalytic subunit. These results suggest that sequence immediately NH 2 -terminal to the consensus inhibition site in RI and RII interacts with different sites at the proximal region of the active
site cleft in the catalytic subunit. These isoform-specific differences would dictate a significantly different domain organization
in the type I and type II holoenzymes.
Cognitive and physical stimulation therapy Loraine, Jeff; Taylor, Susan; McAllister, Maria
Journal of the American Medical Directors Association,
02/2014, Letnik:
15, Številka:
2
Journal Article
Hegel and Canada is a collection of essays that analyses the
real, but under-recognized, role Hegel has played in the
intellectual and political development of Canada. The volume
focuses on the ...generation of Canadian scholars who emerged after
World War Two: James Doull, Emil Fackenheim, George Grant, Henry S.
Harris, and Charles Taylor.
The regulatory (R) subunit of cAMP-dependent protein kinase I has been expressed in Escherichia coli, and oligonucleotide-directed mutagenesis was initiated in order to better understand structural ...changes that are induced as a consequence of cAMP-binding. Photoaffinity labeling of the type I holoenzyme with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371 Bubis, J., & Taylor, S.S. (1987) Biochemistry 26, 3478-3486. The site that was targeted for mutagenesis was Tyr-371. The intention was to establish whether the interactions between the tyrosine ring and the adenine ring of cAMP are primarily hydrophobic in nature or whether the hydroxyl group is critical for cAMP binding and/or for inducing conformational changes. A single base change converted Tyr-371 to Phe. This yielded an R subunit that reassociated with the catalytic subunit to form holoenzyme and bound 2 mol of cAMP/mol of R monomer. The cAMP binding properties of the holoenzyme that was formed with this mutant R subunit, however, were altered: (a) the apparent Kd(cAMP) was shifted from 16 to 60 nM; (b) Scatchard plots showed no cooperativity between the cAMP binding sites in the mutant in contrast to the positive cooperativity that is observed for the wild-type holoenzyme; (c) the Hill coefficient of 1.6 for the wild-type holoenzyme was reduced to 0.99. The Ka's for activation by cAMP were altered in the mutant holoenzyme in a manner that was proportional to the shift in Kd(cAMP).
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