title: Global consequences of activation loop phosphorylation on protein kinase A.
abstract: Phosphorylation of the activation loop is one of the most common mechanisms for regulating protein kinase activity. The catalytic subunit of cAMP-dependent protein kinase autophosphorylates Thr(197) in the activation loop when expressed in Escherichia coli. Although mutation of Arg(194) to Ala prevents autophosphorylation, phosphorylation of Thr(197) can still be achieved by a heterologous protein kinase, phosphoinositide-dependent protein kinase (PDK1), in vitro. In this study, we examined the structural and functional consequences of adding a single phosphate to the activation loop of cAMP-dependent protein kinase by comparing the wild type C-subunit to the R194A mutant either in the presence or the absence of activation loop phosphorylation. Phosphorylation of Thr(197) decreased the K(m) by approximately 15- and 7-fold for kemptide and ATP, respectively, increased the stability of the enzyme as measured by fluorescence and circular dichroism, and enhanced the binding between the C-subunit and IP20, a protein kinase inhibitor peptide. Additionally, deuterium exchange coupled to mass spectrometry was used to compare the structural dynamics of these proteins. All of the regions of the C-subunit analyzed underwent amide hydrogen exchange at a higher or equal rate in the unphosphorylated enzyme compared with the phosphorylated enzyme. The largest changes occurred at the C terminus of the activation segment in the p + 1 loop/APE regions and the alphaH-alphaI loop motifs and leads to the prediction of a coordinated phosphorylation-induced salt bridge between two conserved residues, Glu(208) and Arg(280).
authors: From the Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093.
authors: Jon M
authors: JM
authors: Steichen
authors: From the Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093.
authors: Ganesh H
authors: GH
authors: Iyer
authors: the Department of Medicine and Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093.
authors: Sheng
authors: S
authors: Li
authors: From the Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093.
authors: S Adrian
authors: SA
authors: Saldanha
authors: From the Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093.
authors: Michael S
authors: MS
authors: Deal
authors: the Department of Medicine and Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093.
authors: Virgil L
authors: VL
authors: Woods
authors: From the Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093; Departments of Pharmacology, University of California, San Diego, La Jolla, California 92093; the Departments of Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093. Electronic address: staylor@ucsd.edu.
authors: Susan S
authors: SS
authors: Taylor
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011489:Protein Denaturation; D017510:Protein Folding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
chemical_list: D009842:Oligopeptides; D021122:Protein Subunits; D013912:Threonine; C037642:kemptide; D000255:Adenosine Triphosphate; D014508:Urea; D017868:Cyclic AMP-Dependent Protein Kinases