Global consequences of activation loop phosphorylation on protein kinase A.
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).
Steichen
Jon M
JM
From the Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093.
Iyer
Ganesh H
GH
From the Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093.
Li
Sheng
S
the Department of Medicine and Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093.
Saldanha
S Adrian
SA
From the Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093.
Deal
Michael S
MS
From the Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093.
Woods
Virgil L
VL
the Department of Medicine and Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093.
Taylor
Susan S
SS
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.
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
D009842:Oligopeptides; D021122:Protein Subunits; D013912:Threonine; C037642:kemptide; D000255:Adenosine Triphosphate; D014508:Urea; D017868:Cyclic AMP-Dependent Protein Kinases