title: Catalytic independent functions of a protein kinase as revealed by a kinase-dead mutant: study of the Lys72His mutant of cAMP-dependent kinase.
abstract: A highly conserved lysine in subdomain II is required for high catalytic activity among the protein kinases. This lysine interacts directly with ATP and mutation of this residue leads to a classical "kinase-dead" mutant. This study describes the biophysical and functional properties of a kinase-dead mutant of cAMP-dependent kinase where Lys72 was replaced with His. Although the mutant protein is less stable than the wild-type catalytic subunit, it is fully capable of binding ATP. The results highlight the effect of the mutation on stability and overall organization of the protein, especially the small lobe. Phosphorylation of the activation loop by a heterologous kinase, 3-phosphoinositide-dependent protein kinase-1 (PDK-1) also contributes dramatically to the global organization of the entire active site region. Deuterium-exchange mass spectrometry (DXMS) indicates a concerted stabilization of the entire active site following the addition of this single phosphate to the activation loop. Furthermore the mutant C-subunit is capable of binding both the type I and II regulatory subunits, but only after phosphorylation of the activation loop. This highlights the role of the large lobe as a scaffold for the regulatory subunits independent of catalytic competency and suggests that kinase dead members of the protein kinase superfamily may still have other important biological roles although they lack catalytic activity.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive 0654, La Jolla, CA 92093-0654, USA.
authors.forename: Ganesh H
authors.initials: GH
authors.lastname: Iyer
authors.forename: Siv
authors.initials: S
authors.lastname: Garrod
authors.forename: Virgil L
authors.initials: VL
authors.lastname: Woods
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D008024:Ligands; D010455:Peptides; D006639:Histidine; D006859:Hydrogen; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D008239:Lysine