Differential effects of substrate on type I and type II PKA holoenzyme dissociation.
It has been widely accepted that cAMP activates the protein kinase A (PKA) holoenzyme by dissociating the regulatory and catalytic subunits, thus freeing the catalytic subunit to phosphorylate its targets. However, recent experiments suggest that cAMP does not fully dissociate the holoenzyme. Here, we investigate this mechanism further by using small-angle X-ray scattering to study, at physiological enzyme concentrations, the type Ialpha and type IIbeta holoenzyme structures under equilibrium solution conditions without any labeling of the protein subunits. We observe that while the addition of a molar excess of cAMP to the type Ialpha PKA holoenzyme causes partial dissociation, it is only upon addition of a PKA peptide substrate together with cAMP that full dissociation occurs. Similarly, addition of excess cAMP to the type IIbeta holoenzyme causes only a partial dissociation. However, while the addition of peptide substrate as well as excess cAMP causes somewhat more dissociation, a significant percentage of intact type IIbeta holoenzyme remains. These results confirm that both the type Ialpha and the type IIbeta holoenzymes are more stable in the presence of cAMP than previously thought. They also demonstrate that substrate plays a differential role in the activation of type I versus type II holoenzymes, which could explain some important functional differences between PKA isoforms. On the basis of these data and other recently published data, we propose a structural model of type I holoenzyme activation by cAMP.
Vigil
Dominico
D
Department of Chemistry and Biochemistry and Howard Hughes Medical Institute, University of California at San Diego, La Jolla, California 92037, USA.
Blumenthal
Donald K
DK
Brown
Simon
S
Taylor
Susan S
SS
Trewhella
Jill
J
D000818:Animals; D020134:Catalytic Domain / Q000502:physiology; D002417:Cattle; D000242:Cyclic AMP / Q000737:chemistry; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D009842:Oligopeptides / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D012542:Scattering, Radiation; D012996:Solutions; D013379:Substrate Specificity; D014965:X-Rays
D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D020035:Holoenzymes; D009842:Oligopeptides; D021122:Protein Subunits; D012996:Solutions; C037642:kemptide; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases