Crosstalk between domains in the regulatory subunit of cAMP-dependent protein kinase: influence of amino terminus on cAMP binding and holoenzyme formation.
The regulatory (R) subunit of cAMP-dependent protein kinase os an asymmetric multidomain protein with a dimerization domain at the N-terminus, an autoinhibitors site, and two cAMP binding domains at the C-terminus. Activation of the tetrameric holoenzyme is mediated by the cooperative binding of cAMP to the two cAMP binding sites. To better understand how the various domains influence each other, the N-terminus (delta 1-91) up to the autoinhibitor site was deleted. Not only did this monomeric deletion mutant, purified from Escherichia coli, still bind cAMP and the catalytic (C) subunit with high affinity, holoenzyme formation was actually accelerated by at least 50-fold. MgATP also was not required for rapid reassociation of (delta 1-91)R(cAMP)2 and C. The Kd(cAMP) and the Ka(cAMP) were similar to those for holoenzyme formed with full-length R; however, cooperatively was lost. Thus the N-terminus, either by inter- or intraprotomer contacts, not only impedes holoenzyme formation but also influences the cooperative binding of cAMP. The 1-91 deletion also renders the remaining fragment resistant to proteolytic degradation. Finally, unlike full-length R, the mutant protein can migrate freely into the nucleus. Surface plasmon resonance studies for the first time enabled direct measurements of the association and dissociation rate constants both for the intact R and for (delta 1-91)R. Both displayed very fast on-rates (1 x 10(-5) M-1 s-1 and 1.1 x 10(-5) M-1 s-1, respectively) and extremely slow off-rates (2.3 x 10(5) M-1 and 4.3 x 10(5) M-1, respectively). Thus, unlike the heat-stable protein kinase inhibitor, the region preceding the autoinhibitor site in R does not contribute in a quantitatively significant way to the high-affinity binding of C.
Herberg
F W
FW
Department of Chemistry, University of California, San Diego, La Jolla 92093-0654.
Dostmann
W R
WR
Zorn
M
M
Davis
S J
SJ
Taylor
S S
SS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D051381:Rats; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000201:enzymology
D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases