Study of the conformation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by fluorescence spectroscopy.
DARPP-32 is a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr34 by cAMP-dependent protein kinase. DARPP-32 is also phosphorylated on Ser45 and Ser102 by casein kinase II, resulting in a facilitation of phosphorylation by cAMP-dependent protein kinase. We have studied the conformation of recombinant rat DARPP-32 by steady-state and time-resolved fluorescence. The steady-state emission spectra and quenching of the intrinsic (Trp163) and extrinsic fluorescence (acrylodan or lucifer yellow linked to Cys72) were consistent with a complete exposure of these residues to the aqueous environment. The intrinsic fluorescence of DARPP-32 was resolved into three decay components with lifetimes of 1, 3.4, and 7 ns, with the intermediate lifetime component giving the major contribution. The ratio between the amplitudes associated with the short and long decay constants was decreased upon denaturation. The rotational behavior of DARPP-32 measured by anisotropy decay revealed that Trp163 is located in a highly flexible peptide chain, whereas Cys72 is embedded in a more rigid environment. Phosphorylation by cAMP-dependent protein kinase did not alter any of the fluorescence parameters, whereas only minor effects were associated with casein kinase II phosphorylation. These findings indicate that DARPP-32 contains at least two distinct domains and that phosphorylation has no dramatic effects on its conformation.
Neyroz
P
P
Dipartimento di Biochimica G. Moruzzi, Università di Bologna, Italy.
Desdouits
F
F
Benfenati
F
F
Knutson
J R
JR
Greengard
P
P
Girault
J A
JA
D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism*; D018076:DNA, Complementary; D004298:Dopamine / Q000378:metabolism*; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D005454:Fluorescence Polarization; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000242:Cyclic AMP; D004298:Dopamine