Protein kinase A-catalyzed phosphorylation and its effect on conformation in phytochrome A.
Phytochromes are ubiquitous red/far-red wavelength-sensitive photoreceptors in plants. Oat phytochrome A is a phosphoprotein. Phytochrome A (phyA) possesses two spatially different sites for phosphorylation with cAMP-dependent protein kinase (PKA) [McMichael & Lagarias (1990) Biochemistry 29, 3872-3878]. To assess the modulation of protein conformation by phosphorylation/dephosphorylation and its possible implication in phytochrome-mediated signal transduction, the conformations of phytochrome have been probed by PKA catalyzed phosphorylation. The phosphorylated species were purified and analyzed, along with untreated phytochrome, by limited proteolysis, circular dichroism (CD) and fluorescence quenching measurements. No significant changes in secondary structure of the phyA molecule after its phosphorylation were observed by CD. However, a subtle topographic and/or electrostatic effect of the phytochrome phosphorylation was detected by the time-resolved fluorescence quenching of Trp residues with Cs+ ions. N-Terminal phosphorylation at Ser17 was unique to the Pr form, but both Pr and Pfr phytochromes were phosphorylated at the hinge region to some extent. Phosphorylation at the hinge region resulted in noticeable changes in the proteolytic patterns, inhibiting cleavage near the phosphorylation site and favoring tryptic digestion of the Lys536-Asn537 peptide bond. Phosphorylation at the N-terminus did not cause observable changes in the helical structure of this region, but had an inhibitory effect on proteinase V8 accessibility at a site near the chromophore attachment. The functional relevance of protein phosphorylation of phyA is also discussed.
Lapko
V N
VN
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.
Wells
T A
TA
Song
P S
PS
D000135:Acid Phosphatase / Q000302:isolation & purification / Q000378:metabolism; D000236:Adenoma / Q000201:enzymology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D018554:Avena; D002417:Cattle; D002942:Circular Dichroism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010766:Phosphorylation; D010768:Phosphoserine; D019000:Phosphotyrosine / Q000032:analysis; D010834:Phytochrome / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D051942:Phytochrome A; D011467:Prostate / Q000201:enzymology; D011471:Prostatic Neoplasms / Q000201:enzymology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D014357:Trypsin
D010446:Peptide Fragments; D051942:Phytochrome A; D010834:Phytochrome; D010768:Phosphoserine; D019000:Phosphotyrosine; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D000135:Acid Phosphatase; D014357:Trypsin