Protein kinase A-catalyzed phosphorylation and its effect on conformation in phytochrome A

被引:23
作者
Lapko, VN [1 ]
Wells, TA [1 ]
Song, PS [1 ]
机构
[1] UNIV NEBRASKA,DEPT CHEM,LINCOLN,NE 68588
关键词
D O I
10.1021/bi9529364
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Ser(17) 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 LYs(536)-Asn(537) 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.
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页码:6585 / 6594
页数:10
相关论文
共 64 条
[1]   CALCIUM TARTRATE GEL [J].
AKHREM, AA ;
DROZHDENYUK, AP .
ANALYTICAL BIOCHEMISTRY, 1989, 179 (01) :86-89
[2]   CYCLIC-AMP AS A 2ND MESSENGER IN HIGHER-PLANTS - STATUS AND FUTURE-PROSPECTS [J].
ASSMANN, SM .
PLANT PHYSIOLOGY, 1995, 108 (03) :885-889
[3]  
BENOVIC JL, 1991, J BIOL CHEM, V266, P14939
[4]   BETA-ADRENERGIC-RECEPTOR KINASE - PRIMARY STRUCTURE DELINEATES A MULTIGENE FAMILY [J].
BENOVIC, JL ;
DEBLASI, A ;
STONE, WC ;
CARON, MG ;
LEFKOWITZ, RJ .
SCIENCE, 1989, 246 (4927) :235-240
[5]   PR-SPECIFIC PHYTOCHROME PHOSPHORYLATION IN-VITRO BY A PROTEIN-KINASE PRESENT IN ANTI-PHYTOCHROME MAIZE IMMUNOPRECIPITATES [J].
BIERMANN, BJ ;
PAO, LI ;
FELDMAN, LJ .
PLANT PHYSIOLOGY, 1994, 105 (01) :243-251
[6]   IMPROVED SILVER STAINING OF PLANT-PROTEINS, RNA AND DNA IN POLYACRYLAMIDE GELS [J].
BLUM, H ;
BEIER, H ;
GROSS, HJ .
ELECTROPHORESIS, 1987, 8 (02) :93-99
[7]   PHYTOCHROME SIGNAL-TRANSDUCTION PATHWAYS ARE REGULATED BY RECIPROCAL CONTROL MECHANISMS [J].
BOWLER, C ;
YAMAGATA, H ;
NEUHAUS, G ;
CHUA, NH .
GENES & DEVELOPMENT, 1994, 8 (18) :2188-2202
[8]  
BOYLAN M, 1994, PLANT CELL, V6, P449, DOI 10.1105/tpc.6.3.449
[9]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[10]  
BUSS JE, 1983, METHOD ENZYMOL, V99, P7