Soybean beta-conglycinin alpha subunit is phosphorylated on two distinct serines by protein kinase CK2 in vitro.
Protein kinase CK2 purified from the yeast Yarrowia lipolytica was used to phosphorylate soybean beta-conglycinin alpha subunit. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Glu/Asp/SerP/TyrP. Beta-conglycinin alpha subunit (68 kDa) presents seven consensus sequences, but only 0.5-1 mol P/mol alpha subunit was incorporated by CK2. [32P]Phosphorylated beta-conglycinin alpha subunit was cleaved either by cyanogen bromide or by trypsin. 32P was incorporated into the largest cyanogen bromide fragment only (50 kDa, N-terminal) and only two radiolabeled zones were detected after HPLC of the trypsic digest. The corresponding phosphorylated zones were collected and further analyzed by RP-HPLC coupled to electrospray ionization mass spectrometry (LC-ESMS). Two phosphorylated sites, Ser 75 and Ser 117, were determined after MS-MS analysis of three phosphopeptides identified as 70-89, 116-126, and 116-127 sequences. Over the seven consensus sequences of beta-conglycinin alpha subunit, Ser 75 is the only one which was phosphorylated. Ser 117 was phosphorylated although it is not an expected phosphorylation site according to the canonical consensus sequence criteria as there is no acidic determinant at the +3 position. Both Ser 75 and Ser 117 are located inside very acidic sequences, by contrast with the other unphosphorylated potential sites.
Ralet
M C
MC
Laboratoire de Chimie Biologique, INRA, INA-PG, Thiverval-Grignon, France.
Fouques
D
D
Leonil
J
J
Molle
D
D
Meunier
J C
JC
D000595:Amino Acid Sequence; D052179:Antigens, Plant; D047390:Casein Kinase II; D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000378:metabolism; D005916:Globulins / Q000378:metabolism*; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D055314:Seed Storage Proteins; D012694:Serine / Q000737:chemistry*; D030262:Soybean Proteins; D013025:Glycine max / Q000737:chemistry*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D052179:Antigens, Plant; D005656:Fungal Proteins; D005916:Globulins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D014357:Trypsin