Phosphorylation of nucleolin by a nucleolar type NII protein kinase.
Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.
Caizergues-Ferrer
M
M
Centre de Recherche de Biochimie et de Genetique Cellulaires du CNRS, Toulouse, France.
Belenguer
P
P
Lapeyre
B
B
Amalric
F
F
Wallace
M O
MO
Olson
M O
MO
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002466:Cell Nucleolus / Q000201:enzymology*; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins; D013379:Substrate Specificity; D000097796:Nucleolin
D009687:Nuclear Proteins; D010750:Phosphoproteins; D011494:Protein Kinases; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C026813:protein kinase NII