Characterization and physiological importance of a novel cell cycle regulated protein kinase in Xenopus laevis oocytes that phosphorylates cyclin B2.
We have partially purified a specific cyclin B2 kinase (cyk) from prophase oocytes of Xenopus laevis after an ATP-gamma-S activation step. Phosphopeptide analysis identified Ser53 as the major in vitro phosphorylation site for cyk in cyclin B2. Using a synthetic peptide derived from cyclin B2 encompassing Ser53 (cyktide) as a substrate, cyk was shown to be activated during progesterone-induced maturation, with a peak of activity between 40 and 50% maturation. A sustained high cyk activity was observed in oscillating egg extracts. Microinjection of cyk-phosphorylated cyclin B2 into prophase oocytes accelerated progesterone-induced maturation by about 2 h, indicating that cyclin B2 is a relevant substrate for cyk and that the function of cyk is situated upstream of cdc2-cyclin B activation. Microinjection of cyk-phosphorylated cyktide or a combination of cyk and cyclin B1 into G2 fibroblasts induced significant changes in cell morphology, reminiscent of a premature prophase-like phenotype. Similarly, addition of cyk-phosphorylated cyktide in cyclin B1-dependent interphase extracts resulted in histone H1 kinase activation.
Derua
R
R
Afdeling Biochemie, Faculteit Geneeskunde, K.U. Leuven, Belgium.
Stevens
I
I
Waelkens
E
E
Fernandez
A
A
Lamb
N
N
Merlevede
W
W
Goris
J
J
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002453:Cell Cycle; D002457:Cell Extracts; D002460:Cell Line; D018844:Cyclin-Dependent Kinases / Q000302:isolation & purification / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007399:Interphase; D008845:Microinjections; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D014982:Xenopus laevis / Q000378:metabolism
D002457:Cell Extracts; D016213:Cyclins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011494:Protein Kinases; C102333:histone H1 kinase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018844:Cyclin-Dependent Kinases; D020929:Mitogen-Activated Protein Kinase Kinases