p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes.
p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified kinase) as features of p40MO15 that are required for the generation of active CAK.
CNRS/INSERM, Montpellier, France.
J C
JC
Labbé
A M
AM
Martinez
D
D
Fesquet
J P
JP
Capony
J M
JM
Darbon
J
J
Derancourt
A
A
Devault
N
N
Morin
J C
JC
Cavadore
M
M
Dorée
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009865:Oocytes; D010766:Phosphorylation; D010768:Phosphoserine / Q000302:isolation & purification; D010769:Phosphothreonine / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism*; D014981:Xenopus; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D011994:Recombinant Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase