Specificity of Cdk activation in vivo by the two Caks Mcs6 and Csk1 in fission yeast.
Activating phosphorylation of cyclin-dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk-activating kinases (Caks): the trimeric Cdk7-cyclin H-Mat1 complex in metazoans and the single-subunit Cak1 in budding yeast. Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single-subunit kinase. Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain. However, this apparent redundancy is not observed in fission yeast in vivo. We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6-activating phosphorylation or in Cdc2-activating phosphorylation. Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast. Although Csk1 could activate Mcs6, it was unable to activate Cdc2. Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not. These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6. This demonstrates in vivo specificity in Cdk activation by Caks.
Hermand
D
D
Haartman Institute & Biocentrum Helsinki, University of Helsinki, 00014 Helsinki, HUCH Laboratory Diagnostics, 00029 HYKS, Finland.
Westerling
T
T
Pihlak
A
A
Thuret
J Y
JY
Vallenius
T
T
Tiainen
M
M
Vandenhaute
J
J
Cottarel
G
G
Mann
C
C
Mäkelä
T P
TP
D003001:Cloning, Molecular; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D005816:Genetic Complementation Test; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D016366:Open Reading Frames; D010641:Phenotype; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029702:Schizosaccharomyces pombe Proteins; D013379:Substrate Specificity; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases; C107804:Sck1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase