PHOSPHORYLATION OF THE P34 SUBUNIT OF HUMAN SINGLE-STRANDED-DNA-BINDING PROTEIN IN CYCLIN-A-ACTIVATED G(1) EXTRACTS IS CATALYZED BY CDK CYCLIN-A COMPLEX AND DNA-DEPENDENT PROTEIN-KINASE
Zq. Pan et al., PHOSPHORYLATION OF THE P34 SUBUNIT OF HUMAN SINGLE-STRANDED-DNA-BINDING PROTEIN IN CYCLIN-A-ACTIVATED G(1) EXTRACTS IS CATALYZED BY CDK CYCLIN-A COMPLEX AND DNA-DEPENDENT PROTEIN-KINASE, Proceedings of the National Academy of Sciences of the United Statesof America, 91(18), 1994, pp. 8343-8347
The human single-stranded-DNA-binding protein (HSSB, also called RP-A)
is a trimeric complex (p70, p34, and p14) required for multiple funct
ions in DNA transactions. We report here that the p34 subunit of HSSB
was hyperphosphorylated by kinase activities present in G(1) extract (
obtained from HeLa cells in G(1) phase) preincubated with human cyclin
A. This hyperphosphorylated HSSB product included at least four speci
es of p34 that migrated more slowly through denaturing polyacrylamide
gels than the hypophosphorylated form. Fractionation of cyclin A-activ
ated G(1) extract identified two kinases involved in the hyperphosphor
ylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 prote
in kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activat
ed G(1) extract, p34 was first phosphorylated by cdk-cyclin A prior to
the action of DNA-PK. Addition of p21(cip1), a specific inhibitor of
cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation
of HSSB p34 in G(1) extract preincubated with cyclin A. This suggests
a requirement of the cdk-cyclin A activity for the phosphorylation of
p34 by DNA-PK in G(1) extract.