STUDIES ON THE IN-VITRO PHOSPHORYLATION OF HSSB-P34 AND HSSB-P107 BY CYCLIN-DEPENDENT KINASES - CYCLIN-SUBSTRATE INTERACTIONS DICTATE THE EFFICIENCY OF PHOSPHORYLATION

Cyclin-dependent kinases (Cdks) are required for cell cycle progressio n. Two potentially significant Cdk substrates in human cells are the h uman single-stranded binding protein (HSSB or RPA), which plays an ess ential role in DNA replication, repair, and recombination, and the tum or suppressor p107 which acts to negatively regulate cell growth. In t his report we describe the in vitro phosphorylation of these two prote ins by Cdks in an attempt to understand how cyclin-substrate interacti ons direct phosphorylation efficiencies. We show that cyclin A-Cdk2 ef ficiently phosphorylates the p34 subunit of HSSB (HSSB-p34) alone or a s a part of the heterotrimeric complex. In contrast, cyclin E-Cdk2 tha t is active in phosphorylating histone H1, does not support the phosph orylation of the p34 subunit of HSSB. We provide evidence that this di fferential phosphorylation results from a specific interaction between HSSB-p34 and cyclin Abut not cyclin E. Thus the observed cell cycle-d ependent phosphorylation of HSSB-p34 at the G(1) to S transition is mo st likely catalyzed by cyclin A-Cdk2 initiated by the direct interacti on between cyclin A and the HSSB-p34 subunit. These studies are consis tent with our previous observation that p107, which directly binds cyc lin A, is efficiently phosphorylated by cyclin A-Cdk2 but not cyclin B -associated kinases. Here we further demonstrate that cyclin A only co mplexes with p107 in its unphosphorylated form. These data suggest a c atalytic mechanism by which Cdk acts: substrate targeting by a cyclin- substrate interaction followed by dissociation of the Cdk upon phospha te incorporation allowing the Cdk to become available for the next cyc le of phosphorylation.