KINETICS OF DIMERIZATION AND INTERACTIONS OF P13(SUC1) WITH CYCLIN-DEPENDENT KINASES

The impact of p13(suc1) on the conformation and regulation of cyclin-d ependent kinases (cdks) and cyclins was investigated by spectroscopic and rapid kinetic approaches. In the absence of phosphorylation on cdk s, p13(suc1) formed stable complexes, mainly stabilized by hydrophobic interactions, specifically with cdk2 and cdc2. The presence of cyclin A, associated with cdk2 or cdc2, increased the stability of the inter action between cdk2 and p13(suc1) by a factor of 2. However, cyclin A did not modify the association rate of p13(suc1) to cdk2, but the diss ociation rate, which was decreased 3-fold. Moreover, binding of p13(su c1) to cdk2 resulted in a 2-fold decrease in the release of nucleotide from cdk2, indicating that p13(suc1) induces a marked change in the s tructure of the nucleotide binding site of cdks. On the basis of the s tructure of cdk2/CksHs1 complex and on our kinetic results, we propose that the binding of Cks proteins to C-lobe of cdk2 is stabilized by t he presence of cyclin A and that it may modify the orientation of the loop carrying residues 14 and 15 and their consequent access for depho sphorylation by cdc25 phosphatases. Finally, we have shown that dimeri zation of p13(suc1) in the presence of zinc abolishes its interaction with cdks, which suggests that the binding of p13(suc1) to cdk2. or cd k2/cyclin A may be regulated by dimerization of p13(suc1) in vivo.