TI - Phosphorylation independent activation of human cyclin -dependent kinase 2 by cyclin A in vitro . AB - p33cdk2 is a serine-threonine protein kinase that associates with cyclins A , D , and E and has been implicated in the control of the G1/S transition in mammalian cells . Recent evidence indicates that cyclin-dependent kinase 2 ( Cdk2 ) , like its homolog Cdc2 , requires cyclin binding and phosphorylation ( of threonine-160 ) for activation in vivo . However , the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown . We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts . Recombinant Cdk2 is essentially inactive as a histone H1 kinase ( &lt ; 4 x 10 ( -5 ) pmol phosphate transferred.min-1 x microgram-1 Cdk2 ) . However , in the presence of equimolar cyclin A , the specific activity is approximately 16 pmol.mon-1 x microgram-1 , 4 x 10(5) -fold higher than Cdk2 alone . Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex : the activity of Cdk2T160E was indistinguishable from Cdk2 , whereas that of Cdk2T160A was reduced by five-fold . To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160 , complexes were treated with Cdc2 activating kinase ( CAK ) , purified approximately 12,000-fold from Xenopus eggs . This treatment resulted in an 80-fold increase in specific activity . This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK ( approximately 1600 pmol.mon-1 x microgram-1 ) . Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK . In striking contrast with cyclin A , cyclin B did not directly activate Cdk2 . However , both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK . For the Cdk2/cyclin A complex , both cyclin binding and phosphorylation contribute significantly to activation , although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol . The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed .