Exp Mol Med.  2003 Oct;35(5):421-430.

Biochemical characterizations reveal different properties between CDK4/cyclin D1 and CDK2/cyclin A

• Kim DM
• Yang K
• Yang BS

Abstract

CDK2 and CDK4 known promoter of cell cycling catalyze phosphorylation of RB protein. Enzyme specificity between two CDKs that work at a different cell cycle phase is not clearly understood. In order to define kinase properties of CDK2 and CDK4 in complex with cycline A or cycline D1 in relation to their respective role in cell cycling regulation, we examined enzymatic properties of both CDK4/cycline D1 and CDK2/cycline A in vitro. Association constant, Km for ATP in CDK4/cyclin D1 was found as 418 micrometer, a value unusually high whereas CDK2/cyclin A was 23 micrometer, a value close to most of other regulatory protein kinases. Turnover value for both CDK4/cyclin D1 and CDK2/cyclin A were estimated as 3.4 and 3.9 min(-1)respectively. Kinetic efficiency estimation indicates far over one order magnitude less efficiency for CDK4/cyclin D1 than the value of CDK2/cycline A (9.3 pM(-1)min(-1)and 170 pM(-1)min(-1)respectively). In addition, inhibition of cellular CDK4 caused increase of cellular levels of ATP, even though inhibition of CDK2 did not change it noticeably. These data suggest cellular CDK4/cyclin D1 activity is tightly associated with cellular ATP concentration. Also, analysis of phosphorylated serine/threonine sites on RB catalyzed by CDK4/cyclin D1 and CDK2/cyclin A showed significant differences in their preference of phosphorylation sites in RB C-terminal domain. Since RB is known to regulate various cellular proteins by binding and this binding is controlled by its phosphorylation, these data shown here clearly indicate significant difference in their biochemical properties between CDK4/cyclin D1 and CDK2/cyclin A affecting regulation of cellular RB function.

MeSH Terms

Adenosine Triphosphate/metabolism
Amino Acid Sequence
Baculoviridae/genetics
CDC2-CDC28 Kinases/genetics/isolation&purification/*metabolism
Cyclin A/genetics/isolation&purification/*metabolism
Cyclin D1/genetics/isolation&purification/*metabolism
Cyclin-Dependent Kinases/antagonists&inhibitors/genetics/isolation&purification/*metabolism
Human
Kinetics
Molecular Sequence Data
Phosphorylation
Protein Conformation
Protein p16/metabolism
Recombinant Proteins/genetics/isolation&purification/metabolism