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 microM, a value unusually high whereas CDK2/cyclin A was 23 microM, 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.
Highlights
Cell cycle dependent protein kinase (CDK) family proteins possess a serine/threonine protein kinase activity and phosphorylate serine or threonine residue in front of proline with a consensus sequence of (S/T) P (X) (K/R) (Endicott et al, 1999)
Each cyclin protein appears at a specific cell cycle phase, activates its partnering CDK (Sherr, 1994; Pavletich, 1999)
CDK4 forms a complex with type D cyclins such as D1 and D2 and the cellular amount of cyclin D1 reaches to the highest level when cells are in Mid-G1, CDK4/cyclin D1 activity exists in mid G1 phase
Summary
Cell cycle dependent protein kinase (CDK) family proteins possess a serine/threonine protein kinase activity and phosphorylate serine or threonine residue in front of proline with a consensus sequence of (S/T) P (X) (K/R) (Endicott et al, 1999). Their major cellular role is suggested to be involved in the positive control of cell cycle progress. Each cell cycle dependent kinase needs to form a heteroduplex with a specific cyclin protein to become an active kinase (Sherr, 1994). Each cyclin protein appears at a specific cell cycle phase, activates its partnering CDK (Sherr, 1994; Pavletich, 1999). We carried out comparative study on the enzymatic properties of the purified CDK4/cyclin D1 and CDK2/cyclin A
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