Cyclin-dependent protein kinase 2 (Cdk2) regulates critical events in the progre

Question

Cyclin-dependent protein kinase 2 (Cdk2) regulates critical events in the progression of the cell cycle in mammalian cells. Cdk2 can form a complex with cyclin A and can be phosphorylated by another protein kinase, Civl, to produce P-Cdk2. To determine the roles of cyclin A and phosphorylation in the function of Cdk2, you purify nonphosphorylated and Civ1- phosphorylated Cdk2. You mix these tvsio forms of Cdk2 and cyclin A in various combinations with 32P-ATP and assay for phosphorylation of histone H1 (Figure 3-32). You also measure the binding affinity of various forms of Cdk2 for ATR ADR cyclin A, and histone H1 (Table 3-4).!

A. From Figure 3-32, what is required for Cdk2 to phosphorylate histone H1 efficiently?!

B. How do the requirements identified in part A specifically affect the function of Cdk2 relative to its target, histone H1 (Table 3-4 and Figure 3-32)?!

C. The usual intracellular concentrations of ATP and ADP are in the range 0.1 to 1 mM. Assume that the binding of cyclin A to Cdk2 or P-Cdk2 does not alter the affinities of either form of Cdk2 for ATP and ADP Is it likely that the observed changes in affinity for ATP and ADP are important for Cdk2 function? Why or why not?!

Explanation / Answer

a) Look at lanes 1,3, and 5. Based on the information in those lanes, we need the phosphorylated form of Cdk2 (P-Cdk2) bound to cyclin A to efficiently phosphorylate H1. b) The phosphorylated form of Cdk2 results in observed, though not very strong binding to H1 (Kd = 100; remember that binding affinity is the reciprocal of Kd), while unphosphorylated Cdk2 does not result in any binding (Kd is undefined). Once Cdk2 binds cyclin A, there is considerable binding to H1 (Kd = 1.0). The maximum binding affinity is observed when P-Cdk2 is bound to cyclin A (Kd = 0.7). c) No. The relative binding affinities of Cdk2 for ATP and ADP are about the same in the unphosphorylated and phosphorylated form. In other words, the ratio of Kd for ADP to ATP in Cdk2 and P-Cdk2 are almost exactly the same (Kd ratio = 5.6).

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