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

Question

Cyclin-dependcnt 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 phosphory lation in the function of Cdk2. you purify nonphosphorylated and Civl-phosphorylated Cdk2. You mix these two forms of Cdk2 and cyclin A in various combinations with 32P-ATP and assay for phosphorylation of histone H1 (see Figure below). You also measure the binding affinity of various forms of Cdk2 for ATP, ADP, cyclin A, and histone HI (see Table below). Figure: Phosphorylation of histone H1 by various combinations of Cdk2 and cyclin A. The amount of radioactive phosphate (32P) attached to H1 in lanes 1 and 3 are .3% and .2% (respectively) of that in lane 5. Table: the observed dissociation constants (Kd values) of Cdk2 for ATP, ADP, cyclin A, and histone H1. Based on the Figure, what is required for Cdk2 to phosphorylate histone H1 efficiently? How do the requirements identified in part a) specifically affect the function of Cdk2 relative to its target, histone HI? (Hint: base your answer on the information in both the Figure and the Table) The usual intracellular concentrations of ATP and ADP are in the range 0.1-1 m.M. 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 arc 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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