A peculiar characteristic of protein kinase CK2 is its ability to use GTP as a phosphodonor substrate almost as efficiently as ATP (13). This property has been utilized to highlight the CK2-dependent phosphoproteome in cellular lysates (14). In Figure 1C, we compared the ability of PLK2, PLK3, and CK2 to use either ATP or GTP in the phosphorylation reaction. As expected, GTP can effectively replace ATP for CK2 activity, but this was not the case for PLK2 and PLK3. Therefore using GTP allows discrimination between the activity of CK2 and PLK2/PLK3 kinases. Figure 1D analyzes the possibility that PLK2 and PLK3 are redox-regulated enzymes: the addition of the reducing agent DTT shows a strong increase (up to six times) of α-casein phosphorylation by both kinases (Figure 1D). In silico analysis for cysteines that potentially can form intra or intermolecular disulfide bridge and that are conserved in both PLK2 and PLK3 kinases disclosed two cysteines located in the ATP binding cleft (Cys 96 and 162 for PLK2; Cys 76 and 142 for PLK3) and one in the activation loop (PLK2 Cys 241, PLK3 Cys 221) (Figure S2). Intriguingly these cysteines are conserved also in PLK1 (Cys 67, Cys 133, and Cys 212), highlighting the possibility that this group of kinases presents a similar mechanism of redox regulation.
Therefore, optimal buffer conditions for both PLK2 and PLK3 kinases are 50 mM Tris pH 7,5, 10 mM Mg2+, 1 mM DTT, and no NaCl. This buffer is the same as utilized for CK2, except for the presence of DTT since the activity of this kinase is insensitive to reducing agents (15).
Specific inhibitors could represent useful tools to discriminate between targets affected by either CK2 or PLK2/PLK3. Therefore CK2 inhibitor 4,5,6,7-tetrabromo-1H-benzotriazole (TBB) and the recently developed CK2 inhibitor CX-4945, which is in Phase I clinical trials for treating cancer (16), and BI 2536, a potent inhibitor of PLK kinases (17) were compared for their ability to inhibit CK2, PLK2, and PLK3. TBB is the most widely used CK2 inhibitor and its selectivity has been previously tested at 1 and 10 µM on a panel of 70 kinases which didn't include PLK2/PLK3 kinases (18). Selectivity of CX-4945 has been tested at a single concentration on a panel of 235 kinases, including PLK2 and PLK3, which were only slightly inhibited (≤10% at 0,5 µM) (16). Figure 2A and 2B show the effect of TBB and CX-4945 on kinase activities. TBB was not useful in discriminating between CK2 and PLK since PLK2 and PLK3 were inhibited with a similar IC50 (Figure 2A). In contrast CX-4945 is almost ineffective on PLK2 and PLK3 up to 200 nM concentration while CK2 at this concentration is totally inhibited (Figure 2B). Figure 2C shows the effects of PLK inhibitor BI 2536. The selectivity of this compound against CK2 has never been tested. BI 2536 is absolutely specific, giving clear-cut results: CK2 is fully refractory to its inhibition up to concentrations of 10 µM (not shown), while PLK2 and PLK3 activities are completely suppressed by sub-micromolar concentrations of BI 2536. The structural basis for the cross-selectivity between the two inhibitors could be explained by in silico analysis. The binding mode of BI 2536 is conserved both in PLK2 and PLK3; the sequence similarity of the three polo-like kinases at 4.5Å from BI 2536 is about 80% and all the residues interacting with BI 2536 are conserved. On the other hand, the PLK and CK2 ATP binding clefts are very different (Figure 3A); the CK2 cavity is more hydrophobic and smaller, due to bulkier residues. PLK2/3 present Ala 109/89, Leu 159/139, Cys 96/76, Leu 88/68, Cys 162/142, while in CK2 the corresponding residues are Val 66, Phe 113, Val 51, Leu 59, Val116. For this reason, in the CK2 active site there is not enough space to accommodate BI 2536; in particular CK2 Val 66 and Val 53 fill up the space for of BI 2536 7R-ethyl group, thus preventing the binding of the inhibitor to the hinge region.
On the other hand, the selective CK2 inhibitor CX-4945 binds to the PLK2/3 active site much less efficiently, since a point mutation study on CK2 has revealed that the inhibitor binding is largely dependent on hydrophobic residues, notably V66 and I174 (16), which are replaced in PLK2/3 by smaller and less hydrophobic residues. Moreover, PLK2/3 cannot host the stabilizing water molecule due to the presence of Arg 165/145 instead of Asn 118 in CK2 (Figure 3B)