Collectively, these results indicate that the combined use of CX-4945 and BI 2536 effectively discriminate between CK2 and PLK2/PLK3.
To probe the reliability of this approach, we focused our attention on CHMP3, a protein of the ESCRT-III complex that is required for multivesicular body (MVB) formation (19). CHMP3 phospho S200, identified in different cell lines in large scale mass spectrometry experiments (see www.phosphosite.org for references), is adjacent to a very acidic cluster (PPGAMAApSEDEEEEE) and the kinase responsible has not been identified. As shown in Figure 4, CHMP3 immunoprecipitated from 293T cells is phosphorylated by a kinase that coimmunoprecipitates with the protein. To gain information about the kinase responsible for CHMP3 S200 phosphorylation, use of GTP (instead of ATP) as a phosphate donor and inhibitors BI 2536 and CX-4945 was exploited. The results shown in Figure 4 show that GTP is able to replace ATP in the phosphorylation reaction and that phosphorylation is completely refractory to high concentrations of BI 2536, while potently inhibited by a low concentration of CX-4945. Note that phosphorylation of CHMP3 is completely abrogated by the mutation of a phosphoacceptor residue (S200), which has been shown to be phosphorylated in vivo (see inset in Figure 4). Taken together, these results provide a clear-cut demonstration that the kinase that coimmunoprecipites with and phosphorylates CHMP3 is CK2, not PLK2/PLK3, suggesting that CK2 is also the kinase responsible for CHMP3 phosphorylation in vivo.
In conclusion, the combined use of CX-4945 and BI 2536, in conjunction with GTP as phosphate donor, allows a neat discrimination between acidic phosphosites generated either by CK2 or by PLK2/PLK3.
We thank The Molecular Modeling Section (MMS), coordinated by Professor S. Moro (Padova, Italy). This work was supported by University of Padova Progetto Ateneo CPDA089591/08 to M.S. and by Associazione Italiana per la Ricerca sul Cancro (AIRC) to L.A.P.
The authors declare no competing interests.
Address correspondence to Mauro Salvi, Department of Biomedical Sciences, University of Padova, V.le G. Colombo 3, 35131 Padova, Italy. Email: [email protected]
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