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Tools to discriminate between targets of CK2 vs PLK2/PLK3 acidophilic kinases
M. Salvi1, E. Trashi1, G. Cozza1, A. Negro1, P.I. Hanson2, and L.A. Pinna1
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Supplementary Material

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 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.

Figure 4. Discrimination between acidiphilic kinase activities (Click to enlarge)

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.

Competing interests

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:

1.) Salvi, M., L. Cesaro, and L.A. Pinna. 2010. Variable Contribution Of Protein Kinases To The Generation Of The Human Phosphoproteome: A Global Weblogo Analysis. Biomol. Concepts 2:185-196.

2.) Salvi, M., S. Sarno, L. Cesaro, H. Nakamura, and L.A. Pinna. 2009. Extraordinary pleiotropy of protein kinase CK2 revealed by weblogo phosphoproteome analysis. Biochim. Biophys. Acta 1793:847-859.

3.) Pinna, L.A., and M. Ruzzene. 1996. How do protein kinases recognize their substrates?. Biochim. Biophys. Acta 1314:191-225.

4.) Meggio, F., and L.A. Pinna. 2003. One-thousand-and-one substrates of protein kinase CK2?. FASEB J. 17:349-368.

5.) Johnson, E.F., K.D. Stewart, K.W. Woods, V.L. Giranda, and Y. Luo. 2007. Pharmacological and functional comparison of the polo-like kinase family: insight into inhibitor and substrate specificity. Biochemistry 46:9551-9563.

6.) Hornbeck, P.V., J.M. Kornhauser, S. Tkachev, B. Zhang, E. Skrzypek, B. Murray, V. Latham, and M. Sullivan. 2012. PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res. 40:D261-D270.

7.) Songyang, Z., K.P. Lu, Y.T. Kwon, L.H. Tsai, O. Filhol, C. Cochet, D.A. Brickey, T.R. Soderling. 1996. A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1. Mol. Cell. Biol. 16:6486-6493.

8.) Salvi, M., E. Trashi, O. Marin, A. Negro, S. Sarno, and L.A. Pinna. 2012. Superiority of PLK-2 as α-synuclein phosphorylating agent relies on unique specificity determinants. Biochem. Biophys. Res. Commun. 418:156-160.

9.) Salvi, M., S. Sarno, O. Marin, F. Meggio, E. Itarte, and L.A. Pinna. 2006. Discrimination between the activity of protein kinase CK2 holoenzyme and its catalytic subunits. FEBS Lett. 580:3948-3952.

10.) Lin, Y., L.A. Kimpler, T.V. Naismith, J.M. Lauer, and P.I. Hanson. 2005. Interaction of the mammalian endosomal sorting complex required for transport (ESCRT) III protein hSnf7-1 with itself, membranes, and the AAA+ ATPase SKD1. J. Biol. Chem. 280:12799-12809.

11.) Sarno, S., H. Reddy, F. Meggio, M. Ruzzene, S.P. Davies, A. Donella-Deana, D. Shugar, and L.A. Pinna. 2001. Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 (‘casein kinase-2’). FEBS Lett. 496:44-48.

12.) Meggio, F., N. Grankowski, W. Kudlicki, R. Szyszka, E. Gasior E, and L.A. Pinna. 1986. Structure and properties of casein kinase-2 from Saccharomyces cerevisiae. A comparison with the liver enzyme. Eur. J. Biochem. 159:31-38.

13.) Hathaway, G.M., and J.A. Traugh. 1983. Casein kinase II. Methods Enzymol. 99:317-331.

14.) Venerando, A., M.A. Pagano, K. Tosoni, F. Meggio, D. Cassidy, M. Stobbart, L.A. Pinna, and A. Mehta. 2011. Understanding protein kinase CK2 mis-regulation upon F508del CFTR expression. Naunyn Schmiedebergs Arch. Pharmacol. 384:473-488.

15.) Meggio, F., M. Ruzzene, S. Sarno, M.A. Pagano, and L.A. Pinna. 2000. pCMB treatment reveals the essential role of cysteinyl residues in conferring functional competence to the regulatory subunit of protein kinase CK2. Biochem. Biophys. Res. Commun. 267:427-432.

16.) Battistutta, R., G. Cozza, F. Pierre, E. Papinutto, G. Lolli, S. Sarno, S.E. O'Brien, A. Haddach, K. Anderes, D.M. Ryckman, F. Meggio, and L.A. Pinna LA. 2011. Unprecedented selectivity and structural determinants of a new class of protein kinase CK2 inhibitors in clinical trials for the treatment of cancer. Biochemistry 50:8478-8488.

17.) Steegmaier, M., M. Hoffmann, A. Baum, P. Lénárt, M. Petronczki, M. Krssák, U. Gürtler, P. Garin-Chesa. 2007. BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo. Curr. Biol. 17:316-322.

18.) Pagano, M.A., J. Bain, Z. Kazimierczuk, S. Sarno, M. Ruzzene, G. Di Maira, M. Elliott, A. Orzeszko, G. Cozza, F. Meggio, and L.A. Pinna LA. 2008. The selectivity of inhibitors of protein kinase CK2: an update. Biochem. J. 415:353-365.

19.) Hanson, P.I., S. Shim, and S.A. Merrill. 2009. Cell biology of the ESCRT machinery. Curr. Opin. Cell Biol. 21:568-574.

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