Structural Determinants of Protein Kinase CK2 Regulation by Autoinhibitory Polymerization

CK2 is a Ser/Thr protein kinase essential for cell viability whose activity is anomalously high in several cancers. CK2 is a validated target for cancer therapy with one small molecule inhibitor in phase I clinical trials. This enzyme is not regulated by mechanisms common to other protein kinases, and how its activity is controlled is still unclear. We present a new crystal structure of the CK2 holoenzyme that supports an autoinhibitory mechanism of regulation whereby the β-subunit plays an essential role in the formation of inactive polymeric assemblies. The derived structural model of (down)­regulation by aggregation contributes to the interpretation of biochemical and functional data and paves the way for new strategies in the modulation of CK2 activity and for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the β regulatory subunits

Putative PLK2-substrate localization (A) and functional (B) analysis.

<p>Subcellular localization (A) and functional analysis (B) for each protein have been assigned using GeneCoDis3 webserver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111018#pone.0111018-TabasMadrid1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111018#pone.0111018-NogalesCadenas1" target="_blank">[36]</a>.</p

Workflow for PLK2 peptide substrate identification.

<p>Workflow for PLK2 peptide substrate identification.</p

Logarithmic distribution of quantification values.

<p>A. Distribution of Log2 ratios relative to all phosphopeptides identified in this study. B. Distribution of Log2 ratios relative to all non-phosphopeptides.</p

In vitro phosphorylation of recombinant proteins by PLK2.

<p>A. Increasing amounts of recombinant GST-HDGF (lane 2, 50 ng; lane 3, 100 ng; lane 4 250 ng; lane 5 and 6, 500 ng) were incubated in radioactive mixture in presence (lanes 1–5) of absence (lane 6) of PLK2 recombinant kinase as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111018#s2" target="_blank">Materials and Methods</a>. B–D Increasing amounts of purified proteins (lane 2, 100 ng; lane 3, 250 ng; lane 4 and 5, 500 ng) were incubated in radioactive mixture in presence (lanes 1–4) of absence (lane 5) of PLK2 recombinant kinase as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111018#s2" target="_blank">Materials and Methods</a>. Samples were loaded on SDS-PAGE, stained with colloidal coomassie and ³³P incorporation was analyzed by PhopshorImager. A- Hepatoma-derived growth factor. B- Aromatic L-amino acid decarboxylase (Dopa decarboxylase). C- Annexin A2. D- Prostaglandin E synthase 3 (PTGES3).</p

List of phosphosites identified in this study as PLK2 substrates that are present in Phosphosite database.

<p>List of phosphosites identified in this study as PLK2 substrates that are present in Phosphosite database.</p

Two-sample logo analysis of phosphosites generated by individual kinases vs. random S/T proteome.

<p>PLK2 phosphopeptides identified in this paper (A) or <i>bona fide</i> CK2 (B), PLK1 (C), and CK1δ (D) substrates collected from PhosphositePlus database, have been analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111018#s2" target="_blank">Materials and Methods</a>.</p

Synthesis and Properties of a Selective Inhibitor of Homeodomain–Interacting Protein Kinase 2 (HIPK2)

<div><p>Homeodomain-interacting protein kinase 2 (HIPK2) is a Ser/Thr kinase controlling cell proliferation and survival, whose investigation has been hampered by the lack of specific inhibitors able to dissect its cellular functions. SB203580, a p38 MAP kinase inhibitor, has been used as a tool to inhibit HIPK2 in cells, but here we show that its efficacy as HIPK2 inhibitor is negligible (IC₅₀>40 µM). In contrast by altering the scaffold of the promiscuous CK2 inhibitor TBI a new class of HIPK2 inhibitors has been generated. One of these, TBID, displays toward HIPK2 unprecedented efficacy (IC₅₀ = 0.33 µM) and selectivity (Gini coefficient 0.592 out of a panel of 76 kinases). The two other members of the HIPK family, HIPK1 and HIPK3, are also inhibited by TBID albeit less efficiently than HIPK2. The mode of action of TBID is competitive with respect to ATP, consistent with modelling. We also provide evidence that TBID is cell permeable by showing that HIPK2 activity is reduced in cells treated with TBID, although with an IC₅₀ two orders of magnitude higher (about 50 µM) than in vitro.</p></div

Lorenz curves, Gini coefficients and hit rates for TBID and TBI.

<p>For details see experimental section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089176#pone.0089176-Graczyk1" target="_blank">[25]</a>. Lorenz curves were drawn from the selectivity profile of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089176#pone-0089176-g004" target="_blank">Figure 4</a> and from analogous data published in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089176#pone.0089176-Pagano1" target="_blank">[19]</a> for TBID and TBI, respectively.</p

IC₅₀ (µM) of 2-aryl-4,5,6,7-tetrabromoisoindoline-1,3-dione derivatives (Figure 1) for HIPK2, CK2, PIM1, CK1; TBB and TBI values are drawn from [19].

<p>n.d. = not determined.</p