Figure 3.

BRCA2_48–284 contains conserved CDKs and PLK1 phosphorylation sites and two PLK1 docking sites. (A) BRCA2_48–284 conservation profile, calculated as in Figure 1, shows two conserved patches: BRCA2_63-93 and BRCA2_164–230. These patches contain predicted as well as experimentally identified CDKs and PLK1 phosphorylation sites, marked by black boxes. (B,D) Alignments of the human, mouse, rat, chicken and zebra fish BRCA2 sequences corresponding to the regions boxed in (A) illustrate the conservation of the reported CDKs and PLK1 phosphorylation sites. Arrows point to the CDKs (black) and PLK1 (red) phosphorylation sites in these regions: pThr77 [7,59], pSer193 [7,59,60], pThr207, pThr219 and pThr226 [57]. (C) NMR analysis identified four PLK1-dependent phosphosites: pSer193, pThr207, pThr219 and pThr226 [57]. ¹H-¹⁵N SOFAST-HMQC spectra of BRCA2_167–260 (50 μM) were recorded before (black) and after (red) 12 hrs of in vitro phosphorylation by PLK1 (150 nM). (E) Isothermal Titration Calorimetry (ITC) thermogram revealed binding of a BRCA2 peptide containing pThr207 to the PBD domain of PLK1 (PLK1_PBD; [57]). (F) 3D structure of BRCA2_{200–209(pThr207)} bound to PLK1_PBD identified critical intermolecular interactions involving BRCA2 Ser206 and pThr207 [57]. In BRCA2_48–284, two PLK1 docking sites were identified, centered on either pThr77 [60] or pThr207 [57]. The X-ray structure of BRCA2_{199–210(pThr207)} bound to PLK1_PBD was recently solved (PDB code: 6GY2). Within BRCA2_{199–210(pThr207)} (grey, blue and salmon), side chains of BRCA2 Ser206 (blue) and pThr207 (salmon) directly bind to the two Polo-box motifs of the PBD (green), and drive the specificity of the interaction. PBD residues in interaction with pThr207 and Ser206 are displayed as green sticks, and red dotted lines mark the hydrogen bonds involved in these interactions.