Fig. 2

CK2 phosphorylates Ty1 integrase in vitro and is involved in its phosphorylation in vivo. A Full-length IN (200 ng), IN_578–635 (IN_C) (500 ng) or Maf1 (200 ng) expressed in E. coli were subjected to in vitro radioactive phosphorylation assays with purified yeast CK2 holoenzyme. Incorporation of γ³²P was detected by autoradiography (left panel), the loading of the recombinant proteins was analyzed by Coomassie blue staining (right panel). MW: protein marker. Full length IN, IN_C and Maf1 are indicated with an asterisk (*). B Mapping of CK2 phosphorylation sites in Ty1 IN. A schematic representation of IN with the N-terminal (NTD), the catalytic core (CCD) and the C-terminal domain (CTD) is depicted [25]. Starting and ending residues for the CTD, the IN_C sequence and Ckb2 binding site are indicated. All amino acids within a CK2 consensus site listed in Table S4A (Netphos 3.1 analysis) are indicated with an asterisk (*). The 12 amino acids potentially phosphorylated by CK2 in the endogenous IN, based on Phosphogrid database, are shown in bold (Table S4B). C In vitro kinase assays of IN phosphomutants. Left: Autoradiography (incorporation of γ³²P) and Coomassie blue staining of WT and mutant IN proteins purified from E. coli (200 ng). MW: protein marker. Right: Mutations preventing phosphorylation of specific residues of IN shown in panel B are indicated by the absence of a vertical line in the representation of IN mutants M1 to M6. D Phosphorylation of ectopically IN expressed alone is not dependent on CK2 in vivo. Total protein extracts from ste12Δ or cka2Δ cbk2Δ ste12Δ cells expressing WT or IN^M6 (as indicated in panel C) from pTet-off-IN plasmids were separated by SDS-PAGE in the absence (upper panel) or the presence of Phos-Tag™ (lower panel). Pgk1 is a loading control. The presence of IN and Pol intermediates produced from endogenous Ty1 elements was detected by Western blot using rabbit anti-IN polyclonal antibodies. The black circle corresponds to the dephosphorylated form of IN. E Phosphorylation of IN produced from an ectopic Ty1 element depends in part on CK2 in vivo. Western blot analysis as described in panel D of total protein extracts prepared from cells expressing ectopic Ty1 or Ty1^M6 mutant from pTet-off plasmids as indicated. Asterisks indicate Pol intermediates from endogenous and ectopic Ty1 elements. IN expressed from pTet-off-IN was used as a control. In Phos-Tag™ SDS-PAGE, the smear above IN indicates the presence of several forms of phosphorylated IN or Pol intermediates. A similar profile was reproduced with cell extracts from 3 independent experiments