Figure 2.

SIRT2 is phosphorylated at serine 331 in cells and interacts with Cdk complexes. (A) HEK293 cells were transiently transfected with plasmids encoding 10 μg HA-SIRT2, 10 μg HA-SIRT2-S331A, and 5 μg p27, as indicated, and metabolically labeled with [³²P]orthophosphate. SIRT2 was then immunoprecipitated via its HA-tag. The top shows an autoradiography, the bottom shows Western blots for HA-SIRT2 (mAb 3F10) and p27^KIP1 (C-19). (B) N-TAP-SIRT2 was purified from HEK293 cells. The fragmentation spectrum of parent ion m/z 1144.5163, 2 + (mass accuracy, 2.8 ppm) is shown. Mascot searches against the Uniprot database identified that this peptide unambiguously phosphorylated at position Ser-331 with a Mascot ion score of 68 (Expectance value, 1.9*E-4). The phosphopeptide was sequenced eight times from two unique overlapping peptide sequences because of the presence of miscleaved tryptic sites. The y-ion fragmentation ladder starting from the C terminus is shown in red. The phosphorylation site was mapped by the detection of the y⁺ ₃ fragment ion in the linear ion trap at m/z 411.3, which corresponds to the sequence pSPK. (C) HEK293 cells were transiently transfected with a plasmid encoding 10 μg HA-SIRT2 and treated with 200 μM hydroxyurea, 400 ng/ml nocodazole, or 200 ng/ml colcemide for 20 h and then metabolically labeled with [³²P]orthophosphate. The SIRT2 analysis was performed as described in A. White lines indicate that intervening lanes have been spliced out. (D) Bacterially expressed and purified GST-SIRT2 or GST was phosphorylated by recombinant cyclin E–Cdk2 in the presence or absence of ATP as indicated. Half of the reactions were analyzed by Coomassie blue (CB) staining, the other half were subjected to Western blot analysis using the mAb 6B5^P that is specific for Ser-331 phosphorylated SIRT2. (E) HEK293 cells were treated as indicated with hydroxyurea (HU) for 16 h and with roscovitine (Rosc) for the last 2 h. SIRT2 was immunoprecipitated using the polyclonal serum 748 from RIPA lysates of 6 × 10⁶ cells. The immunoprecipitated proteins were phosphatase or mock treated and the proteins were analyzed by Western blotting with the indicated mAbs. Equal aliquots of the lysates were taken before immunoprecipitation and α-tubulin expression was determined. (F) Primary HDFs were serum starved and then treated with 10% serum for the indicated times. Parallel samples were analyzed for BrdU incorporation, indicating that the cells started to enter S-phase 18 h after serum addition. SIRT2 was immunoprecipitated from lysates of roughly 4 × 10⁶ cells per sample and detected with the indicated mAbs. (G) HEK293 cells were cotransfected with Flag-SIRT2, cyclin E, and Cdk2 construct. Their expression was visualized in total cell lysates by Western blotting (right). Flag-SIRT2 was immunoprecipitated and the association of coexpressed cyclin E and Cdk2 analyzed by Western blotting (left). (H) The experimental setup and analysis was as in D. Constructs expressing Flag-SIRT2, HA-Cdk5, and myc-p35 were used. (I) Low-stringency lysates of P14 mouse brain were generated and Cdk5 was immunoprecipitated. Coimmunoprecipitated SIRT2 was visualized by Western blotting. (J) The experiment was performed as in F. SIRT2 was immunoprecipitated with two different polyclonal antisera (T749 and T809). For a control, p35 and Cdk5 were immunoprecipitated. Cdk5 coimmunoprecipitated with SIRT2 was monitored by Western blotting.