FIGURE 3.

Plk3 phosphorylates HIF-1α on Ser⁵⁷⁶ and Ser⁶⁵⁷. A, both Plk3 and Plk3-K91R were expressed in Sf9 cells and purified using NTA-Ni resin. Purified Plk3 and Plk3-K91R were assayed for their kinase activities toward HIF-1α in vitro in a kinase buffer containing [γ-³²P]ATP. A representative autoradiogram is shown. B, kinase assays were carried out in the presence of various components as indicated. After the reaction, the samples were analyzed on SDS-PAGE followed by Coomassie Blue staining. A lane of “hot” sample (autoradiogram on the left) that ran on the same gel is also shown. C, HIF-1α sequence representation shows the relative positions of serine phosphorylation sites Ser⁵⁷⁶ (upper panel) and Ser⁶⁵⁷ (lower panel) to HIF-1α domains. ODDD, oxygen-dependent degradation domain; N-TAD, N-terminal transactivation domain; NES, nuclear export signal; NLS, nuclear localization signal; C-TAD, C-terminal transactivation domain. The nuclear export signal in the lower panel is highlighted by underlining. Ser⁶⁴¹ and Ser⁶⁴³ are known to be phosphorylated by ERKs. D, amino acid alignment is shown of HIF-1α molecules from different species in the region that were targeted by Plk3. The conserved serine residues are highlighted.