Abstract
The mammalian polo-like kinase (Plk) plays a critical role in M-phase progression. Plk is phosphorylated and activated by an upstream kinase(s), which has not yet been identified in mammalian cells. Phosphopeptide mapping and phosphoamino acid analyses of Plk labeled in vivo and phosphorylated in vitro by Xenopus polo-like kinase kinase-1 (xPlkk1) or by lymphocyte-oriented kinase, its most closely related mammalian enzyme, indicate that Thr-210 is a major phosphorylation site in activated Plk from mitotic HeLa cells. Although the amino acid sequence surrounding Ser-137 is similar to that at Thr-210 and is conserved in Plk family members, Ser-137 is not detectably phosphorylated in mitotic mammalian cells or by xPlkk1 in vitro. Nevertheless, the substitution of either Thr-210 or Ser-137 with Asp (T210D or S137D) elevates the kinase activity of Plk. The kinase activity of the double mutant S137D/T210D is not significantly different from that of T210D or S137D, demonstrating that substitution of both residues does not have an additive effect on Plk activity. Expression of the S137D mutant construct arrested HeLa cells in early S-phase with slightly separated centrosomes, whereas cells expressing wild type and T210D were arrested or delayed in M-phase. These data indicate that the Ser-137 may have an unexpected and novel role in the function of Plk.
Highlights
Progression through the mammalian cell cycle depends on the periodic control of various cyclin-dependent kinases
Mammalian polo-like kinase (Plk) Is Phosphorylated and Activated during Mitosis—To analyze the modification and activation of Plk during the cell cycle, HeLa cells were synchronized in G1/S phase with mimosine and transferred into fresh medium with nocodazole to allow entry into M phase
We have demonstrated that Thr-210 is the major in vivo phosphorylation site of activated mammalian Plk during M phase
Summary
Polo-like kinase; Plx, Xenopus Plk; xPlkk, Xenopus Plk kinase-1; LOK, lymphocyte-oriented kinase; HA, hemagglutinin; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; PBS, phosphate-buffered saline; WT, wild type; HPLC, high pressure liquid chromatography. The original studies on Thr-210 in Plk demonstrated that substitution of aspartate at this site significantly elevates protein kinase activity [20] This residue is in the activation loop between protein kinase subdomains VII and VIII, and phosphorylation within this loop results in the activation of several kinases including Cdc and Mek1 [1, 21]. Substitution of Ser-137, which is embedded in a similar sequence as Thr210, with aspartate activates Plk, this residue is not phosphorylated in vitro by xPlkk or in vivo in mitotic cells and does not appear to be involved in activation of Plk during mitosis. Experiments with Ser-137 mutants suggest that this site has the potential to have a biologically significant role in regulating Plk activity during other stages of the cell cycle
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