Phosphorylation of threonine 210 and the role of serine 137 in the regulation of mammalian polo-like kinase.

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.