Identification of phosphorylated residues that affect the activity of the mitotic kinase Aurora-A

Abstract

The activity of the kinase Aurora-A (Aur-A) peaks during mitosis and depends on phosphorylation by one or more unknown kinases. Mitotic phosphorylation sites were mapped by mass spec sequencing of recombinant Aur-A protein that had been activated by incubation in extracts of metaphase-arrested Xenopus eggs. Three sites were identified: serine 53 (Ser-53), threonine 295 (Thr-295), and serine 349 (Ser-349), which are equivalent to Ser-51, Thr-288, and Ser-342, respectively, in human Aur-A. To ask how phosphorylation of these residues might affect kinase activity, each was mutated to either alanine or aspartic acid, and the recombinant proteins were then tested for their ability to be activated by M phase extract. Mutation of Thr-295, which resides in the activation loop of the kinase, to either alanine or aspartic acid abolished activity. The S349A mutant had slightly reduced activity, indicating that phosphorylation is not required for activity. The S349D mutation completely blocked activation, suggesting that Ser-349 is important for either the structure or regulation of Aur-A. Finally, like human Aur-A, overexpression of Xenopus Aur-A transformed NIH 3T3 cells and led to tumors in nude mice. These results provide further evidence that Xenopus Aur-A is a functional ortholog of human Aur-A and, along with the recently described crystal structure of human Aur-A, should help in future studies of the mechanisms that regulate Aur-A activity during mitotic progression.

Fig 1.

Identification of sites phosphorylated on Aur-A by M phase extracts. (A) Activation of recombinant Aur-A. His-tagged recombinant Aur-A protein was expressed in Sf9 cells in the presence of okadaic acid (Aur-A^OA, lane 1) or Ser/Thr kinase inhibitors (Aur-A^KI, lanes 2–4) and purified. Aur-A^KI samples were incubated with either G₂-arrested oocyte extract (lane 3) or M phase extract (lane 4), repurified on Ni-NTA beads, and assayed for in vitro kinase activity toward MBP. Samples were separated by SDS/PAGE, quantified (Upper), and visualized by autoradiography (Lower). (B) Location of phosphorylated residues. (C) Sequences surrounding phosphorylated sites.

Fig 2.

Effects of N-terminal truncation and of Ser-53, Thr-295, and Ser-349 mutations on Aur-A kinase activity. (A and B) Equimolar amounts of wild-type Aur-A and the indicated mutants were expressed in Sf9 cells in the presence of okadaic acid, purified, and assayed for in vitro kinase activity toward MBP. Autoradiograms of the ³²P-labeled MBP products are shown. (C) Activity of Aur-A mutants after incubation in M phase extract. His-tagged Aur-A proteins, either wild type or mutant, were expressed in Sf9 cells, dephosphorylated with λ-phosphatase, incubated with or without M phase extracts, repurified, and assayed for kinase activity toward MBP, as above.

Fig 3.

Effect of kinase inhibitors on the ability of M phase extracts to activate Aur-A. M phase extracts were first incubated in the absence or presence of individual kinase inhibitors, PKI (PKA inhibitor), roscovitine (cyclin B/cdc2 inhibitor), staurosporine (broad range Ser/Thr kinase inhibitor), and apigenin (CK2 inhibitor), and then tested for their ability to activate His-tagged Aur-A as in Fig. 2C.

Fig 4.

Thr-295 and Ser-349 mutations do not interfere with Cdh1-dependent destruction of Aur-A. M phase extracts containing cycloheximide were incubated with Cdh1 or Cdc20, as indicated, and mixed with [³⁵S]methionine-labeled Aur-A in vitro translation products. Calcium was added to initiate M phase exit, and samples were taken at the indicated times. Samples were analyzed by SDS/PAGE followed by autoradiography.

Fig 5.

Xenopus Aur-A is oncogenic. (A) Aur-A transforms NIH 3T3 cells. NIH 3T3 cells were transfected with empty vector, Aur-A, or catalytically inactive K169R Aur-A, and stably transfected cell lines were established. Cells from each line were plated on soft agar and assayed for growth 20 days later. Colonies were counted and compared in the bar graph (A), where efficiency of colony formation by Aur-A was normalized to 100%. (B) Cells overexpressing wild-type Aur-A are tumorigenic in nude mice. Cells (10⁶) from each of the cell lines described above were injected s.c. in nude mice. Two mice were injected with each cell line at two locations right behind the front leg, and two additional mice were injected at three locations with the three constructs per mouse.