Evidence toward a dual phosphatase mechanism that restricts Aurora A (Thr-295) phosphorylation during the early embryonic cell cycle.
The mitotic kinase Aurora A (AurA) is regulated by a complex network of factors that includes co-activator binding, autophosphorylation, and dephosphorylation. Dephosphorylation of AurA by PP2A (human, Ser-51; Xenopus, Ser-53) destabilizes the protein, whereas mitotic dephosphorylation of its T-loop (human, Thr-288; Xenopus, Thr-295) by PP6 represses AurA activity. However, AurA(Thr-295) phosphorylation is restricted throughout the early embryonic cell cycle, not just during M-phase, and how Thr-295 is kept dephosphorylated during interphase and whether or not this mechanism impacts the cell cycle oscillator were unknown. Titration of okadaic acid (OA) or fostriecin into Xenopus early embryonic extract revealed that phosphatase activity other than PP1 continuously suppresses AurA(Thr-295) phosphorylation during the early embryonic cell cycle. Unexpectedly, we observed that inhibiting a phosphatase activity highly sensitive to OA caused an abnormal increase in AurA(Thr-295) phosphorylation late during interphase that corresponded with delayed cyclin-dependent kinase 1 (CDK1) activation. AurA(Thr-295) phosphorylation indeed influenced this timing, because AurA isoforms retaining an intact Thr-295 residue further delayed M-phase entry. Using mathematical modeling, we determined that one phosphatase would be insufficient to restrict AurA phosphorylation and regulate CDK1 activation, whereas a dual phosphatase topology best recapitulated our experimental observations. We propose that two phosphatases target Thr-295 of AurA to prevent premature AurA activation during interphase and that phosphorylated AurA(Thr-295) acts as a competitor substrate with a CDK1-activating phosphatase in late interphase. These results suggest a novel relationship between AurA and protein phosphatases during progression throughout the early embryonic cell cycle and shed new light on potential defects caused by AurA overexpression.
From the Department of Biology, Indiana University, Bloomington, Indiana 47405-7003.
Qing
Q
Kang
From the Department of Biology, Indiana University, Bloomington, Indiana 47405-7003.
Jeyaraman
J
Srividhya
From the Department of Biology, Indiana University, Bloomington, Indiana 47405-7003.
Joseph
J
Ipe
From the Department of Biology, Indiana University, Bloomington, Indiana 47405-7003 jpomeren@indiana.edu.
Joseph R
JR
Pomerening
D000818:Animals; D064096:Aurora Kinase A / Q000235:genetics / Q000378:metabolism*; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D010766:Phosphorylation / Q000502:physiology; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D029867:Xenopus Proteins; D013912:Threonine; D064096:Aurora Kinase A; D016203:CDC2 Protein Kinase; D054637:Dual-Specificity Phosphatases
Aurora A; CDK1; Cell Cycle; Cell Signaling; Cyclin-dependent Kinase (CDK); Early Embryonic Cell Cycle; Embryo; Phosphatase