Zorba Adelajda, Buosi Vanessa, Kutter Steffen, Kern Nadja, Pontiggia Francesco, Cho Young-Jin, Kern Dorothee
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, United States.
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, United States
Elife. 2014 May 27;3:e02667. doi: 10.7554/eLife.02667.
We elucidate the molecular mechanisms of two distinct activation strategies (autophosphorylation and TPX2-mediated activation) in human Aurora A kinase. Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation. We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays. We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX2(1-25). While autophosphorylation is the key regulatory mechanism in the centrosomes in the early stages of mitosis, allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules. The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here, may have implications for understanding regulation of other protein kinases.DOI: http://dx.doi.org/10.7554/eLife.02667.001.
我们阐明了人类极光激酶A（Aurora A kinase）中两种不同激活策略（自磷酸化和TPX2介导的激活）的分子机制。经典的别构激活发挥作用，即激活环磷酸化或TPX2与保守疏水凹槽的结合会使平衡大幅向活性构象移动。我们通过将X射线晶体学与功能测定相结合，在长寿命二聚体中证明分子间自磷酸化，从而解决了关于自磷酸化机制的争议。然后，我们通过活性测定以及去磷酸化的极光激酶A与TPX2（1 - 25）的结构域交换二聚体的晶体结构，研究了TPX2介导的别构激活。虽然自磷酸化是有丝分裂早期中心体中的关键调节机制，但TPX2对去磷酸化的极光激酶A的别构激活可能在纺锤体微管中起作用。本文提出的关于自磷酸化以及TPX2结合介导的别构激活的机制见解，可能对理解其他蛋白激酶的调节有启示意义。DOI: http://dx.doi.org/10.7554/eLife.02667.001
