Ran modulates spindle assembly by regulating a subset of TPX2 and Kid activities including Aurora A activation

Abstract

Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin β, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 we identified nuclear targeting information, microtubule-binding and Aurora A binding activities. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevented Aurora A activation and recruitment to microtubules. In addition we identified nuclear targeting information in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly.

Characterization of the TPX2 Domains Involved in Microtubule Nucleation and Spindle Assembly inXenopusEgg Extracts

“…These experiments indicated the presence of a microtubule binding activity in the N-terminal half of TPX2 as previously found for human TPX2 (Trieselmann et al, 2003). We then decided to use a novel method called fluorescence loss after photoconversion (FLAP; our unpublished data) to measure the affinities of the different GFP-fragments for microtubules.…”

RASSF1A interacts with and activates the mitotic kinase Aurora-A

“…The temporal and spatial distribution of Aurora-A activity is at least partially controlled by Aurora-A activators. For instance, activation of Aurora-A by TPX2 is required for bipolar-spindle assembly (Trieselmann et al, 2003;Tsai et al, 2003), whereas activation of Aurora-A by Ajuba is essential for mitotic entry . Activation of Aurora-A by HEF-1 in a nonmitotic pathway is mandatory for the disassembly of the primary cilium (Pugacheva et al, 2007).…”

“…Several activators of Aurora-A have been identified, which include TPX2, Ajuba, HEF1, Bora and PAK1 (Kufer et al, 2002;Hirota et al, 2003;Crane et al, 2004;Satinover et al, 2004;Pugacheva and Golemis, 2005;Zhao et al, 2005;Hutterer et al, 2006). Activation of Aurora-A by TPX2 is required for bipolar-spindle assembly (Trieselmann et al, 2003;Tsai et al, 2003), whereas activation of Aurora-A by Ajuba is essential for mitotic entry . It is tempting to propose that Aurora-A activators may at least partially control the temporal and spatial distribution of Aurora-A activity along the cell division cycle.…”

TPX2 in human clear cell renal carcinoma: Expression, function and prognostic significance

“…Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein, and functions to regulate the construction of the mitotic spindle by promoting microtubule nucleation from chromatin and stabilization of the spindle microtubules (7,8). It has been demonstrated that TPX2 overexpression induces the amplification of centrosomes and results in DNA polyploidy (9).…”