TPX2

Targeting protein for Xklp2 is a protein that in humans is encoded by the TPX2 gene. It is one of the many spindle assembly factors that play a key role in inducing microtubule assembly and growth during M phase.

Key domains of TPX2

TPX2 has been reported to have two NLS-containing domains that mediate its localization to microtubules; one in the amino-terminal domain, and the other in the carboxy-terminal domain. In addition to an NLS, the carboxy-terminal domain of TPX2 consists of tandem repeats that cover over two-thirds of the protein and are computationally predicted to consist of predominantly alpha-helical content. This region can be further divided into five clusters of conserved residues separated by unstructured regions: α3-7. α3-6 all contain a central α-helical region that is followed by a characteristic "FKARP" motif. α7 is longer and exhibits a long α-helical stretch that is computationally predicted to form a coiled coil. Lastly, the final 35 amino acids of the carboxy-terminus of TPX2 are responsible for interacting with tetrameric kinesin Eg5.
TPX2 contains one KEN box motif at amino acid 87 and three D-box motifs at amino acids 119, 341, and 708. Both motif types have been suspected to be important in regulation and degradation of TPX2 by the APC/C, as typically mutations in these motifs render substrates resistant to ubiquitination by the APC/C. However, in vitro ubiquitination assays have shown that only the first 83 amino acids of the N-terminal region of TPX2 along with the KEN box are pertinent for recognition by Cdh1, an activator of the APC/C.

Role in microtubule assembly

TPX2 has been shown in several biochemical assays to behave as a microtubule-associated protein and co-localize with spindle microtubules during M-phase. It plays a role in microtubule nucleation and is regulated by importin proteins.
TPX2 serves as a complement, depleting importin α affinity in order to allow RanGTP-induced microtubule nucleation. This has been demonstrated both in vitro in Xenopus laevis egg extracts, and with the human homologue in vivo in HeLa cells. TPX2 is also important in activating and recruiting Aurora A kinase, a kinase responsible for phosphorylating TPX2 and essential for cell proliferation. In the presence of nuclear import factor importin α, TPX2 is bound and prevented from binding Aurora A kinase, though it is still able to bind microtubules via its amino-terminal domain. This leads to inhibition of M phase microtubule nucleation. In contrast, TPX2 is freed from inhibition by displacement of importin α via RanGTP, though RanGTP is not required for free TPX2 activity, as TPX2 has been shown to induce microtubule assembly in the absence of exogenous and depletion of endogenous RanGTP. This suggests that TPX2 is downstream of RanGTP activity, but whether TPX2 is directly regulated by RanGTP still remains to be determined.
The mechanism by which TPX2 promotes microtubule nucleation has yet to be determined. One proposed mechanism has been based on TPX2's role in directly suppressing tubulin subunit off-rates at the microtubule tip during microtubule assembly and disassembly, verified by fluorescence microscopy. This is made possible partially by TPX2's role in sequestering free tubulin subunits and nucleating small multi-subunit tubulin complexes, which inadvertently also slows the rate of growth by decreasing the effective free tubulin concentration. TPX2's stabilization of the microtubule in its polymer form therefore contributes to microtubule nucleation. Computational simulations speculate that TPX2 suppresses tubulin subunit kinetics at the microtubule tip by randomly increasing the bond stability between adjacent tubulin subunits.
In addition, TPX2 has been shown to be important in chromatin-dependent spindle assembly. Even with duplicated centrosomes, TPX2 has been demonstrated to be required for formation of a stable bipolar spindle with overlapping antiparallel microtubule arrays. More specifically, TPX2 contributes to microtubule branching during spindle assembly by cooperating with augmin in order to amplify microtubule mass and preserve its polarity. Branching nucleation by TPX2 is observed without RanGTP, though more fan-shaped microtubule structures are formed when both RanGTP and TPX2 are present. The rate of branched formation is also enhanced in the presence of both components compared to Ran alone.
The TPX2 region necessary for branching microtubule nucleation resides in its carboxy-terminal half, with TPX2 domains α5-7 as the minimal necessary requirement and domains α3-4 serving as contributors to nucleation efficiency by enabling earlier induction at faster rates. The amino-terminal half of TPX2 also increases the efficiency of the reaction. TPX2 α5-7 is different from the remainder of the protein in that it contains conserved regions in its amino acid sequence that share sequence similarity with two known γ-TuRC nucleation activator motifs: SPM and γ-TuRC. The SPM-like motif is found within the α5 domain, while the γTuNA-like motif is found to start in the α5 domain and stretch into the SPM-like motif. Without these two motifs, no microtubule nucleation was observed in vitro, though microtubule binding ability was maintained. However, these two motifs are not the only essential ones in microtubule branching nucleation; the FKARP motifs of α5 and α6 are also essential for stimulating this process. Furthermore, the α-helical region stretch of domain α7 and the C-terminal residues that interact with Eg5 are critical for microtubule branching nucleation as well. While α5-7 domains are important in this process, none of them have intrinsic microtubule nucleation activity.
In terms of binding to and bundling microtubules, at least any of the three domains α3-7 of TPX2 are necessary for significant binding and bundling in vitro. Furthermore, it is likely that the domains cooperatively mediate microtubule binding and bundling, as successive addition or subtraction of a domain does not result in a linear change in microtubule binding and bundling capacity.

Activation and reciprocation through Aurora A kinase

TPX2 recruits and activates Aurora A kinase by utilizing its short 43 amino acid long amino-terminal sequence to bind the catalytic domain of Aurora A, locking the kinase into its active conformation. More specifically, this interaction positions the activation segment of the kinase into a more favorable conformation for substrate binding and swings the crucial phosphothreonine residue, a target usually exposed and accessible for deactivation of Aurora A kinase by PP1, into a buried position, thereby locking Aurora A into an active conformation. Notably, this recognition between TPX2 and Aurora A is analogous to that between the cAMP-dependent protein kinase catalytic core and its flanking region, suggesting a recurring theme in kinase regulation. Activated Aurora A in turn phosphorylates TPX2, but it is still unclear how Aurora A's phosphorylation of TPX2 affects its activities.

Role in cleavage arrest and interaction with Eg5

When four-fold TPX2 over the endogenous level was injected into a blastomere with a two-cell embryo, cleavage arrest was induced. This arrest has been attributed to the amino acids 471-715 of the carboxy-terminus of the TPX2 protein, with the last 35 amino acids being absolutely necessary for inducing cleavage arrest. During cytokinesis failure, cycles of DNA synthesis and mitosis continue. Notably, spindle poles fail to segregate, leading to a failure to establish a bipolar spindle, a spindle midzone, and a central spindle complex. Because cleavage furrow ingression is primarily triggered by signals from the spindle midzone, these biological phenotypes could account for the failure of this event due to the inability to activate the spindle checkpoint. Instead of a bipolar spindle, both spindle poles are in apposition, with an impairment of pushing forces generated by interpolar microtubules.
The mechanistic cause behind cleavage arrest is attributed to TPX2's ability to directly bind motor protein Eg5, which requires the last 35 amino acids of the TPX2 carboxy-terminus for its interaction. When Eg5 was co-injected with TPX2 in vivo, cleavage furrow arrest was blocked and ingression was observed. This suggests that the carboxy-terminus of TPX2 regulates spindle pole movement via an Eg5-dependent mechanism.

Binding with Xlp2

When bound to microtubules, TPX2 recruits a plus-end directed motor protein, Xlp2, a protein that is required in early mitosis and localizes to spindle poles, to microtubule minus ends of asters. Like TPX2's localization to microtubules, this recruitment is also RanGTP independent.

Regulation of TPX2 in the cell cycle

Monitoring TPX2 gene mRNA expression during cell cycle progression in synchronized HeLa cells revealed that TPX2 expression is high in G2/M phase, decreases dramatically upon G1 phase entry, increases upon entry into S phase, and peaks again at the next G2/M phase. This is correlated by results showing an increased stability of TPX2 in S-phase extracts compared to that of TPX2 in mitotic extracts, indicated by a significant increase in TPX2 half-life. The drop in TPX2 is consistent with the drastic reorganization in structure and dynamics of the mitotic spindle.
Overall, TPX2 has been shown through in vivo experiments to be regulated by the APC/C^Cdh1 pathway. The instability and drop in TPX2 at mitotic exit is dependent on both the anaphase-promoting complex/cyclosome and an ubiquitin ligase integral in mitotic progression, along with APC/C's activator protein, Cdh1. This is a result of TPX2 being bound directly by Cdh1, and not Cdc20 or any other substrate of APC/C^Cdh1, and designated for degradation by APC/C. Moreover, the Cdh1-TPX2 binding interaction produces the TPX2 stability seen during mitosis up until mitotic exit: The amino-terminal region of Cdh1 can act as a dominant negative mutant when expressed in mammalian cells, stabilizing APC/C^Cdh1 substrates such as TPX2 by competitive binding.