A Microtubule‐Facilitated Nuclear Import Pathway for Cancer Regulatory Proteins

Abstract

Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormonerelated protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time. We examine 10 nuclear-localizing proteins with diverse IMP-dependent nuclear import pathways, our results indicating that the cytoskeleton does not have a general mechanistic role in nuclear localization sequence-dependent nuclear protein import. Of the proteins examined, only the p110 Rb tumor suppressor protein Rb, together with p53 and PTHrP, was found to require MT integrity for optimal nuclear import. Fluorescence recovery after photobleaching experiments indicated that the MT-dependent nuclear transport pathway increases both the rate and extent of Rb nuclear import but does not affect Rb nuclear export. Dynamitin overexpression experiments implicate the MT motor dynein in the import process. The results indicate that, additional to IMP/diffusion-dependent processes, certain cancer regulatory proteins utilize an MT-enhanced pathway for accelerated nuclear import that is presumably required for their nuclear functions.Key words: actin filaments, FRAP, importins, microtubules, nuclear import, retinoblastoma protein Transport into the nucleus through the nuclear envelopelocalized nuclear pore complexes (NPCs) is dependent on modular nuclear localization sequences (NLSs) (1-3) within cargoes that are typically recognized by members of the importin (IMP) superfamily. Most commonly, the IMPa/b1 heterodimer or IMPb1 alone (4-6) act to effect NLSdependent translocation through the NPC, and release within the nucleus. Understanding of the detailed mechanisms of nuclear protein import, however, is largely based on the study of semi-intact cell systems (7-10), where cytoskeletal elements are either lacking or severely damaged, meaning that the role of the cytoskeleton has been largely overlooked. Many viruses are known to exploit the microtubule (MT) network for efficient nuclear targeting (11), while there is evidence for negative regulation of the nuclear import of several viruses through association with the actin cytoskeleton (12). Intriguingly, IMPa has been demonstrated to associate with MTs in mammalian and plant cells (13,14), while NLS-comprising peptides have been reported to invoke active transport along MTs (15). Significantly, a role for the MT network in facilitating nuclear import (16) is strongly implicated in the case of the tumor suppressor protein p53 and the cancer-related signaling molecule parathyroid hormone-related protein (PTHrP) (17,18). Treatment with the MT-depolymerizing agent nocodazole (NCZ) reduces both p53 and PTHrP nuclear accumulation in vivo (17,18), with application of the flu...

Kinetic stabilization of microtubule dynamic instability by benomyl increases the nuclear transport of p53

“…In contrast, low concentration of benomyl caused a strong increase in the nuclear localization of p53 in the presence of cisplatin. Although p53 is shown to be transported into the nucleus through the dynein/dynamitin complex [23,24], it can bind to microtubules directly and it was shown to bind preferentially to polymerized microtubules than to tubulin dimers [24]. Therefore, it is quite possible that the binding of p53 to the stabilized microtubules in the presence of low concentration of benomyl might prevent its degradation; however, its transport into the nucleus needs the dynein/ dynamitin motor complex [23,24].…”

“…Kinetic suppression of microtubule dynamics increased the association of DLC with microtubules Since p53 is reported to be transported into the nucleus through the microtubular tracks by the dynein/dynamitin motor complex [23,24], we examined the localization of the motor protein DLC on microtubules under normal conditions and under the suppressed dynamic instability conditions using EGFP transfected MCF-7 cells and confocal microscopy.…”

“…Microtubules are the targets for various successful anticancer drugs like vincristine, vinblastine, paclitaxel and several other drugs like epothilone, estramustine, and noscapine, which are in different stages of clinical trials [18][19][20]. Microtubule targeted agents have also been shown to alter the expression [21] and translocation of the tumor suppressor protein, p53 into the nucleus [22,23] suggesting that the nuclear accumulation of p53 is linked with the assembly dynamics of microtubules. Further, the translocation of p53 into the nucleus is thought to occur through the microtubule tracks [22][23][24] and low concentration of microtubule targeted drugs is shown to increase the nuclear accumulation of p53 [22].…”

“…Microtubule targeted agents have also been shown to alter the expression [21] and translocation of the tumor suppressor protein, p53 into the nucleus [22,23] suggesting that the nuclear accumulation of p53 is linked with the assembly dynamics of microtubules. Further, the translocation of p53 into the nucleus is thought to occur through the microtubule tracks [22][23][24] and low concentration of microtubule targeted drugs is shown to increase the nuclear accumulation of p53 [22].…”

A spatial model of cellular molecular trafficking including active transport along microtubules

“…The microtubules, together with the cytoplasmic filaments, constitute the cytoplasm's dynamic structure that maintains cell shape (cytoskeleton). They facilitate nuclear import of some proteins and viruses by providing a preferential way directed towards the nuclear envelope (see [51] and references therein). This is a typically spatial phenomena that cannot be taken into account in compartmental models.…”