Abstract
Although the microtubule (MT) cytoskeleton has been shown to facilitate nuclear import of specific cancer-regulatory proteins including p53, retinoblastoma protein, and parathyroid hormone-related protein (PTHrP), the MT association sequences (MTASs) responsible and the nature of the interplay between MT-dependent and conventional importin (IMP)-dependent nuclear translocation are unknown. Here we used site-directed mutagenesis, live cell imaging, and direct IMP and MT binding assays to map the MTAS of PTHrP for the first time, finding that it is within a short modular region (residues 82-108) that overlaps with the IMPβ1-recognized nuclear localization signal (residues 66-108) of PTHrP. Importantly, fluorescence recovery after photobleaching experiments indicated that disruption of the MT network or mutation of the MTAS of PTHrP decreases the rate of nuclear import by 2-fold. Moreover, MTAS functions depend on mutual exclusivity of binding of PTHrP to MTs and IMPβ1 such that, following MT-dependent trafficking toward the nucleus, perinuclear PTHrP can be displaced from MTs by IMPβ1 prior to import into the nucleus. This is the first molecular definition of an MTAS that facilitates protein nuclear import as well as the first delineation of the mechanism whereby cargo is transferred directly from the cytoskeleton to the cellular nuclear import apparatus. The results have broad significance with respect to fundamental processes regulating cell physiology/transformation.
Highlights
Summary of Kd and Bmax values obtained from ALPHAScreen assays using human IMP1
Values shown are the average of three individual experiments ϮS.E. according to Fig. 6
The absence of IMP1 in the PTHrP MT fraction indicates that PTHrP is able to bind both IMP1 and MTs it does not associate with IMP1 when bound to MTs in intact cells, consistent with the mutual exclusivity of binding observed in the in vitro assays (Fig. 7, A–C). These results indicate that PTHrP-(82–108) is likely to be the MTAS that mediates interaction of PTHrP with MTs in vivo and in vitro
Summary
Generation of Expression Constructs for GFP-PTHrP Fusion Cassettes and Site-directed Mutagenesis—PTHrP coding sequences were inserted into the C terminus of enhanced green nucleolar to cytoplasmic fluorescence ratio; Fn/c, nuclear to cytoplasmic fluorescence ratio; T-ag, SV40 large tumor antigen; NHS, N-hydroxysuccinimide; P, pellet; S, supernatant; DLC, dynein light chain. PTHrP was preincubated with 3 M IMPs at room temperature for 30 min prior to MT incubation, or IMPs were added 10 min prior to imaging following 1-h preincubation of PTHrP with MTs. In some cases, MTs were visualized by CLSM using a Bio-Rad MRC-600 microscope with a 60ϫ oil immersion lens, and image analysis on the digitalized confocal files was performed using ImageJ 1.38 where a line plot was used to quantify the specific MT-associated fluorescence with background fluorescence subtracted [35]. COS-7 cells transfected to express recombinant GFP-PTHrP constructs, GFP alone, and/or human myc-IMP1 were lysed using MT stabilizing buffer containing 100 M GTP, 1 mM ATP, and 10 l/ml protease inhibitor mixture by incubation for 10 min at 37 °C. In experiments where cells were cotransfected to express GFPPTHrP-(66 –108) (or BASIC-1mut) and IMP1, the ratio of MT-associated to non-associated PTHrP protein (P/S) was calculated, and to quantify the effect of IMP1 coexpression, results were shown as a percentage of the P/S ratio from cells expressing GFP-PTHrP-(66 –108) alone
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