Nuclear Transport of Parathyroid Hormone (PTH)-Related Protein Is Dependent on Microtubules

Abstract

PTH-related protein (PTHrP) was first discovered as a circulating factor secreted by certain cancers and is responsible for the syndrome of humoral hypercalcemia of malignancy induced by various tumors. The similarity of its N terminus to that of PTH enables PTHrP to share the signaling properties of PTH, but the rest of the molecule possesses distinct functions, including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import is mediated by importin beta1. In this study we use the technique of fluorescence recovery after photobleaching to demonstrate the ability of PTHrP to shuttle between cytoplasm and nucleus and to visualize directly the transport of PTHrP into the nucleus in living cells. Endogenous and transfected PTHrP was demonstrated to colocalize with microtubule structures in situ using various high-resolution microscopic approaches, as well as in in vitro binding studies, where importin beta1, but not importin alpha, enhanced the microtubular association of PTHrP with microtubules. Significantly, the dependence of PTHrP nuclear import on microtubules was shown by the inhibitory effect of pretreatment with the microtubule-disrupting agent nocodazole on nuclear-cytoplasmic flux. These results indicate that PTHrP nuclear/nucleolar import is dependent on microtubule integrity and are consistent with a direct role for the cytoskeleton in protein transport to the nucleus.

Mechanism of Microtubule-facilitated “Fast Track” Nuclear Import

“…Image analysis was carried out on the digitalized confocal files using the ImageJ 1.38 public domain software (National Institutes of Health) (9,(21)(22)(23)). An area of the nucleolus or nucleus (not including the nucleolus) and cytoplasm was selected to determine the relative nucleolar (Fnu), nuclear (Fn), and cytoplasmic (Fc) concentrations of GFP fusion proteins after correction for background fluorescence, enabling the nucleolar (Fnu/c) or nuclear (Fn/c) to cytoplasmic fluorescence ratios to be calculated (10,24,25). Results were expressed for a minimum of 40 cells, and experiments were repeated at least twice.…”

“…The clear implication of these data is that GFP-PTHrP-(66 -108) specifically binds IMP␤1 with high affinity with both KKKK 91 and KKRR 106 required for this interaction. 91 and KKRR 106 -To test whether PTHrP can interact directly with intact MTs and determine the possible role of IMP␤1 in this interaction, tubulin was polymerized in vitro as described previously (10). The in vitro polymerized MT filaments were then incubated with bacterially expressed GFP-PTHrP fusion proteins (amino acids 38 -108, 66 -108, 82-108, and 66 -94) as well as mutant derivatives thereof and negative controls (GFP and GFP-T-ag NLS).…”

“…Nuclear protein import is generally accepted to be independent of the cytoskeleton (9), conventionally being mediated by soluble transport factors (importins (IMPs)), 3 which promote the passage of cargoes bearing nuclear localization signals (NLSs) through the nuclear envelope-localized nuclear pore complex. It is becoming increasingly apparent, however, that specific cancer-related proteins, such as the tumor suppressors p53 (4) and retinoblastoma protein (Rb) (9), and the paracrine/autocrine signaling molecule parathyroid hormone-related protein (PTHrP) (10), rely on the MT network for efficient nuclear import as do certain transcription factors (e.g. STATs 1 and 5) (11,12).…”

“…PTHrP is a crucial factor in the early stages of development of various tissues as well as in the biology of adult tissues (13,14). Importantly, it is present as a circulating factor in numerous types of tumors (15) with its malignant actions linked to its nuclear localization (2,3), which significantly is known to be dependent on the IMP family member IMP␤1, as well as MT integrity (10). Specific nuclear localizing proteins, such as PTHrP, appear to possess the ability to interact with MTs for facilitated transport through the cytoplasm prior to conventional IMP-dependent import through the nuclear pore complex into the nucleus (9,16,17).…”

Binding of p110 Retinoblastoma Protein Inhibits Nuclear Import of Simian Virus SV40 Large Tumor Antigen

“…The nuclear to cytoplasmic ratio (Fn/c) was determined as previously (6, 9, 10, 18, 27, 29 -32) using the Image J 1.40o public domain software (NIH), from single cell measurements for the nuclear (Fn) and cytoplasmic (Fc) fluorescence, subsequent to the subtraction of fluorescence due to autofluorescence/background. Fluorescence Recovery after Photobleaching (FRAP)-FRAP was performed essentially as previously (33)(34)(35), whereby bleaching of nuclear fluorescence and monitoring of the return of fluorescence to the nucleus enables the nuclear import rate to be determined. Briefly, COS-7 cells transiently expressing GFP-T-ag derivatives were visualized using an Olympus Fluoview 1000 microscope (100ϫ oil immersion lens).…”