Recent progress indicates that there are multiple pathways of nucleocytoplasmic transport which involve specific targeting sequences, such as nuclear localization sequences (NLSs), and cytosolic receptor molecules of the importin/karyopherin superfamily which recognise and dock the NLS‐containing proteins at the nuclear pore. This first step of nuclear import/export is of central importance, with the affinity of the importin‐targeting sequence interaction a critical parameter in determining transport efficiency. Different importins possess distinct NLS‐binding specificities, which allows the system to be modulated through differential expression of the importins themselves, as well as through competition between different importins for the same protein, and between different proteins for the same importin. The targeting sequence‐importin interaction can also be influenced directly by phosphorylation increasing the affinity of the interaction with importins or by targeting sequence masking through phosphorylation or specific protein binding. Targeting sequence recognition thus appears to represent a key control point in the regulation of nuclear transport. BioEssays 22:532–544, 2000. © 2000 John Wiley & Sons, Inc.

Parathyroid hormone-related protein (PTHrP), expressed in a range of tumors, has endocrine, autocrine/ paracrine, and intracrine actions, some of which relate to its ability to localize in the nucleus. Here we show for the first time that extracellularly added human PTHrP (amino acids 1-108) can be taken up specifically by receptor-expressing UMR106.01 osteogenic sarcoma cells and accumulate to quite high levels in the nucleus and nucleolus within 40 min. Quantitation of recognition by the nuclear localization sequence (NLS)-binding importin subunits indicated that in contrast to proteins containing conventional NLSs, PTHrP is recognized exclusively by importin ␤ and not by importin ␣. The sequence of PTHrP responsible for binding was mapped to amino acids 66 -94, which includes an SV40 large tumor-antigen NLS-like sequence, although sequence determinants amino-terminal to this region were also necessary for high affinity binding (apparent dissociation constant of ϳ2 nM for importin ␤). Nuclear import of PTHrP was assessed in vitro using purified components, demonstrating that importin ␤, together with the GTPbinding protein Ran, was able to mediate efficient nuclear accumulation in the absence of importin ␣, whereas the addition of nuclear transport factor NTF2 reduced transport. The polypeptide ligand PTHrP thus appears to be accumulated in the nucleus/nucleolus through a novel, NLS-dependent nuclear import pathway independent of importin ␣ and perhaps also of NTF2.Parathyroid hormone-related protein (PTHrP) 1 is expressed in a range of tumors and is an endocrine agent in humoral hypercalcemia of malignancy. It is also expressed in many normal tissues where it exerts autocrine/paracrine or intracrine actions (1-6). The structural similarities to parathyroid hormone (PTH) at the amino terminus of PTHrP are sufficient to confer functions similar to those of PTH, mediated by the shared PTH/PTHrP receptor and its ability to activate adenylate cyclase. Although both PTH and PTHrP promote bone resorption and reduce renal calcium excretion (1, 7), roles in the regulation of placental calcium transport to the fetus (1, 7, 8), osteoclast inhibition (9, 10), and the regulation of cell growth and apoptosis (2, 11, 12) have been ascribed to distinct regions of PTHrP.We and others have recently shown that PTHrP is expressed in a cell cycle-dependent manner (13, 14) as well as being localized to the nucleus/nucleolus at G 1 (14). Regulation of the nuclear localization of PTHrP appears to be mediated through phosphorylation by the cyclin-dependent kinases p33 cdk2 and p34 cdc2 . 2 These observations are consistent with the idea that cell cycle-dependent regulation of nuclear localization of PTHrP is central to the control of growth and apoptosis (2, 4). Of significance in this regard is our observation 2 that within amino acids 61-93, PTHrP retains a putative CcN motif, originally described for the SV40 large tumor antigen (T-ag; Refs. 16 and 17), comprising consensus protein kinase CK2 (S 61 DDE and ET 78 KYE-"C") and con...

Although the specific role of transcription factors (TFs) is nuclear, surprisingly little is known in quantitative terms regarding the pathways by which TFs localize in the nucleus. In this study, we use direct binding assays, native gel electrophoresis, and fluorescence polarization measurements to show for the first time that the cAMP-response element binding protein (CREB) and related AP-1 and jun and fos constituents are recognized by importin beta1 (Impbeta) with nanomolar affinity. We reconstitute the nuclear import of these TFs in vitro, demonstrating dependence on cytosolic factors, and show that this is due to the requirement for Impbeta, since antibodies to Impbeta, but not to importin alpha (Impalpha), inhibit nuclear accumulation significantly. We show that Impbeta is necessary and sufficient for docking of CREB at the nuclear envelope; that Ran is essential for CREB nuclear import is demonstrated by the reduction of nuclear accumulation effected by RanGTPgammaS but not RanGDP, and by dissociation of the Impbeta-CREB-GFP complex by RanGTPgammaS but not RanGDP as demonstrated using fluorescence polarization assays. The results support the existence of an Impbeta1- and Ran-mediated nuclear import pathway for CREB and related constitutively nuclear TFs, which is Impalpha-independent and thus distinct from import pathways utilized by inducible TFs.

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