Topical reviewA Model for the Cytoplasmic Trafficking of Signalling Proteins Involving the hsp90-Binding Immunophilins and p50cdc37
Introduction
A major unsolved problem that pertains to all signalling pathways for hormones, drugs, and growth factors that act via effects on gene transcription relates to how signalling proteins, such as steroid receptors, STAT proteins, and MAP kinases, move through the cytoplasm and nucleus to their sites of action. This problem has been approached almost solely by studying the factors required for import and export of model proteins across the pores in the nuclear envelope (for review, see Mattaj and Englmeier [1]), rather than the factors involved in protein movement within the cytoplasm or within the nucleus itself. Although movement of vesicles is known to occur on cytoskeletal tracks in a process requiring molecular motors (for review, see Refs. 2, 3), essentially nothing is known about the cytoplasmic movement of soluble proteins that are not associated with vesicles.
It could be that signalling protein solutes move through the cytoplasm by diffusion and then become trapped at their sites of action by protein-protein interactions, such as those involved in the formation of multiprotein signalosome complexes at the internal surface of the cell membrane or those involved in the binding of a localisation signal by a signal recognition protein. Alternatively, protein solutes may utilise a movement machinery to traverse the cytoplasm, in which case, movement would be likely to involve molecular motors and cytoskeletal tracts much like vesicular movement. Because this kind of movement machinery operates bidirectionally, specific protein interactions would be required to determine the direction of signal protein movement.
There is evidence that short-range trafficking of large solutes through the cytoplasm involves both diffusion and movement along the cytoskeleton. For example, studies with microinjected fluorescein-labelled dextrans indicate that solutes up to ā¼500 kDa, a mass that would include large multiprotein complexes, can diffuse within the cytoplasm and within the nucleus [4]. However, microinjection studies in axons show that protein solutes containing a nuclear localisation signal (NLS) utilise a microtubule-based machinery for targeted directional movement through the axoplasm [5]. The axon is a specialised cytoplasmic extension where movement by random diffusion alone would not permit delivery of protein solutes over long distances. However, in the cytoplasm of the nerve cell body or in the cytoplasm of nonneuronal cells like lymphocytes or fibroblasts, signal proteins undergo a relatively short-range transport that could involve both random diffusion and movement by an organised transport system. Studies of vesicle movement have shown that microtubules are used but are not required for such short-range transport (for review, see Bloom and Goldstein [6]).
In this paper, we will review studies from our laboratory on the movement of the glucocorticoid receptor (GR), a steroid receptor that undergoes a hormone-dependent shift in localisation from the cytoplasm to the nucleus. Like other steroid receptors and several protein kinases involved in signalling (e.g., Src, Raf), the function of the GR is regulated by the multiprotein hsp90-based chaperone system (for review, see Refs. 7, 8). We will review here evidence for two mechanisms of GR movement. In cells with an intact cytoskeletal system, movement by diffusion is limited, and rapid receptor movement utilises an hsp90-dependent machinery and occurs along cytoskeletal tracts. However, in cells in which the cytoskeleton is disrupted, the receptor appears to move by diffusion alone. This review is intended to be speculative and heuristic, and our goal is to expose the reader to a model in which the hsp90-binding immunophilins and p50cdc37 target the retrograde (toward the nucleus) or anterograde (toward the plasma membrane) direction of signalling protein movement.
Section snippets
The gr as a model for studying cytoplasmic trafficking
On the basis of biochemical partitioning studies, Allan Munck proposed nearly 30 years ago that the GR undergoes an energy-dependent cycling between the cytoplasm and the nucleus [9] (see Orti et al. [10] for review). Subsequent immunofluorescence localisation studies in heterokaryons demonstrated that unliganded steroid receptors constantly shuttle into and out of the nucleus 11, 12, 13, 14 (see Guichon-Mantel and Milgrom [15] and DeFranco et al. [16] for review). In hormone-free cells,
Steroid receptor and protein kinase heterocomplexes with hsp90
Hsp90 is the most abundant of the heat shock, or stress, proteins. It is a ubiquitous and highly conserved molecular chaperone that is essential for eukaryotic cell survival (for review, see Buchner [38]). Steroid and aryl hydrocarbon (Ah) receptors exist in the cytosol of hormone-free cells as large multiprotein complexes containing hsp90 7, 8. Hsp90 is bound as a dimer to the receptor hormone binding domain (HBD), and for the GR, the HBD must be bound to hsp90 for there to be a high affinity
Assembly of heterocomplexes with hsp90
A model of the heterocomplex assembly mechanism that is derived from studies with purified components is presented in Fig. 1. The first step appears to be the formation of an hsp90Ā·HopĀ·hsp70Ā·hsp40 complex that we call a foldosome [88]. These complexes can be prepared simply by mixing purified components or they can be immunoadsorbed from reticulocyte lysate with an antibody against Hop [84]. When mixed with immunoadsorbed, hsp90-free GR, the immunoadsorbed hsp90Ā·HopĀ·hsp70Ā·hsp40 complex converts
Involvement of hsp90 in gr movement
Several years ago, we proposed that the steroid receptors shuttle through the cytoplasm in heterocomplex form and suggested that hsp90 and the immunophilins might play an active role in the movement process 111, 112. Subsequently, Kang et al. [113] targeted hsp90 to the nucleus by preparing a fusion with the NLS of nucleoplasmin, and then showed that coexpression of the hsp90-NLS and cytoplasmic steroid receptor mutants devoid of an NLS resulted in complete nuclear localisation of the
The tpr acceptor site on hsp90
The immunophilins FKBP52, FKBP51, and CyP-40 were discovered because of their presence in steroid-receptor heterocomplexes. FKBP52 was first reported by Faber's laboratory to be a 59-kDa protein that was a common component of untransformed steroid receptor heterocomplexes [125]. It was subsequently cloned and shown to be an FK506-binding immunophilin 47, 48, 49, 50 and a stress protein [126]. FKBP52 was known to exist in heterocomplexes with hsp90 independent of steroid receptors 127, 128, and
How signalling proteins determine the composition of their hsp90 heterocomplexes
If a common TPR acceptor site on hsp90 were the only determinant of heterocomplex composition, then the signalling proteins that form stable complexes with hsp90 would all form the same spectrum of heterocomplexes. In such a case, the composition of those heterocomplexes would be determined by the relative abundance of the various hsp90-binding TPR domain proteins and the relative affinities of TPR domains for the acceptor site. However, it is clear that the signalling proteins themselves
Potential roles of immunophilins and p50cdc37 in targeting signalling protein movement
In considering a model for short-range movement of signalling proteins utilising an organised movement system along microtubules, the major missing elements have been the connector proteins that link the signalling proteins to the movement machinery. On the basis of its structure and cellular localisation, we proposed several years ago that FKBP52 might perform this role for the GR [124]. In the hinge region between Domain I and Domain II [129], FKBP52 possesses a conserved sequence of eight
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