A Model for the Cytoplasmic Trafficking of Signalling Proteins Involving the hsp90-Binding Immunophilins and p50cdc37

doi:10.1016/S0898-6568(99)00064-9

Cellular Signalling

Volume 11, Issue 12, December 1999, Pages 839-851

https://doi.org/10.1016/S0898-6568(99)00064-9 Get rights and content

Abstract

A number of transcription factors and protein kinases involved in signal transduction exist in heterocomplexes with the ubiquitous and essential protein chaperone hsp90. These signalling protein·hsp90 heterocomplexes are assembled by a multiprotein chaperone system comprising hsp90, hsp70, Hop, hsp40, and p23. In the case of transcription factors, the heterocomplexes with hsp90 also contain a high molecular weight immunophilin with tetratricopeptide repeat (TPR) motifs, such as FKBP52 or CyP-40. In the case of the protein kinases, the heterocomplexes contain p50^cdc37. The immunophilins bind to a single TPR acceptor site on hsp90, and p50^cdc37 binds to an adjacent site so that binding is exclusive for p50^cdc37 or an immunophilin. Direct interaction of immunophilins with the transcription factors or p50^cdc37 with the protein kinases leads to selection of different heterocomplexes after their assembly by a common mechanism. Studies with the glucocorticoid receptor, for which translocation from the cytoplasm to the nucleus is under hormonal control, suggest that dynamic assembly of the heterocomplexes is required for rapid movement of the receptor through the cytoplasm along cytoskeletal tracts. As for the similar short-range trafficking of vesicles along microtubules, there must be a mechanism for linking the signalling protein solutes to the molecular motors involved in movement. We present here a model in which the immunophilins and p50^cdc37 target, respectively, the retrograde or anterograde direction of signalling protein movement by functioning as connectors that link the signalling proteins to the movement machinery.

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 ∼500 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 p50^cdc37 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 hsp90·Hop·hsp70·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 hsp90·Hop·hsp70·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 p50^cdc37 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

References (168)

G.M. Langford
Curr. Opinion Cell Biol.
(1995)
A. Munck et al.
J. Steroid Biochem.
(1972)
D.B. DeFranco et al.
Vitam. Horm.
(1995)
G. Akner et al.
J. Steroid Biochem. Mol. Biol.
(1991)
J. Barsony et al.
J. Biol. Chem.
(1992)
G. Akner et al.
J. Steroid Biochem. Mol. Biol.
(1995)
D. Szapary et al.
J. Steroid Biochem. Mol. Biol.
(1994)
J. Buchner
Trends Biochem. Sci.
(1999)
E.H. Bresnick et al.
J. Biol. Chem.
(1989)
L.C. Scherrer et al.
J. Biol. Chem.
(1990)

R.S. Sikorski et al.

Cell

(1990)

T. Ratajczak et al.

J. Biol. Chem.

(1996)

A.W. Yem et al.

J. Biol. Chem.

(1992)

M.-C. Lebeau et al.

J. Biol. Chem.

(1992)

D.F. Smith et al.

J. Biol. Chem.

(1993)

D.F. Smith et al.

J. Biol. Chem.

(1993)

T. Ratajczak et al.

J. Biol. Chem.

(1993)

L.J. Kieffer et al.

J. Biol. Chem.

(1993)

M.-S. Chen et al.

J. Biol. Chem.

(1996)

A.M. Silverstein et al.

J. Biol. Chem.

(1997)

J.S. Brugge et al.

Cell

(1981)

A. Ziemiecki et al.

Biochem. Biophys. Res. Commun.

(1986)

T. Cutforth et al.

Cell

(1994)

L.F. Stancato et al.

J. Biol. Chem.

(1993)

M. Wartmann et al.

J. Biol. Chem.

(1994)

L.F. Stancato et al.

J. Biol. Chem.

(1997)

L.A. Carver et al.

J. Biol. Chem.

(1994)

T. Hunter et al.

Trends Cell Biol.

(1997)

N. Grammatikakis et al.

J. Biol. Chem.

(1995)

K.A. Hutchison et al.

J. Biol. Chem.

(1992)

K.D. Dittmar et al.

J. Biol. Chem.

(1996)

K.D. Dittmar et al.

J. Biol. Chem.

(1997)

K.D. Dittmar et al.

J. Biol. Chem.

(1997)

K.D. Dittmar et al.

J. Biol. Chem.

(1998)

H. Kosano et al.

J. Biol. Chem.

(1998)

W.B. Pratt et al.

Trends Endocrinol. Metab.

(1998)

K.A. Hutchison et al.

J. Biol. Chem.

(1994)

L.F. Stancato et al.

J. Biol. Chem.

(1996)

J.P. Grenert et al.

J. Biol. Chem.

(1997)

D.O. Toft

Trends Endocrinol. Metab.

(1998)

W. Sullivan et al.

J. Biol. Chem.

(1997)

K.A. Hutchison et al.

J. Biol. Chem.

(1994)

B. Honoré et al.

J. Biol. Chem.

(1992)

B.D. Johnson et al.

J. Biol. Chem.

(1998)

S. Chen et al.

J. Biol. Chem.

(1998)

I.W. Mattaj et al.

Annu. Rev. Biochem.

(1998)

R.B. Vallee et al.

Annu. Rev. Neurosci.

(1991)

O. Seksek et al.

J. Cell Biol.

(1997)

R.T. Ambron et al.

J. Neurosci.

(1992)

G.S. Bloom et al.

J. Cell Biol.

(1998)

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Topical reviewA Model for the Cytoplasmic Trafficking of Signalling Proteins Involving the hsp90-Binding Immunophilins and p50cdc37

Abstract

Introduction

Section snippets

The gr as a model for studying cytoplasmic trafficking

Steroid receptor and protein kinase heterocomplexes with hsp90

Assembly of heterocomplexes with hsp90

Involvement of hsp90 in gr movement

The tpr acceptor site on hsp90

How signalling proteins determine the composition of their hsp90 heterocomplexes

Potential roles of immunophilins and p50cdc37 in targeting signalling protein movement

Curr. Opinion Cell Biol.

J. Steroid Biochem.

Vitam. Horm.

J. Steroid Biochem. Mol. Biol.

J. Biol. Chem.

J. Steroid Biochem. Mol. Biol.

J. Steroid Biochem. Mol. Biol.

Trends Biochem. Sci.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

Biochem. Biophys. Res. Commun.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Endocrinol. Metab.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Endocrinol. Metab.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Annu. Rev. Biochem.

Annu. Rev. Neurosci.

J. Cell Biol.

J. Neurosci.

J. Cell Biol.

Topical review
A Model for the Cytoplasmic Trafficking of Signalling Proteins Involving the hsp90-Binding Immunophilins and p50^cdc37

Potential roles of immunophilins and p50^cdc37 in targeting signalling protein movement