Curtin Conference

Immunology and Cell Biology (2000) 78, 447–451; doi:10.1046/j.1440-1711.2000.00928.x

Cellular function of p70S6K: A role in regulating cell motility

Leise A Berven1 and Michael F Crouch1

1Molecular Signalling Group, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia

Correspondence: Leise A, Berven, Division of Neuroscience, John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia. Email: leise.berven@anu.edu.au

Received 11 April 2000; Accepted 11 April 2000.

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Abstract

The 70 kDa ribosomal S6 kinase (p70S6K) is activated by numerous mitogens, growth factors and hormones. Activation of p70S6K occurs through phosphorylation at a number of sites and the primary target of the activated kinase is the 40S ribosomal protein S6, a major component of the machinery involved in protein synthesis in mammalian cells. In addition to its involvement in regulating translation, p70S6K activation has been implicated in cell cycle control and neuronal cell differentiation. Recent data obtained in this laboratory suggests that p70S6K may also function in regulating cell motility, a cellular response that is important in tumour metastases, the immune response and tissue repair. The present paper reviews the regulation and cellular function of p70S6K and proposes a novel function of p70S6K in regulating cell motility.

Keywords:

cytoskeleton, migration, p70S6K

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Regulation of p70S6K

The 70 kDa ribosomal S6 kinase (p70S6K) is activated through the phosphoinositide (PI) 3-kinase-regulated pathway (Figure 1). This pathway is responsible for the generation of inositol lipids, which are key mediators of intracellular signalling. Multiple isoforms of PI 3-kinase have been identified and classed according to substrate specificity and structure.1 Class I PI 3-kinase is involved in receptor-induced hormonal responses and the mechanism of activation of this enzyme differs depending on the particular extracellular stimuli. For tyrosine kinase-coupled receptor systems, p110 catalytic subunits alpha, beta and delta are stimulated through interaction with the p85 adaptor subunit, which binds selectively through its SH2 domain to phosphorylated tyrosines on the activated receptor.2 For G-protein-coupled receptor systems, the signalling for PI 3-kinase activation is less clear and several mechanisms have been suggested. A number of reports have identified a p110gamma catalytic subunit, which is activated through binding either directly to the betagamma subunits of activated trimeric G-proteins3 or through interaction with a p101 adaptor protein.4, 5 Other studies have suggested alternative mechanisms involving G-protein-mediated activation of receptor tyrosine kinase activity6 or through the p110beta isoform, which can also be activated directly by G-protein betagamma subunits.7

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

A model of the p70S6K signalling pathway. P70S6K is activated by phosphorylation at multiple sites via phosphoinositide (PI) 3-kinase-dependent kinases, which are activated in response to stimulation of G-protein coupled receptors (GPCR) or tyrosine kinase receptors. The main physiological target of activated p70S6K is S6, which is involved in upregulating protein synthesis. Other targets are unidentified, but may include those which are involved in regulating cell motility. PIP3, phosphatidylinositol 3,4,5-triphosphate, PIP2, phosphatidylinositol 3,4-bisphosphate; GPCR, G-protein-coupled receptor; PKC, protein kinase C; PH, pleckstrin homology domain; PDK1, PIP3-dependent protein kinase; mTOR, target of rapamycin; FRAP, FKBP12–rapamycin-associated protein; TOP, terminal oligopyrimidine.

Full figure and legend (40K)

For both receptor systems, PI 3-kinase catalyses the phosphorylation of phosphoinositides at the 3'-OH position. For hormone-induced responses in vivo, phosphatidylinositol 4,5-bisphosphate (PI-4,5P2) is the primary substrate for PI 3-kinase, leading to the production of phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5P3). Phosphatidylinositol 3,4,5P3 is also rapidly dephosphorylated by 5'-phosphatases, giving rise to the production of phosphatidylinositol 3,4-bisphosphate (PI-3,4P2).8 Although the mechanism regulating the relative levels of these two phospholipids is complex, both are thought to act as second messengers in cellular responses such as mitosis, apoptosis, membrane trafficking, motility, differentiation and oncogenic transformation by targeting certain pleckstrin homology (PH) domain-containing proteins to the plasma membrane.9

Two isoforms of p70S6K have been identified: a 70 kDa cytoplasmic form and a 85 kDa nuclear form. The sequence of the longer form includes a 23 amino acid nuclear localization sequence at the amino terminus. The molecular sequences of these two isoforms are otherwise the same and can be jointly referred to as p70S6K or S6K1. Recently, a second functional homologue, S6K2, has been identified. This second S6 kinase shows similar sensitivity to rapamycin and PI 3-kinase inhibitors and is upregulated in p70S6K1-deficient mice.10, 11 The identification of S6K2 has raised the possibility that some functional responses of S6K1 may be caused by activation of S6K2 or even other unidentified S6 kinases.

Activation of p70S6K occurs through a complex series of phosphorylation events on eight or more serine or threonine residues. As reviewed by Dufner and Thomas,12 these phosphorylation sites have been identified as S404, S411, S418, S424 and T421 on the C-terminal autoinhibitory domain and T229, S371 and T389, which are critical for catalytic activity. In addition, site-specific mutagenesis studies have shown that phosphorylation of sites T229, T389, S404 and S411 is inhibited by rapamycin or wortmannin.13 Autoinhibitory sites are thought to be phosphorylated by members of the mitogen-activated protein kinase (MAPK) family, p38 and extracellular signal-regulated kinases (ERK).14 Phosphorylation at these sites induces a conformational change that allows phosphorylation of catalytic sites. Numerous signalling proteins, such as phosphoinositide-dependent kinase-1 (PDK1),15, 16 Akt,17 atypical protein kinase C (PKC),18 cyclin-dependent kinases,19 and mammalian target of rapamycin (mTOR; or FRAP or RAFT1),20 have been implicated in phosphorylation of these sites, but the mechanism here is not well understood and identification of kinases that directly phosphorylate p70S6Kin vivo is of much current interest. Full activation of p70S6K appears to require phosphorylation of T229 by PDK1, a serine/threonine protein kinase that contains a PH domain at its C-terminus and binds PI-3,4,5P3 with high affinity,21 and phosphorylation of T389, possibly by mTOR.20, 22 Akt, a PH domain-containing protein that binds PI-3,4,5P3, is also phosphorylated at a similar site by PDK1.17 Akt has been shown to effect p70S6K activation,23 although recent studies indicate that Akt-dependent phosphorylation of p70S6K is mediated by an unknown intermediate kinase.24 It seems likely that the complexity of signalling between Akt, p70S6K and PDK1 may be due, in part, to the relative availability of PI-3,4,5P3 and PI-3,4P2 to bind Akt or PDK1.

Inactivation of p70S6K by dephosphorylation at specific sites is possible either through rapamycin treatment or amino acid deprivation,12 although the mechanism for inactivation is not clear in either case. Mechanisms for dephosphorylation of p70S6K may involve either: (i) inhibition of mTOR or other p70S6K kinases, thus reducing phosphorylation on regulatory sites; and/or (ii) activation of a serine/threonine protein phosphatase that dephosphorylates p70S6K at these regulatory sites. Although many studies have shown that rapamycin inactivates p70S6K, presumably through inhibition of mTOR, evidence that mTOR directly phosphorylates p70S6Kin vivo has not been conclusive. Thus, support for the second mechanism has been demonstrated recently in studies using phosphatase-specific inhibitors, which identified protein-serine/ threonine phosphatase 2A (PP2A) as a likely p70S6K-specific phosphatase.25, 26

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Cellular function of p70S6K

Protein synthesis

The physiological target of growth factor-activated p70S6K is the 40S subunit of the S6 ribosomal protein. Phosphorylation of S6 induces, by an unknown mechanism, the selective translation of 5' TOP mRNA, a subset of mRNA that contains an oligopyrimidine tract at its 5' untranslated region. The 5' TOP mRNA encode components of the translational machinery such as elongation factors, ribosomal proteins and poly(A)-binding protein and thus play a key role in modulating translational efficiency.

Several steps in the assembly of translational protein complexes have been shown to involve or require the mTOR-regulated pathway and these have been reviewed recently.27 Rapamycin, which binds and inhibits the kinase activity of mTOR, blocks translation of 5' TOP mRNA (via p70S6K inactivation) as well as the phosphorylation of 4E-BP1, a protein that binds to the translational initiation factor eIF4E. Phosphorylation of 4E-BP1 promotes initiation of translation by causing dissociation of the eIF4E/ 4E-BP1 complex to allow formation of eIF4F, a multiprotein complex that binds to several initiation factors on stimulation. In addition, rapamycin has been shown to inhibit the activation of eEF2, the elongation factor that is required for the translocation step of elongation in which the ribosome moves relative to the mRNA. Thus, protein synthesis is controlled by the p70S6K/mTOR pathway on multiple levels: by direct activation of specific proteins of the translational apparatus through mTOR activation and by p70S6K-mediated upregulation in the synthesis of these proteins.

Cell cycle control

Rapamycin inhibits cell proliferation in T lymphocytes and other cell types by arresting cells in G1 phase. In fibroblasts, rapamycin delays G1–S phase transition, suggesting that in these cells additional pathways are involved in controlling cell growth.28 Evidence that rapamycin-induced effects on cell cycle are due to inactivation of p70S6K has also been shown by microinjection of anti-p70S6K antibodies, which block progression of fibroblasts into S phase.29 Because cell cycle progression involves not only activation of protein synthesis but also the ordered activation of cyclins and cyclin-dependent kinases (cdk), it seems possible that induction of cyclin and cdk is regulated by p70S6K or another rapamycin-sensitive pathway via its function in activating protein synthesis, as described earlier. Recently, studies in endothelial cells have shown that rapamycin-induced inhibition of protein synthesis is associated with inhibition of cyclin D1 and p21 synthesis. Cyclin D1 and p21 are critical members of the cyclin-cdk complex in G1 phase, which is required for phosphorylation of retinoblastoma (Rb) protein and entry into S phase.30 It has been suggested that this effect can be explained by the presence of a 5' TOP sequence in cyclin D1 mRNA, although it has been proposed that this tract is not functional in vivo.31 Studies in fibroblasts also suggest that this is not a general mechanism for the inhibitory effects of rapamycin on cell cycle progression. For example, we have found in our laboratory that rapamycin has little effect on DNA synthesis in Swiss 3T3 cells, but partially blocks cell proliferation, suggesting that p70S6K or another mTOR-regulated pathway is involved downstream of S phase.32 In addition, it has also been shown that, while PI 3-kinase inhibition causes G1 arrest by blocking cyclin D1 activation and phosphorylation of Rb, rapamycin has little inhibitory effect on these events.33 The potential role of other S6 kinases in cell cycle control has been highlighted with the identification of S6K2, as discussed earlier.10, 11

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Cell migration: A novel function for p70S6K activation?

Movement of cells across a substratum is controlled by coordinated assembly and rearrangements of the cytoskeleton involving actin polymerization and depolymerization, extension of the cytoskeletal structures at the leading edge and translocation of the cell through the forward motion of the cell at the leading edge and detachment at the trailing edge. The mechanical motion of migrating fibroblasts is characterized by a number of morphological features. (Figure 2). Briefly, most motile fibroblasts are fan-shaped or polarized with a broad flattened region at the anterior side called a lamella. New contacts with the substratum are made through small membrane protrusions of the cytoplasm, called lamellipodia, at the leading edge. Precursors of lamellipodia are structures lying within lamella called microspikes, filopodia or actin ribs, all of which describe short rib-like actin filaments present at the leading edge. These cellular structures are formed in response to actin polymerization and the formation of actin stress fibres, the basic component of the cytoskeleton necessary for focal contacts (adhesion), migration and maintaining cellular shape. At the rearward side of the cell are tails that anchor the cell to the substratum and that are released in a controlled manner as the cell migrates forward.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Swiss 3T3 cells labelled with phalloidin-Texas red to reveal the actin cytoskeleton. Cells were grown on coverslips to subconfluency, starved overnight, then activated with serum for 4 h. Fixed cells were stained with phalloidin-Texas red and visualized using confocal microscopy. The scale bar represents 50 mum. Details of methods have been described previously.35

Full figure and legend (240K)

The signalling pathways that underlie formation of these cytoskeletal structures are complex. Studies have shown that receptor activation of cell migration is biochemically regulated by signalling proteins and second messengers such as PI-3,4P2, phospholipase C, PKC, Ca2+, PI 3-kinase, Rho and Rac GTPases and MAPK.34

p70S6K has been shown to associate with Rac1 and cdc42.35 Both are small GTPases involved in the regulation of membrane ruffling, migration and actin polymerization. In this study, dominant-negative Rac1 overexpression in several cell types prevented growth factor-induced p70S6K activity, suggesting that Rac1 activates p70S6K. In addition, activation of p70S6K by expression of an activated allele of Rac1 is inhibited by rapamycin and the PI 3-kinase inhibitor wortmannin. The results of both experiments support a role for p70S6K in regulation of the cytoskeleton, except that the authors indicate that rapamycin had no effect on membrane ruffling or stress fibre formation. Further studies showing that Rac1-mediated activation of p70S6K is unrelated to cytoskeleton reorganization have not been published.

In contrast, several studies conducted in our laboratory have shown a role for p70S6K in cytoskeleton regulation and, potentially, cell migration. First, we have shown that p70S6K colocalizes with actin stress fibres, suggesting that p70S6K activation plays a role in actin polymerization. Second, thrombin stimulation causes a shape change effect that is characterized by elongation and organization of stress fibres and this effect is inhibited by treatment with rapamycin.32 Nitric oxide donors have been found to increase growth factor-stimulated p70S6K activity and this potentiation of p70S6K activity is associated with a prolonged shape change effect and enhancement of tails, both morphological features that may be enhanced in rapidly migrating cells.36 Last, in migrating cells, we have found that p70S6K localizes to the actin arc (LA Berven and MF Crouch, unpubl. data, 2000), an actin-based structure that is located near the edge of the leading edge and that passes back over the apical surface of the cell as the cell moves forward.37 Based on these morphological and biochemical results, we propose that p70S6K is involved in regulating the migration of 3T3 fibroblasts and thus present a potentially novel function of p70S6K.

How does p70S6K function to activate or enhance migration? Clearly, p70S6K is important for regulation in translation. Thus, growth factor-induced translocation of p70S6K to the actin cytoskeleton and leading edge of the cell followed by localized synthesis of key protein regulators of filopodia or lamella extension is one possible mechanism. In support of this idea, it has been shown that p70S6K may be targeted to nerve endings via its interaction with neurabin, an F-actin binding protein that is highly expressed in nerve tissue.38 In this study, p70S6K and neurabin were shown to colocalize in brain sections by in situ hybridization and were both enriched in the synaptosomal fraction in rat brain. Although localized protein translation in nerve terminals has not been established, it is possible that p70S6K functions to increase synthesis of proteins required for the assembly of actin cytoskeletal structures that are involved in neurite outgrowth or growth cone formation. Thus, it is possible that the function of p70S6K at the synapse may be analogous to its role in migration.

Recently, it has been shown that mTOR interacts with gephyrin,39 a protein that is necessary for the clustering of glycine receptors at the postsynaptic terminals in spinal cord neurons.40 In these studies, expression of mTOR mutants that were unable to bind gephyrin failed to activate the downstream targets of mTOR, p70S6K and 4E-BP1. Furthermore, while mTOR expressed in HeLa cells appears uniformly distributed throughout the cytoplasm, coexpression of mTOR with gephryin causes aggregation of mTOR at polarized regions of the cell, suggesting that gephyrin may influence mTOR (and consequently p70S6K) signalling through its role in clustering receptors or other signalling molecules that contain a gephyrin-binding domain. The idea that receptor clustering may play a role in mediating cytoskeletal rearrangements is supported by studies that show that Rho mediates clustering of monocyte binding receptors in endothelial cells, an effect that may enhance their association with the actin cytoskeleton and that may facilitate monocyte migration across the endothelium.41 Our studies suggest that a similar phenomenon may be involved in the mechanism that regulates migration and other cellular processes in fibroblasts. These results are described in detail by Crouch et al. in this issue of Immunology and Cell Biology.42

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