Nuclear localization of p85 : functional requirement for ... - Europe PMC

The EMBO Journal vol.13 no.7 pp.1557-1565, 1994

Nuclear localization of p85 entry into S phase

:

functional requirement for

C.Reinhard, A.Fernandez1, N.J.C.Lambl and G.Thomas Friedrich Miescher Institute, PO Box 2543, 4002 Basel, Switzerland and ICBRM CNRS-INSERM, BB 5051, F-340033 Montpellier Cedex, France

Communicated by G.Thomas

Immunolocalization of a newly described isoform of p70s6k, termed p85s6k, demonstrated a predominantly nuclear location in rat embryo fibroblasts (REF-52), a compartment in which growth factor-mediated phosphorylation of S6 has recently been reported. Microinjection of expression vectors encoding either p85s6k or a fusion protein containing only the putative nuclear localization motifs led to the exclusive accumulation of both products in the nucleus. Consistent with such a localization, microinjection of affinitypurified anti-p85s6k IgG into the nucleus, but not the cytoplasm, blocked serum-induced initiation of DNA synthesis. Co-injection into the nucleus of the anti-p85s6k IgG with activated p7Os6k, which lacks the antigenic epitope, rescued the S phase block, arguing that the antibody exerts its effects through inhibiting p85s6k function. The results indicate a novel role for S6 phosphorylation in the nucleus distinct from that in the cytoplasm, a role essential for mitogenesis. Key words: G1I/S transition/microinjection/p70s6k/p85s6k

Introduction Stimulation of cells to proliferate leads to the activation of number of obligatory biochemical events which culminate in DNA synthesis and cell growth (Pardee, 1989). Proliferation is initiated through the binding of specific growth factors to receptors which are either linked to G protein or tyrosine kinase signalling pathways (Boume et al., 1990; Cantley et al., 1991). A common feature of both pathways is the intracellular propagation of the response through a family of recently described Ser/Thr kinases (Kozma and Thomas, 1992; Blenis, 1993). One of the early obligatory steps in the proliferative response is a 2- to 3-fold increase in the rate of initiation of protein synthesis, which appears to be controlled by the phosphorylation of a number of key translational components, including initiation factor eIF-4F and ribosomal protein S6 (Hershey, 1989; Morley and Thomas, 1991). In the case of S6, multiple, ordered phosphorylation at five distinct sites residing at the carboxy tail of the protein either triggers or facilitates this process (Morley and Thomas, 1991). The kinase largely responsible for linking receptor activation to S6 phosphorylation has been identified as an Mr 70 000 polypeptide (Jeno et al., 1988, 1989), termed p70s6k (Reinhard et al., 1992; Thomas, 1992). The enzyme

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Oxford University Press

is highly specific for S6 (Jeno et al., 1989), having a Km of 0.2 /AM, with substrate recognition dependent on a block of arginines lying upstream of the first site of phosphorylation (Flotow and Thomas, 1992). In turn, activation of the kinase is associated with multiple phosphorylation (Ballou et al., 1988) at four residues which are clustered in a putative autoinhibitory domain (Ferrari et al., 1992, 1993). Activation is biphasic and there appears to be at least two independent signalling pathways involved (Susa et al., 1989, 1992), each of which is distinct from the p42rapk/p44rakk signalling pathway (Ballou et al., 1991; Blenis et al., 1991). Although the identification of the p70s6k kinase has remained elusive, the four major phosphorylation sites associated with p70s6k activation display Ser/Thr-Pro motifs, typical of cell cycle-regulated enzymes (Ferrari et al., 1992, 1993; Thomas, 1992). Recently, sufficient amounts of activated p70s6k were isolated from the livers of rats treated with cycloheximide (Kozma et al.., 1989; Price et al., 1989; Lane and Thomas, 1991) to obtain protein sequence data and clone p70s6k. Two cDNA clones were isolated: clone 1 encoded a protein of 502 amino acids (Kozma et al.., 1990) and clone 2 encoded the identical protein with a 23 amino acid extension at its amino-terminus (Banerjee et al., 1990; Reinhard et al., 1992). Northern blot analysis with a common cDNA probe revealed four distinct transcripts in rat liver (Kozma et al., 1990). Using specific probes it was possible to assign each clone to a corresponding transcript and to show that both clones were derived from a single gene (Reinhard et al., 1992). In vitro, both clones produced p70s6k, but clone 2 also produced a protein of Mr 85 000, termed p85s6k. This novel isoform was shown to be present in trace amounts in highly purified preparations of p7056k (Grove et al., 1991; Reinhard et al., 1992) and to be activated by mitogenic stimulation (Reinhard et al., 1992). The 23 amino acid extension of p85s6k contains sequence motifs similar to those involved in nuclear targeting, a cellular compartment where S6 has recently been shown to be phosphorylated in response to mitogens (Franco and Rosenfeld, 1990). These findings have raised questions concerning the intracellular localization of both kinases, whether the recently reported inhibitory effects on cell growth elicited by antibody injection into the cytoplasm can be solely ascribed to p70s6k function (Lane et al., 1993), and the importance of having two isoforms of the same kinase generated from a common gene. Here we have used immunofluorescence to localize the endogenous p85s6k, as well as overexpressed p85s6k, in rat embryo fibroblast (REF-52) cells. Using a similar approach, we have determined whether the putative p85s6k nuclear targeting and enhancer sequences can direct a heterologous fusion protein to the nucleus. Finally, we have assessed the functional role of p85s6k in mitogenesis through the microinjection of affinity-purified anti-p85s6k antibodies into either the nucleus or cytoplasm. -

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Results

number of p85s6k molecules per REF-52 cell was roughly estimated by Western blot analysis to be 300. -

Abundance of p85s6k The recently described p85s6k isoform is identical in sequence to p70s6k, except for a 23 amino acid extension at its amino-terminus (Figure IA). This isoform of the kinase was initially detected as a minor component which copurified with rat p70s6k (Grove et al., 1991; Reinhard et al., 1992), suggesting that the enzyme had largely been lost during purification or that this isoform was less abundant in cells than the p7Os6k. To distinguish between these possibilities, REF-52 cell extracts were subjected to Western blot analysis employing an antibody termed Ml, which was generated against a carboxyl peptide sequence common to both isoforms [Figure 1A and Lane et al. (1993)]. The results show that the antibody recognizes both kinase isoforms (Figure 1B, lane 1) and that each reaction is blocked by competing peptide (Figure 1B, lane 2). Of the six peptide antibodies we have generated against p7Os6k/ p85s6k, this is the only antibody which detects the p85s6k on Western blots of total REF-52 cell extracts. Based on this analysis, p85s6k is represented at 15- to 20-fold fewer copies per REF-52 cell than p70s6k, which itself is a rare enzyme (Jen6 et al., 1989). Employing purified baculovirus recombinant p7Os6k (Kozma et al., 1993) as a standard, the A

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Affinity-purified anti-p85s6k antibodies The amino-terminus of p85s6k reveals motifs implicated in classical, as well as recently described bipartite, nuclear localization signals [Figure 1A, Dingwall and Laskey (1991)

and Garcia-Bustos et al. (1991)]. In addition, p85s6k harbours a casein kinase II consensus sequence at Ser4O, whose phosphorylation is known to enhance the rate of nuclear import of SV40 T-antigen (Rihs et al., 1991). To determine whether this sequence targeted p85s6k to the nucleus, affinity-purified IgG, C3a, were prepared from a polyclonal serum, C3, directed against a 15 amino acid peptide spanning residues Asp8 to Asp23 of p85s6k [Figure 1A and Reinhard et al. (1992)]. These antibodies specifically recognize baculovirus recombinant p85s6k on Western blots (Figure IC, lanes 1 and 3) and immunoprecipitate S6 kinase activity (Figure ID, lanes 1 and 3). Both reactions were blocked by competing peptide (Figure 1C and D, lanes 2 and 4) and the corresponding negative IgG, C3 -, failed to recognize p85s6k in either assay (Figure IC and D, lane 5). It should be noted that although these antibodies efficiently immunoprecipitate p85s6k, they inhibit total kinase activity in the immune complex assay by >60%. The C3a antibodies also immunoprecipitated p85s6k activity from REF-52 cells, but did not detect p85s6k by Western blot analysis of these same extracts (data not shown), consistent with all of the antibodies we have generated against p7os6kIp8Ss6k, except M1 (Figure 1B), as well as the low p85s6k copy number. Although these antibodies did not recognize p85s6k on Western blots of total cell extracts from REF-52 cells, it was reasoned that they might still be useful in immunofluorescence and functional studies. Localization of p85s6k

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Fig. 1. Characterization of p70s6k and p85s6k antibodies. (A) The amino-tenninal extention unique to the p85s6k isoform and two internal sequences common to both isoforms are shown in single-letter code, clusters of basic amino acids representing putative nuclear targeting sequences (stippled), peptide sequences used to generate polyclonal antisera either specific for p85s6k (C3) or p70s6k and p85s6k (Al and Ml), common catalytic domain (black), carboxyl-terminal regulatory domain harbouring the Ser/Thr-Pro phosphorylation sites (hatched area). (B) A Western blot of 50 /tg total REF-52 cell extract was incubated with Ml antiserum (Lane et al., 1993) in the absence or presence of competing peptide (lanes 1 and 2, respectively). The position of both isoforms is indicated on the left. (C) Western blot analysis using anti-p85s6k antibodies. Baculovirus recombinant p85s6k-infected Sf9 cell extracts (1 /Lg) were incubated with total C3 IgG (lanes 1 and 2), affinity-purified C3a IgG (lanes 3 and 4) or negative C3- IgG (lane 5). Competing peptide was added at 5 /4M to reactions depicted in lanes 2 and 4. (D) Baculovirus recombinant p85s6k was immunoprecipitated from 5 jig of infected Sf9 cell extracts employing the C3 IgG (lanes 1 and 2), affinity-purified C3a (lanes 3 and 4) or the C3- IgG (lane 5) was assayed in the immune complex assay for S6 kinase activity. Competing peptide was added at 5 jiM to assays depicted in lanes 2 and 4.

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The localization of endogenous p85s6k was examined by indirect immunofluorescence in REF-52 cells employing either C3a or C3- IgG. In addition, a second monospecific antibody directed against a common p7Os6k/p85s6k amino acid sequence, termed Al (Figure IA), was tested, as well as its corresponding negative IgG, Al-, depleted of all anti-p70s6k/p85s6k antibodies by affinity purification (Lane et al., 1993). Whereas the C3a antibody showed strong preferential staining of nuclei (Figure 2A), the Al antibody stained both compartments (Figure 2C), as would be expected if the p7Os6k isoform resides in the cytoplasm. It should be noted that because the p7Os6k and p85S6k isoforms are relatively rare enzymes, long exposure times were required to detect either kinase (see the legend to Figure 2). Employing confocal laser microscopy, it was found that > 97 % of C3a IgG staining was nuclear, with the remaining cytoplasmic staining accounted for by the secondary antibody (Materials and methods). No staining could be detected with either the C3- or Al - IgG (Figure 2B and D, respectively). These results strongly indicated that p85s6k resides largely, if not exclusively, in the nucleus.

Localization of overexpressed p85s6k Extensive attempts to corroborate a nuclear location for p85s6k employing a number of standard biochemical

Nuclear p85s6k is required for entry into S phase

protocols failed (data not shown, see Discussion). Therefore, an expression plasmid under the control of the SV40 early promotor was prepared which only expressed p85s6k

(Materials and methods). This plasmid, pSV85, was then microinjected together with a mouse marker antibody into the nuclei of quiescent REF-52 cells which had been

Fig. 2. Immunolocalization of endogenous p85s6k. Exponentially growing REF-52 cells were cultured on coverslips as described (Materials and methods) and immunostained with the following antisera: (A) C3a IgG; (B) C3- IgG; (C) Al purified total IgG; (D) negative Al - IgG. The localization of nuclei in cells shown in (A)-(D) is depicted by Hoechst (33258) staining in (E)-(H), respectively. Exposure times for the C3 and Al antibodies were 70 and 15 s, respectively.

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stimulated for 2 h with 10% (v/v) fetal calf serum (FCS). After 17 h, cells were fixed and the localization of p85s6k was determined by immunofluorescence employing either the C3a or Al antibody. The results show strong nuclear staining with either the C3a or the Al antibody in cells in which the plasmid expressed protein (Figure 3A and D, respectively), indicating the accumulation of p85s6k exclusively in the nucleus. This is readily visible by comparing the staining of p85s6k in the plasmid-injected cell (Figure 3A) with uninjected cells (Figures 2A and 3A). In contrast, the Al antibody also exhibited cytoplasmic staining (Figure 3D), reflecting the presence of endogenous p7Os6k. The data taken together strongly indicate that p85s6k is a resident nuclear protein.

Nuclear localization of a p85s6k - CAT fusion protein To determine whether the putative nuclear targeting and enhancer sequences at the amino-terminus were sufficient to localize p85s6k to the nucleus, a cDNA fragment encoding the first 48 amino acids, with the second translational start site removed, were fused to CAT within a pSVLderived expression vector (Materials and methods). Either this plasmid or a plasmid expressing CAT alone were microinjected together with a rabbit marker antibody into the nucleus of quiescent REF-52 cells 2 h following stimulation with 10% (v/v) FCS. After 17 h, cells were fixed and stained with a monoclonal antibody directed against CAT (Davies et al., 1986). Immunostaining of cells which expressed the p85s6k-CAT fusion protein with the CAT

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Fig. 3. Immunolocalization of overexpressed p85s6k. A plasmid expressing only the p85S6k isoform together with an inert mouse marker IgG was microinjected into serum-stimulated REF-52 cells (Materials and methods). Plasmid-injected cells were identified by staining of the marker antibody (B). Staining with C3a IgG (A) shows a cell overexpressing p85s6k (marked by an arrow). Cells injected with the same plasmid (E) were also stained with total Al IgG (D) to identify cells overexpressing p85s6k (arrow). (C) and (F) show Hoechst (33258) staining of the corresponding fields.

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Nuclear p85S6k is required for entry into S phase

monoclonal antibody revealed almost exclusive accumulation of the p85s6k-CAT fusion protein in the nucleus (Figure 4A). In contrast, the CAT protein expressed alone was found throughout the cell (Figure 4D), as was a p7Os6k-CAT fusion protein containing only the casein kinase 11 phosphorylation site, p7Os6k (Metl -Gly24) (data not shown). These results argue that the 23 amino acid terminal extension of p85s6k is sufficient to localize it to the nucleus. Nuclear injection of C3a IgG blocks G1 progression In a number of recent studies, antibodies have been used to assess the importance of their antigenic target in vivo (Mulcahy et al., 1985; Girard et al., 1991; Lane et al., 1993; Pagano et al., 1993; Twamley-Stein et al., 1993). To

determine whether the C3a antibody could alter the mitogenic phenotype, it was microinjected into either the nucleus or the cytoplasm of quiescent REF-52 cells, which were then stimulated with serum to proliferate. In cells whose nuclei were injected with C3a antibodies (Figure 5A), S phase entry was blocked, as measured by the incorporation of 5-bromodeoxyuridine (BrdU) into DNA (Figure 5B). In contrast, no inhibition of entry into S phase was detected when the C3a antibody was injected up to a concentration of 3 mg/ml into the cytoplasm (Figure 5A and B), nor when the C3- antibody was injected into either cell compartment (Figure 5C and D). Similarly, no effect was observed when a polyclonal IgG to the myosin light chain kinase (Lamb et al., 1988) was injected at a 3-fold higher concentration into either cell compartment (data not shown). The *__ill __ : ' _'............................:..JS1 .:.=WM'= . >f¢

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Fig. 4. Imnmunolocalization of a p85S6k-CAT fusion protein. Plasmids expressing a p85S6k-CAT fusion protein (A) or the CAT protein alone (D) together with an inert rabbit marker IgG were injected into serum-stimulated REF-52 cells (Materials and methods). Subsequent staining with monoclonal anti-CAT antiserum allows the localization of the CAT protein (A-D). Cells injected with the plasmid construct are identified by staining of the mouse marker antibody (B and E). The nuclei of A and B and D and E are revealed by Hoechst (33258) staining (C and F, respectively).

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Fig. 5. Injection of affinity-purified p85S6k antibody into the nucleus blocks G1/S transition. Affinity-purified C3a IgG (A and B) and negative C3IgG (C and D) were microinjected into quiescent REF-52 cells, followed by 10% (v/v) serum stimulation for 32 h in the presence of BrdU. Panels A and C show staining for rabbit IgG. Panels B and D show the corresponding cells stained for BrdU incorporation. The injected compartment is marked by white arrows. Table I. Effect of anti-p85s6k injection on S phase entry

Antibody C3a C3-

% cells

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Nucleus Cytoplasm Nucleus Cytoplasm

22/3, 28/4 19/17, 50/46 26/22, 34/32 34/30, 11/9

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Injected

Each set of numbers represents an independent experiment performed as described in the legend to Figure 5.

quantitation of the results obtained with the C3a and C3 antibody in Figure 5 are presented in Table I. Further studies also showed that the C3a antibody could exert its inhibitory effect on S phase entry at any point shortly prior to the initiation of DNA synthesis (data not shown). That the inhibitory effects of the C3a antibody were observed only when the antibody was injected into the nucleus provides compelling evidence for the functional localization of p85s6k in this compartment of the cell.

p7&S6k rescues the C3a antibody inhibitory block

The results above did not exclude targets other than p85s6k whose function might be inhibited by the C3a IgG. The epitope used to generate a specific antibody was necessarily restricted to a short domain (Figure IA) and covers a sequence which may be involved in localizing the kinase to the nucleus, a sequence which could be postulated to be

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present in other proteins. It should be noted that a search of a number of databases revealed no other protein which shared this sequence and a second antibody generated against a peptide spanning Arg7 to Argl7 of p85s6k exerted a similar inhibitory effect on S phase entry (data not shown). To address rigorously the question of specificity would require that the antibody inhibitory effect be rescued by a form of p85s6k lacking the antigenic epitope. The p7Os6k isoform meets this criterion (Figure IA), and it was reasoned that the absence of a nuclear localization signal could be overcome by microinjection of an activated form of the enzyme into the nucleus. The most active preparations of p7Os6k are derived from rat liver (Kozma et al., 1989); however, prior to microinjection trace amounts of p85s6k (Reinhard et al., 1992) had to be removed by passing the purified preparation of rat p7Os6k kinase over a C3a antibody affinity column. After such treatment, no detectable

Nuclear p85s6k is required for entry into S phase

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Fig. 6. Co-injection of anti-p85S6k IgG with active p7oS6k rescues G1 progression. Quiescent REF-52 cells were microinjected ' with C3- IgG (1), C3a IgG (2), C3a together with active rat liver p7OS (3) or C3a together with heat-inactivated rat liver p70S6k (4). Entry iinto S phase was monitored by BrdU incorporation. Each value repressents the average of at least two independent experiments in which -40 cells were injected per experiment.

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p85s6k activity was present in the preparatio in (data not shown). Co-injection into the nucleus of REF -52 cells of active p7Os6k with the C3a inhibitory antibody enabled 65 % of the cells to enter S phase, whereas only 109GO entered in the presence of the heat-inactivated p7Os6k (Figure 6). Microinjection of the p7Os6k alone into the nucleus or cytoplasm had no effect on DNA synthesis (data not shown). It should also be noted that the C3a antibody inhibitory effect was not rescued by injection of rat liver p7C)s6k into the cytoplasm, nor by co-injection of recombinant p)70s6k whose specific activity is significantly lower than thait of the rat enzyme (Ferrari et al., 1993; Kozma et al., 1 993). These fmdings strongly support the conclusion th,at the C3a antibodies specifically recognize the nuclear p85;s6k, and that these antibodies block a physiological process M vhich can be functionally restored by active p70s6k.

Discussion Recently, it was reported that a nuclear prote-in, initially termed BRP for basic regulated phosphoprotei n (Murdoch et al., 1983), which became phosphorylated in response to mitogenic stimulation, was equivalent to 40' 3 ribosomal protein S6 (Franco and Rosenfeld, 1990). Signiificantly, S6 was found in a free form associated with chromtatin as well as in nucleoli, and the tryptic-phosphopeptide ma]ps generated from either cytoplasmic or nuclear S6 were relported to be identical. One hypothesis put forward for this observation was the possible existence of a mitogen-activeated nuclear S6 kinase. Initial attempts to localize the pt 85s6k to the nucleus by standard cell fractionation procedur es failed, as in a number of other cases (for a discussion see Gordon et al., 1981; Bensch et al., 1982; Krek et al., 1992). However, the results obtained by immunc)localization of either endogenous (Figure 2), overexpre: ssed p85s6k (Figure 3) or p85s6k -CAT fusion protein (Figure 4) indicated that this form of the kinase is a resi(dent nuclear

protein. This finding is also supported by the fact that the C3a antibody blocked entry into S phase only when injected into the nucleus (Figure 6 and Table I). Consistent with these results, the unique 23 amino acid extension of rat p85s6k contains two possible nuclear localization motifs (Dingwall and Laskey, 1991; Garcia-Bustos et al., 1991): the classical SV40 targeting sequence and the recently described bipartite nuclear targeting motif. The bipartite nuclear targeting sequence is not conserved in rabbit and human p85s6k (Harmann and Kilimann, 1990; Grove et al., 1991) and, thus, nuclear localization is most likely conferred by the first cluster of basic amino acids, Arg2-Arg7. Site-directed mutagenesis studies should resolve which of these motifs is being employed to direct p85s6k to the nucleus (Cochrane et al., 1990). Increased S6 phosphorylation is argued to exert its effect on the activation of protein synthesis and the pattern of translation (see Hershey, 1989; Morley and Thomas, 1991). This argument is supported by demonstration that cytoplasmic injection of any of three inhibitory antibodies against three distinct

sequences

shared by

p70s6k/p85s6k

blcseu-nueaciainoprtisyteiadety block serum-induced activation of protein synthesis and entry into S phase (Lane et al., 1993). However, the absence of protein synthesis in the nucleus suggests a unique role for p85s6k and nuclear phosphorylated S6 in GI progression. One intriguing possibility is that nuclear S6 phosphorylation is involved in regulating ribosome biogenesis. The protein is an early assembler into 80S preribosomal particles (Todorov et al., 1983), and ribosome biogenesis is dramatically up-regulated in a number of systems following anabolic stimulation (Emerson, 1971; Morgan and Peters, 1971). Alterations in this process may be crucial for mitogenesis. Another possibility is the recognition and selective transport of essential mRNA transcripts. The large increase in nuclear mRNA export following mitogenic stimulation (Johnson et al., 1975) appears to be exerted at the processing level of specific transcripts. In turn, phosphorylated ribosomes can selectively interact with different mRNA transcripts (Palen and Traugh, 1987). It may be that such an interaction is involved in the export of essential mRNA transcripts involved in cell growth. Finally, it cannot be excluded that the p85s6k has an essential as yet unidentified nuclear target. The inhibitory effect of the C3a antibody can be rescued by active p7Os6k lacking the antigenic epitope (Figure 6), implying that the C3a IgG blocks p85s6k function and does not exert this effect by simply altering the microenvironment in which the kinase resides. Indeed, microinjection of the Al antibody into the nucleus elicits a similar inhibitory effect on S phase entry (data not shown). Employing a similar antibody inhibitory approach, others were able to rescue the block in S phase entry by injecting plasmids which overexpressed the antigen (Pagano et al., 1993; TwamleyStein et al., 1993). Employing this approach to rescue p7Os6k/p85s6k function, we found that overexpression of either kinase blocked entry into S phase. This effect could be attributed to overexpression of kinase activity or that the regulatory domain of p70s6k/p85s6k (Ferrari et al., 1992, 1993) may act as a sink for an upstream kinase which has other essential substrates besides p70s6k/p85s6k. It should also be noted that the C3a antibodies only exhibit their inhibitory effects when injected into the nucleus, indicating

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that inhibition of S phase entry through cytoplasmic injection of anti-p7Os6k/p85s6k antibodies is most likely elicited through inhibition of p7Os6k. This finding, combined with the fact that the block of S phase entry upon C3a antibody nuclear injection is rescued by microinjection of exogenous p7Os6k into the nucleus, but not the cytoplasm or by the endogenous enzyme, indicates that p7Os6k may be confined to the cytoplasm. Consistent with this result, quantitative confocal microscopy employing the Al antibody suggests that there are 10-12 more copies of the antigen in the cytoplasm than in the nucleus, which closely agrees with the ratio of p70s6k to p85s6k in these cells, as estimated by Western blot analysis. In addition, microinjection of p7Os6k expression vectors into REF-52 cells leads to initial accumulation of the overexpressed protein in the cytoplasm, although as more protein is produced it can also be detected in the nucleus (C.Reinhard, A.Fernandez, G.Thomas and

N.J.C.Lamb, unpublished). The mechanism by which the inhibitory antibodies block entry into S phase can only be speculated. The IgG used to block either p7Os6k function (Lane et al., 1993) or p85s6k (Table I) function inhibits S6 kinase activity in immunocomplex assays to different degrees. It is possible that the formation of the immune complex between p7Os6k or p85s6k and inhibitory IgG might impair the ability of the kinase to interact with S6, which itself is part of a large multimeric particle. It is also possible that the association of the antibody with the antigen in the subcellular microenvironment of the kinase places constraints on the ability of the enzyme to access the substrate. In this regard, it will be important in

the future to determine whether the earlier demonstrated cytoplasmic antibody block of p7Os6k (Lane et al., 1993) can be rescued by a kinase lacking the antigenic epitope. Differential compartmentalization of p7Os6k and p85s6k raises the question as to whether both isoforms lie on the same signalling pathway. Although we have not identified the phosphorylation sites associated with mitogenic activation of p85s6k, active baculovirus-expressed recombinant p7Os6k and p85s6k display the same tryptic phosphopeptide pattern (Kozma et al., 1993) as the mitogen-activated p7Os6k (Ferrari et al., 1992). Furthermore, a number of recent reports demonstrate that the immunosuppressant, rapamycin, selectively blocks p7Os6k/p85s6k activation without affecting p74raf, p42mapk/p44mapk or pgorsk activation (Calvo et al., 1992; Chung et al., 1992; Kuo et al., 1992; Price et al., 1992). The simplest interpretation of the results is that a common kinase, present in both cell compartments, activates p70s6k and p85s6k. This view is supported by the fact that we observe no redistribution of p85s6k following serum stimulation of quiescent cells (data not shown). In addition, cytoplasmic injection of the C3a antibody, which binds next to the nuclear targeting sequence and which might be expected to block the shift of cytoplasmically activated p85s6k to the nucleus, does not block entry into S phase. The regulation of S6 phosphorylation is controlled through a complex network of regulatory events (Kozma and Thomas, 1992). This complexity is further magnified by its apparent modulation in two distinct cellular compartments. It will now be necessary to elucidate the mechanisms by which p70s6k and p85s6k functions are co-ordinately integrated within these compartments, as well as the differential regulation of their two transcripts, one of which expresses

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only p7Os6k and the other which produces both isoforms. Finally, an obvious challenge in the near future will be to discern the functional role of S6 phosphorylation in the nucleus.

Materials and methods Recombinant plasmids

To generate plasmids that express only p85S6k, the second methionine of the p2BK cDNA, the translational start site for p70s6k (Reinhard et al., 1992), was mutated by polymerase chain reaction (PCR) (Ho et al., 1989) to alanine. The final PCR product was digested with NotI and SpeI and the resulting 240 bp fragment was subcloned into p2B4 (Reinhard et al., 1992). This clone was then digested with XbaI and PstI and the insert introduced into the same restriction sites of pSVK3 (Pharmacia) to form pSV85. For the chimeric plasmid expressing the p85S6k-CAT fusion protein, the first 150 nucleotides of pSV85 were obtained by PCR and inserted into pSC70 (Bugler et al., 1990) after digestion with XhoI and ApaI.

Antibody purification To obtain affinity-purified IgG, an anti-peptide serum specific for p85s6k, C3 (Reinhard et al., 1992), was first purified over a protein A - Sepharose CL-4B (Pharmacia) column (Harlow and Lane, 1988) and then passed three times over a peptide affinity column containing the p85s6k antigenic peptide coupled to CNBr-Sepharose (Pharmacia). The final flowthrough, depleted of all specific IgG, was used as 'negative' control serum (C3-) and the affinity-purified C3 antibody (C3a) was eluted as described (Harlow and Lane, 1988). Both antibody preparations were dialysed overnight against phosphate-buffered saline (PBS), concentrated in a Centricon 30 (Amicon) to > 1 mg/ml and stored at -70°C. The specificity of the antibody preparations was tested with baculovirus recombinant p85s6k (Kozma et al., 1993) by either immunoprecipitation of 5 jig Sf9 baculovirus-infected cell extracts or on Western blots containing 1 Ag/lane of the same extract. All procedures were carried out as previously described (Reinhard et al., 1992). Cell culture and immunofluorescence Rat embryo fibroblasts REF-52 (McClure et al., 1982) were seeded on acid-

treated glass coverslips and grown in DMEM supplemented with 7.5 % (v/v) FCS (Lamb et al., 1988). Quiescent cells were obtained after incubation in serum-free DMEM for 24-36 h (Lamb et al., 1990). For immunofluorescence studies, cells were fixed in 3.7% (v/v) formaldehyde and permeabilized in acetone at -20°C for 30 s (Girard et al., 1991). Incubation with the primary antibodies, diluted 1: 100 in PBS containing 1 % (w/v) bovine serum albumin (BSA), was carried out for 1 h at 37°C. The secondary fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibody (Cappel-Organon Technica) was diluted 1:20 in PBS-BSA and applied for 30 min at 370C. Confocal scanning To determine more precisely the cellular locale of p85s6k, cells stained for C3a IgG as described in Figure 3 were analysed by confocal laser scanning microscopy. Single 200 nm confocal sections were made through the nuclei of stained cells using a Leica CLSM. To confirm that the sections were within the nuclear envelope, cells were either counterstained for nuclear lamins or co-stained with a DNA-reactive dye ToPro (Molecular Probes, OR). Single data sets representing 10 x 10 x 0.2 ,tm image files were directly collected into a shared memory interface between the CLSM and an Iris Indigo (Silicon Graphics, Mountain View, CA) running VoxelView Ultra (Vital Images, Fairfield, IA). Sections were analysed directly without further image processing using VoxelAnalyser. Measurements were made on both single points (Seed) and areas. Areas were chosen randomly inside the nuclear envelope and in the cytoplasm. Statistics were collected and areas were corrected such that similar volumes (cytoplasmic and nuclear) were compared. To obtain a representative quantification, 247 independent cells were analysed. The value for background staining was obtained by analysing cells stained with the secondary antibody alone.

Microinjection and BrdU labelling Stimulated cells injected with antibodies were monitored for entry into S phase by BrdU staining as previously described (Girard et al., 1991). Plasmids expressing p85S6k CAT or p85S6k-CAT fusion proteins were coinjected into the nucleus with either mouse or rabbit IgG, respectively. Following fixation of cells (see above), cells were stained as previously described (Girard et al., 1992).

Nuclear p85S6k is required for entry into S phase

Acknowledgements We would like to thank Drs K.Ashbridge, S.C.Kozma, H.A.Lane, R.Pearson and H.Suidan for their critical reading of the manuscript. We would also like to thank C.Wiedmer for her editing and typing expertise, I.Obergfoell for photography and Dr Ya-zhou Sun for his help in preparing plasmid constructs. F.Almaric kindly provided the CAT vector and A.Matus the CAT monoclonal antibody.

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Received on December 23, 1993; revised on January 24, 1994

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