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Cytoskeletal polarization of T cells is regulated by an immunoreceptor tyrosine-based activation motif-dependent mechanism.

Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell-cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex-dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-zeta and CD3epsilon, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-zeta and once in CD3epsilon, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.

did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-and CD3 ⑀ , were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-and once in CD3 ⑀ , revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements. P olarization of a T cell response towards a triggering antigen-presenting cell (APC) 1 is thought to contribute to the specificity of the immune response. Upon encountering an APC, T cells rapidly undergo cytoskeletal polarization, which includes the formation of a tight collar of polymerized actin at the T cell-APC interface and the reorientation of the microtubule-organizing center (MTOC) towards the bound APC (Geiger et al., 1982;Ryser et al., 1982). Whereas F-actin accumulation at the cell-cell interface was suggested to stabilize and favor continuous T cell antigen receptor (TCR)-antigen interactions (Valitutti et al., 1995 b ), MTOC reorientation is thought to position the T cell secretory apparatus into close proximity with the APC. Indeed, several studies showed that repositioning of the MTOC was accompanied by the polarized concentration of cytokines and cytotoxic mediators at the T cell-APC/target interface (Kupfer and Dennert, 1984;Kupfer et al., 1991Kupfer et al., , 1994. Moreover, Poo et al. (1988) have demonstrated that limited TCR crosslinking in a cloned helper T cell line triggered the release of cytokines preferentially over the area of receptor crosslinking. Thus, the engagement of only a fraction of TCRs on a helper T cell results in the polarized delivery of cytokines towards the site of the stimulus.
Cytoskeletal polarization of a T cell is dependent on the presence of specific antigen (Kupfer and Singer, 1989), suggesting the involvement of a TCR-mediated signaling pathway. A variety of cellular responses, including gene activation, proliferation, and secretion of cytokines and cytotoxic mediators, have been reported to be initiated by TCR engagement. Interestingly, activation of Jurkat T cells with immobilized TCR-specific antibodies could induce actin polymerization and formation of F-actin-rich pseudopods, suggesting that TCR-mediated signaling pathways may indeed regulate cytoskeletal organization (Melamed et al., 1991). Furthermore, a recent report implies a role for the small GTPase CDC42 in APC-mediated T cell polarization (Stowers et al., 1995). Members of the Rho subfamily of small GTPases (Rho, Rac, CDC42) are evolutionary conserved regulators of the actin cytoskeleton in a variety of different cell systems (Hall, 1994). Consequently, TCRmediated signals might activate small GTPases by an as yet unidentified mechanism. However, T cell-APC conjugate formation involves the clustering and engagement of various other T cell surface receptors besides TCR-antigen interactions, including the CD8/CD4 coreceptors and integrins (Kupfer and Singer, 1989;Kupfer et al., 1987). It is conceivable that triggering of these additional receptors is required for or at least contributes to APC-induced T cell polarization.
The TCR is coupled to intracellular signaling pathways by two noncovalently associated signal-transducing complexes, namely the CD3 ( ␥ , ␦ , and ⑀ ) and the TCRchains. Signaling through these complexes requires a conserved sequence motif (YxxLx(6-8)YxxL), termed the immunoreceptor tyrosine-based activation motif (ITAM) (Irving and Weiss, 1991;Romeo and Seed, 1991). The CD3 and TCR-chains each contain one and three ITAMs, respectively. Upon TCR engagement, the tyrosine residues in the ITAMs are rapidly phosphorylated by the Src-family protein tyrosine kinase (PTK) Lck (van Oers et al., 1996) and serve as docking sites for src-homology-domain 2 (SH2) containing signaling molecules such as the PTKs of the Syk/zeta-associated protein 70 (ZAP-70) family. After recruitment, Syk/ZAP-70 is activated by phosphorylation and contributes to the initiation of downstream signaling events such as calcium mobilization and activation of the Ras pathway. Single CD3 and TCRchains can independently activate a variety of qualitatively similar proximal and distal signaling events (Irving and Weiss, 1991;Letourneur and Klausner, 1991;Romeo and Seed, 1991;Shinkai et al., 1995), suggesting that the presence of multiple ITAMs in the TCR mainly serves the purpose of signal amplification. However, amino acids surrounding the ITAMs differ in the CD3/TCR chains and have been shown to bind to distinct SH2-containing signaling molecules (Osman et al., 1995(Osman et al., , 1996Songyang et al., 1993). This suggests that, in addition to signal amplification, distinct ITAMs might couple the receptor to qualitatively different signaling pathways. For example, chimeric proteins containing the cytoplasmic tail of CD3 ⑀ or TCRdiffer in their ability to induce apoptosis in a murine T cell line (Combadiere et al., 1996). Furthermore, the third TCR-ITAM and the CD3 ⑀ ITAM have been implicated in coupling the TCR to the actin cytoskeleton in an activation-dependent manner (Rozdzial et al., 1995). Thus, individual subunits might couple the TCR to effector molecules involved in the regulation of cytoskeletal changes.
To investigate the role of TCR-mediated signaling pathways in APC-induced T cell polarization, we set up a conjugation assay using Jurkat T cells or mutants derived from it as effector cells, with antibody-coated, cell-sized la-tex beads as APCs. We show here that TCR cross-linking alone is sufficient to induce MTOC polarization and actin polymerization at the T cell-bead interface. Cytoskeletal reorientation required the presence of at least one intact ITAM. In support of this result, no cytoskeletal changes could be observed in the Lck-deficient Jurkat mutant JCaM1.6. Furthermore, our studies demonstrate that MTOC reorientation occurs in the absence of Ras activation. Our results imply that APC-induced T cell polarization is a TCR-mediated event that is coupled to the receptor by the same signaling motif as TCR-mediated gene activation, but diverges in its distal signaling requirements, since Ras is not required. The assay described in this study might be a useful tool to characterize signaling molecules involved in T cell polarization.

Cells and Reagents
The Jurkat T cell subclone E6, JCaM1.6 (Goldsmith et al., 1988) and large T antigen (TAg) Jurkat cells (kindly provided by Dr. G. Crabtree, Stanford University, Palo Alto, CA; Clipstone and Crabtree, 1992) were maintained in RPMI 1640 medium supplemented with 5% fetal calf serum (Intergen Co., Purchase, NY), 2 mM glutamine, 50 U/ml penicillin, and 50 g/ml streptomycin. Jurkat-derived stable clones were cultured in complete medium containing 10% FCS and 2 mg/ml G418 (GIBCO BRL, Gaithersburg, MD). JCaM1.6/Lck cells (Straus and Weiss, 1992) were maintained in complete medium containing 10% FCS and 250 g/ml hygromycin (GIBCO BRL). Polyl -lysine was obtained from Sigma Chemical Co. (St. Louis, MO). The MAPKK inhibitor PD 098059 and Wortmannin were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). Polystyrene latex microspheres (diameter 6 m) were purchased from Polysciences Inc. (Warrington, PA). Antibodies were absorbed to the beads as previously described (Mescher, 1992). Briefly, 5-10 g of purified antibody were mixed with 10 7 polystyrene beads in a final volume of 1 ml PBS, and incubated for 90 min at room temperature with constant tumbling. Beads were then blocked in 1.5 ml of PBS/1% BSA for 30 min. After three washes in PBS, latex beads were resuspended in PBS and stored at 4 Њ C. Efficient antibody absorption was verified by flow cytometry.

Chimera Construction and Clones
Stable clones expressing the TT-⑀ chimeras were kindly provided by Dr.
Nicolai S.C. van Oers. Plasmids encoding for chimeric proteins containing the external and transmembrane portions of CD25 fused to the cytoplasmic tails of either CD3 ⑀ (TT-⑀ ) or TCR-(TT-) were a gift from Dr. R.D. Klausner (National Cancer Institute, Bethesda, MD; Letourneur and Klausner, 1991). A truncated version of TT-⑀ was generated by PCR mutagenesis of the original TT-⑀ To construct (kindly provided by Dr. R.D. Klausner) using the following primers: 5 Ј primer (within Tac): 5 Ј ACAG-ATCTCCAGGTAGCAGTG 3 Ј ; 3 Ј primer (within CD3 ⑀ ): 5 Ј TCG-ATATCACCTATTCTTGAGCCCACT 3 Ј . Wild-type TT-⑀ in pCDL SR ␣ was replaced by the BglII/EcoRV-digested PCR fragment. The new truncated TT-⑀ encompasses the three first amino acids of the CD3 ⑀ cytoplasmic tail. All constructs were subcloned into pcDNA3 (Invitrogen Corp., San Diego, CA) and transfected into Jurkat T cells by electroporation. Briefly, 10 7 cells were transfected with 20 g of plasmid in a Gene Pulser (Bio-Rad Laboratories, Hercules, CA) at 250 V and a capacitance of 960 F. After transfection, cells were grown for 2 d in complete RPMI before plating out in medium containing 2 mg/ml of G418 (GIBCO BRL). Clones were obtained by limiting dilution. Generation of the CD8clone has been described elsewhere (Irving and Weiss, 1991). Stable cell lines expressing the wild-type CD8-T76 truncation or the CD8-4F mutant were generated as described elsewhere (Irving and Weiss, 1991). Construction of CD8-T76 and CD8-4F vectors was based on modifications of the original CD8plasmid. The CD8-T76 truncation mutant was created by PCR mutagenesis from the CD8wild type using the following oligonucleotides: 5 Ј primer (within CD8): 5 Ј CACCATCGCGTCGCA-GC3 Ј ; T76 truncation: 5 Ј GATCTCGAGAGGATCCTATTACATCCC-AATCTCACTG 3 Ј .
After PCR amplification, the CD8-T76 truncated fragment was digested with EcoRV and BamHI, and substituted for the wild-type CD8in the ptf neo CD8-expression vector (Irving and Weiss, 1991). The mutation was confirmed by sequencing. The CD8-T76 mutant truncated the chain two amino acids after the second ITAM. The CD8-4F mutant was created from the CD8-T76 truncation by sequential PCR mutagenesis. The outside primers and fragment substitution were the same as for the creation of the CD8-T76 truncation. After PCR mutagenesis, the construct was sequenced to verify that only the intended mutations existed.
To mutate the first tyrosine to phenylalanine of the first ITAM of CD8-, the following primers were used: 5 Ј CAGCTCTTTAACGAGCTCAA 3 Ј , and 3 Ј TCTTGGTCGAGAAATTGCTC 5 Ј .

Conjugate Formation and Immunofluorescence Microscopy
Jurkat T cells or derived clones and mutants were mixed with antibodycoated latex beads at a 2:1 ratio. After centrifugation for 5 min at 100 g , the cell-bead mixture was incubated for an additional 5-25 min at 37 Њ C. Conjugates were then resuspended, plated onto polyl -lysine-treated slides, and fixed for 30 min in 3.4% paraformaldehyde at room temperature. Fixed cells were permeabilized for 4 min in PBS/0.1% Triton X-100 and blocked for 10 min in PBS/0.2% BSA. Cells were labeled for 30 min with antibody diluted in PBS/0.2% BSA, followed by two washes of 10 min each. After secondary antibody labeling, cells were incubated for 10 min in 33 nM rhodamin-phalloidin. After three final washes, slides were mounted. Samples were either viewed under a laser scanning microscope (LSM 410; Carl Zeiss, Inc., Thornwood, NY) (1st experiment) or a conventional Microphot-FXA microscope (all other experiments). The MTOC was scored as reoriented if it was located in close proximity to the T cell plasma membrane between the T cell nucleus and the bead contact area. At least 100 conjugates were scored in each experiment.

Transient Transfection Conjugate Formation Assay
Cell (10 7 ) were transiently cotransfected by electroporation, as described above, with 30 g of the indicated TT chimera and 40 g of a vector containing no insert or expressing dominant-negative mutants of ZAP-70 and Ras. After 36 h, dead cells were eliminated by centrifugation over a Ficoll gradient (Sigma Chemical Co.). After two washes, conjugates were formed and analyzed as described above.

Anti-TCR-coated Latex Beads Induce MTOC Reorientation and Polarized Actin Polymerization in Jurkat T Cells
Previous T cell polarization studies performed in a B cell-T cell system demonstrated the dependence of cytoskeletal rearrangements during conjugate formation on the presence of antigen, implying the participation of the TCR in these events (Kupfer and Dennert, 1984). The involvement of accessory molecules including integrins and coreceptors such as CD4/CD8 made it difficult to evaluate to what extent TCR-linked pathways were involved and sufficient for T cell cytoskeletal polarization. To address this question, we set up an in vitro assay using Jurkat T cells as effectors, and antibody-coated, cell-sized latex microspheres as APCs. Previous studies have demonstrated that such artificial APCs, either coated with TCR-specific antibodies or antigen-major histocompatibility complexes (MHC), could induce early and late signaling events, such as tyrosine phosphorylation and exocytosis, in T cells (Anel et al., 1994;Mescher, 1992). In contrast to soluble or platebound TCR-specific antibodies, the use of latex beads allowed us to study signaling events in response to a focal stimulus. In a first set of experiments, we investigated whether triggering of the TCR alone was sufficient to induce reorientation of the microtubule cytoskeleton and polarized actin polymerization. Latex beads coated with the CD3 ⑀ -specific antibody Leu4 were mixed with Jurkat T cells at a 1:2 ratio and incubated for 30 min at 37 Њ C. Formed conjugates were fixed and stained for tubulin and F-actin. The position of the MTOC within the T cells was analyzed microscopically. Only MTOCs directly underlying the bound latex bead were scored positive. In the majority of all conjugates (73 Ϯ 2%), the T cell MTOC was facing the latex bead contact area (Fig. 1 a ). Polymerized actin was concentrated in a dense collar at the bead attachment site. F-actin-rich pseudopods protruded from the T cell surface, and in many cases surrounded the bead ( Fig.  1 b ). Similar results were obtained at shorter incubation times (10-20 min) and with latex beads coated with the Jurkat Ti ␤ -specific antibody C305 (data not shown). In contrast, the position of the MTOC was random when conjugates between Jurkat T cells and polyl -lysine-treated beads were analyzed (Fig. 1 a). Furthermore, no F-actin accumulation at the bead contact site could be observed (Fig. 1 b).
To determine whether cytoskeletal polarization was specifically associated with TCR cross-linking, antibodies directed against a variety of T cell surface molecules (CD11a/ LFA-1, transferrin receptor [TfR] and CD2) and poly-l-lysine were bound to latex beads and used in the conjugate formation assay. All surface molecules analyzed were expressed at comparable levels in Jurkat T cells and similar numbers of bead-cell conjugates were formed (data not shown). As summarized in Fig. 2, TCR/CD3-specific beads displayed MTOC polarization in %08ف of all scored conjugates, whereas beads coated with either anti-CD11a, anti-TfR, anti-CD2, or poly-l-lysine were randomly positioned (31 Ϯ 5, 26 Ϯ 2, 39 Ϯ 4, and 26 Ϯ 6% of scored conjugates, respectively), demonstrating that cytoskeletal polarization is specifically associated with TCR/CD3 cross-linking.

Clustering of Chimeras Containing the Cytoplasmic Domain of CD3⑀ or TCR-Can Induce MTOC Reorientation and Actin Polymerization
Several studies support the view that the presence of multiple ITAMs in the TCR might serve the purpose of signal amplification (Irving et al., 1993;Shinkai et al., 1995). However, single TCR subunits can associate with distinct SH2-containing signaling molecules and show qualitative differences in the induction of certain cellular responses (Combadiere et al., 1996;Osman et al., 1996). This suggests that distinct TCR subunits could couple the receptor to qualitatively different signaling pathways. To evaluate the ability of single TCR chains to induce cytoskeletal changes, we analyzed Jurkat T cells stably expressing a chimera between the extracellular and transmembrane domain of CD25 (Tac) with either the cytoplasmic tail of TCR-(TT-), CD3⑀ (TT-⑀), or a truncated form of CD3⑀ (TT-⑀T) containing only the first three amino acids of the cytoplasmic tail. An ITAM motif is present once in the CD3⑀ chain and three times in the TCR-chain. The ITAMs in both chains differ in their nonconsensus sequences and were shown to bind signaling molecules with different affinities (Osman et al., 1996). As demonstrated previously (Letourneur and Klausner, 1991;Shinkai et al., 1995), TT chimeras are expressed as monomeric molecules and do not associate with endogenous CD3 subunits. Clones with similar surface expression of the chimeric proteins (Fig. 3) were chosen and mixed with anti-Tac-coated latex beads for 10 min. The percentage of conjugate formation was comparable (data not shown). As shown in Fig. 4 A, clustering of the cytoplasmic tail of either TCR-or CD3⑀ was sufficient to induce MTOC reorientation. The extent of MTOC reorientation triggered by either chimera was comparable and was similar to levels induced by crosslinking of the complete TCR-CD3 complex. Furthermore, both chimeras could induce actin polymerization (Fig. 4, B and C). In contrast, no cytoskeletal polarization was de- Figure 2. MTOC reorientation in Jurkat cells is specifically associated with TCR/CD3 cross-linking. Jurkat T cells were mixed at a 1:2 ratio with latex beads coated either with poly-l-lysine or antibodies against CD11a/LFA-1, the transferrin receptor, and CD2. Conjugates were stained with antitubulin antibody. Each column represents the average of at least four individual experiments in which Ͼ100 conjugates were scored for MTOC reorientation. MTOCs positioned between the bead contact site and the T cell nucleus, in close proximity to the T cell plasma membrane, were scored as positive for reorientation. tected towards beads bound to the truncated TT-⑀T chimera. These results suggest that both the and ⑀ chains can trigger qualitatively similar signals, which are normally generated by the TCR and induce T cell polarization. Furthermore, the presence of one ITAM motif was sufficient to induce cytoskeletal rearrangements, and the number of ITAMS had no obvious quantitative effect on the induction of cytoskeletal polarization. However, in transient expression experiments where chimera surface expression was lower, the TT-⑀ chimera induced MTOC reorientation less efficiently than TT-(see below and data not shown). This result supports the view that multiple ITAMs can act as signal amplifiers under suboptimal activation conditions.

MTOC Reorientation and Actin Polymerization Require the Presence of an Intact ITAM
ITAM phosphorylation is essential for signaling through the TCR in many cellular responses. Phosphorylated ITAMs serve as docking sites for downstream signaling molecules such as ZAP-70 (Osman et al., 1996). Furthermore, phosphorylated ITAMs can mediate the activationinduced association of TCR components with the actin cytoskeleton. In particular, the third ITAM of the TCR-chain has been reported to be crucial for F-actin interaction (Rozdzial et al., 1995). To investigate the role of ITAM phosphorylation and F-actin binding in coupling the TCR to T cell polarization, we analyzed stable clones expressing a chimera between the extracellular/transmembrane domain of human CD8 and either the entire cytoplasmic tail of TCR-(CD8-), the cytoplasmic tail of TCR-truncated after the second ITAM (CD8-T76), or the CD8-T76 chimera in which all four ITAM tyrosines were mutated to phenylalanine (CD8-4F) (Fig. 5 A). Clones with comparable surface expression were used (Fig. 6). As shown before for the TT-expressing clone, cross-linking of the CD8-chimera efficiently induced MTOC reorientation and actin polymerization at the bead attachment site (Fig. 5, B and C). No obvious qualitative difference was observed for the CD8-T76 chimera, which lacks the third ITAM, suggested to mediate F-actin binding. Focal actin polymerization was as frequently detected  with CD8-T76 as in the CD8-chimera (Fig. 5 C). As judged by immunofluorescence microscopy, no significant difference in the extent of F-actin accumulation was observed (Fig. 5 D). Our data suggest that the observed clustering of F-actin at the site of TCR engagement occurs in the absence of the third chain ITAM. In contrast, analysis of CD8-4F demonstrated the strict dependence of both MTOC reorientation and actin polymerization on the tyrosine phosphorylation of the ITAM motif. Mixing of clone CD8-4F with anti-CD8-coated latex beads still yielded conjugates, but MTOC positioning was random (38 Ϯ 7%) (Fig. 5 B). The frequency and extent of actin polymerization was also dramatically reduced (Fig. 5, C  and D). These results indicate that ITAM phosphorylation is required for TCR-induced cytoskeletal polarization.
To corroborate this finding, we analyzed cytoskeletal rearrangements in the mutant Jurkat cell line JCaM1.6, which lacks functional Lck. Src family kinases and in particular Lck have been shown to be responsible for ITAM phosphorylation in T cells van Oers et al., 1996). Consistent with the failure to phosphorylate and CD3 chains, TCR-ligation of JCaM1.6 fails to induce tyrosine phosphorylation, calcium influx, or CD69 expression (Straus and Weiss, 1992). Analyzed in our conjugate formation assay, JCaM1.6 failed to polarize towards CD3-specific latex beads. MTOC positioning was random (30 Ϯ 4% of scored conjugates) and no actin polymerization was detected (Fig. 7, A and B). Reconstitution of JCaM1.6 with wild-type murine Lck (JCaM1.6/Lck) restored TCR-induced cytoskeletal changes comparable with that observed in the parental Jurkat cell line (Fig. 7,  A and B). Similar results were obtained with the mutant Jurkat cell line J45.01, which lacks functional CD45 (data not shown). CD45 is a transmembrane tyrosine phosphatase required for the activation of Src family kinases (Weiss and Littman, 1994). Taken together, these results support the view that ITAM phosphorylation and subse-quent recruitment of downstream signaling molecules to the TCR are a prerequisite for cytoskeletal alterations.

Overexpression of a Dominant-Negative ZAP-70 Mutant Impairs MTOC Reorientation
A crucial step in TCR-mediated signaling is the recruitment of ZAP-70 to phosphorylated ITAMs after TCR engagement. Recruitment leads to tyrosine phosphorylation and activation of ZAP-70, which, in turn, contributes to the activation of both early and late signaling cascades. Several studies suggest that ZAP-70 plays in important role in TCR-mediated signaling. For example, lack of functional ZAP-70 in humans and mice results in severe developmental and functional defects in the T cell compartment (Arpaia et al., 1994;Chan et al., 1994;Elder et al., 1994). Furthermore, introduction of a dominant-negative mutant form of ZAP-70 into T cells suppresses TCRmediated tyrosine phosphorylation of several signaling molecules and activation of NF-AT, a nuclear factor essential for inducible interleukin 2 gene expression (Qian et al., 1996). To assess the role of the ZAP-70-ITAM interaction in TCR-mediated MTOC reorientation, we transiently cotransfected Jurkat cells with the activating chimera TT⑀ and the dominant-negative ZAP-70 mutant SH2 (NϩC) (Qian et al., 1996). SH2 (NϩC) is thought to inhibit TCR  signaling by blocking phosphorylated ITAMs (Qian et al., 1996). Transfected cells were mixed with anti-Tac latex beads 36 h later, and conjugates were formed for 10 min at 37ЊC. Similar to our studies in stable clones, engagement of TT-⑀ induced MTOC reorientation, although to a lesser extent (67 Ϯ 1% of the conjugates). As expected, engagement of transiently expressed TT-⑀T failed to induce MTOC reorientation (20 Ϯ 6%) and served as a negative control. Coexpression of dominant-negative ZAP-70 markedly reduced MTOC reorientation by ,%06-05ف whereas expression of a mutant SH2 (NϩC) form (SH2 (N ϩ*C), (Qian et al., 1996), unable to bind to ITAMs, had only minimal effects (Fig. 8). Expression of both mutants did not affect TT-⑀ surface expression (data not shown). Moreover, comparable results were obtained in Jurkat TAg cells (data not shown). These results demonstrate that engagement of transiently expressed activating chimeras can induce MTOC reorientation. Moreover, a dominant-negative mutant of ZAP-70 encompassing the two SH2 domains impairs both TCR-mediated MTOC reorientation (shown here) and NF-AT gene activation (Qian et al., 1996). This suggests that both responses are mediated by the same proximal signaling events requiring molecules that interact with phosphorylated ITAMs.

MTOC Reorientation Occurs in the Absence of Ras Activation
Two major signaling pathways, namely the Ras and the PLC-␥1 pathway, are activated after TCR engagement. Activation of the small GTPase Ras by the TCR induces the extracellular signal-regulated kinase (ERK 1,2) cascade involving several cytoplasmic serine/threonine kinases. ERKs phosphorylate transcription factors of the AP-1 family and are necessary for TCR-mediated NF-AT gene activation and T cell development Izquierdo-Pastor et al., 1995;Swan et al., 1995). In certain cell types, ERKs are associated with microtubules (Morishima- Kawashima and Kosik, 1996;Reszka et al., 1995) and phosphorylate microtubule-associated proteins (MAPs). MAPs stimulate the polymerization of new and preexisting microtubules. Interestingly, these microtubulepromoting properties are lost after in vitro phosphorylation by ERKs (Hoshi et al., 1992). Thus, TCR-induced ERK activation could influence microtubule dynamics. To address this question, we took advantage of the recently described compound PD 098059, a selective inhibitor of the ERK-activating enzyme MAPK/ERK kinase (Dudley et al., 1995). To characterize the effect of PD 098059 on TCR-induced ERK activation, Jurkat T cells were preincubated for 30 min with increasing concentrations of PD 098059 and were stimulated for 10 min by the addition of C305-coated latex beads. Cells were then lysed and analyzed for tyrosine phosphorylation by immunoblotting. As shown in Fig. 9 A, incubation with PD 098059 resulted in a dose-dependent inhibition of ERK phosphorylation in response to TCR engagement, whereas the phosphorylation status of various other proteins remained unchanged. The identity and quantity of ERK detected was confirmed by stripping and reblotting the same membrane with a phospho-ERK-specific antibody (data not shown). This experiment demonstrates that PD 098059 indeed specifically inhibits ERK phosphorylation. However, the inhibitor had no significant effect on MTOC reorientation ( Fig. 9 B), even at concentrations that substantially reduced ERK phosphorylation. This suggests that ERKs are not involved in the regulation of cytoskeletal events associated with MTOC reorientation.
Besides ERKs, the small GTPase Rac-1 and phosphatidylinositol 3 kinase (PI-3 kinase) have been recently identified as Ras effector molecules (Genot et al., 1996;Rodriguez-Viciana et al., 1994). Interestingly, both PI-3 kinase and CDC42, a close family member of Rac-1, have been shown to be involved in B cell-induced MTOC reorientation of a murine T cell hybridoma (Stowers et al., 1995). Thus, TCR-mediated Ras activation might induce MTOC reorientation via ERK-independent pathways. To investigate this possibility, we transiently coexpressed the activating TT-⑀ chimera with the dominant-negative Ras mutant Ha-N17Ras in TAg Jurkat cells. Earlier studies have demonstrated that overexpression of this Ras mutant efficiently inhibits TCR-mediated events such as NF-AT/IL-2 gene activation or T cell development Rayter et al., 1992). However, when analyzed in the polarization assay, the mutant did not significantly affect MTOC reorientation (Fig. 9 C), although high expression levels could be detected by Western blotting (Fig. 9 D). Furthermore, MTOC reorientation in Jurkat T cells pretreated with the specific PI-3 kinase inhibitor Wortmannin was comparable to cells pretreated with the vehicle alone (Fig. 10). These results further substantiate our inhibitor experiment and suggest that Ras-mediated effector pathways are not involved in TCR-mediated microtubule reorganization. In contrast to earlier studies (Stowers et al., 1995), inhibition of PI-3 kinase did not affect MTOC reorientation in our assay system. This discrepancy might reflect differences in the assay systems used.

Discussion
Upon binding to an APC, T cells undergo rapid and dra- Figure 8. Expression of dominant-negative ZAP-70 mutant impairs ITAM-mediated MTOC reorientation. Jurkat T cells were cotransfected with TT-⑀T or TT-⑀ (30 g), together with 40 g of empty vector or plasmids encoding different ZAP-70 mutants. 36 h later, cells were mixed with anti-Tac latex beads and incubated for 10 min at 37ЊC. Conjugates were fixed in paraformaldehyde and stained with antitubulin antibody. Percent MTOC inhibition equals XϪN/MϪN, where X is the percent MTOC reorientation triggered by TT-⑀ in the presence of dominant-negative ZAP-70, M is the percent MTOC reorientation triggered by TT-⑀ alone, and N is the percent MTOC reorientation triggered by TT-⑀T. Results are the average of three independent experiments. The averaged percent MTOC reorientation for each condition was 20 Ϯ 6% for TT-⑀T plus vector, 67 Ϯ 1% for TT-⑀ plus vector, 40 Ϯ 6% for TT-⑀ plus SH2 (NϩC), and 64 Ϯ 5% for TT-⑀ plus SH2 (Nϩ*C).
The Journal of Cell Biology, Volume 140, 1998 868 matic cytoskeletal changes that ensure the polarized delivery of an effector response. To date, T cell polarization has been mainly studied in a B cell-T cell system, where complex receptor-ligand interactions complicate the identification of the participating signaling pathways. In this report, we describe a simple bead-based conjugate formation assay to study this response. Jurkat T cells were chosen as model effector cells. Although this leukemic cell line may differ in many aspects from primary T cells, it has been a powerful tool in dissecting signaling pathways involved in T cell activation. Jurkat T cells can be easily manipulated by transient transfection approaches, and several TCR-signaling mutants have been generated and characterized. APCs have been replaced by antibody-coated latex beads. Since antibody-induced TCR cross-linking may induce stronger signals than TCR engagement by peptide-MHC complexes, our assay system may underscore the need for accessory molecules such as adhesion receptors or coreceptors. Furthermore, T cell activation with immobilized antibodies may not exactly reflect the role of receptor membrane motility and recycling in the generation of an intracellular signal. Nevertheless, the assay system should be useful to characterize the minimal requirements for polarized cytoskeletal rearrangements and to identify keyregulators of this mechanism. Our analysis provides evidence that TCR engagement alone is sufficient to induce MTOC reorientation and focal F-actin accumulation, suggesting that TCR-dependent signaling pathways can regulate cytoskeletal dynamics.
The induction of actin polymerization through antigen receptor activation has been previously observed in a variety of cell types including T cells, B cells, and macrophages (Greenberg et al., 1991;Melamed et al., 1991;Parsey and Lewis, 1993), but so far the underlying signaling pathways have been elusive. In contrast to cell activation by soluble factors, antigen-induced T cell activation is triggered by spatially constrained ligands; thus, TCR-induced cytoskeletal changes could influence the cellular response in several ways. First, cytoskeleton-dependent changes in cell shape and motility could increase the frequency of APC contacts, eventually resulting in the formation of stable conjugates (Negulescu et al., 1996). Second, actin polymerization could be required for the rapid reorganization and concentration of accessory molecules and receptors at the T cell-APC contact site, which would further increase conjugate stability and result in the formation of signaling  complexes (Monks et al., 1997;Shaw and Dustin, 1997). Focally clustered F-actin could also serve as a spatial cue, recruiting proteins involved in MTOC reorientation. Such a role for F-actin has been postulated in the mechanism of spindle rotation during the embryogenesis of Caenorhabditus elegans (Waddle et al., 1994). Third, reorientation of the MTOC and organellar reorganization could ensure the delivery of a polarized immune response by effector T cells to a specific target cell in crowded environments such as lymphoid organs.
Two complexes within the TCR, namely the TCR-and CD3 chains, couple the receptor via ITAMs to the intracellular signaling pathways. Interestingly, individual phosphorylated ITAMs bind differentially to SH2-containing signaling molecules in vitro (Osman et al., 1995(Osman et al., , 1996 and could therefore activate distinct signaling pathways (Combadiere et al., 1996;Letourneur and Klausner, 1992). In our experiments, TCR-and CD3⑀ were equally able to trigger MTOC reorientation and actin polymerization in stable clones. However, when transiently expressed at lower levels, CD3⑀ induced MTOC reorientation less efficiently compared with , supporting the concept that multiple ITAMs can serve as signal amplifiers. ITAM-mediated signal amplification may be crucial when MTOC reorientation is induced by a physiological stimulus; i.e., a peptide-MHC complex. Earlier studies have demonstrated that MTOC reorientation in antigen-specific T cell-B cell conjugates was highly antigen-dose dependent and occurred inefficiently at low antigen levels (Kupfer et al., 1987). Antibody-induced TCR cross-linking, as used in our study, may induce a more potent signal than TCR engagement by peptide-MHC complexes. Thus, the requirements for multiple ITAM-containing TCR/CD3-chains may differ between the in vitro system and the in vivo situation.
A signaling molecule that binds preferentially to ITAMs, but not to the ⑀ ITAM in vitro, is the p85 regulatory subunit of PI-3 kinase (Osman et al., 1996). Interestingly, inhibition of PI-3 kinase with the specific inhibitor Wortmannin completely abolished MTOC reorientation and actin polymerization in a B cell-T cell hybridoma conjugate assay (Stowers et al., 1995). This result suggests that PI-3 kinase is a regulator of T cell polarization. Tested in our system, a TT-⑀ chimera readily induces cytoskeletal polarization. This indicates that PI-3 kinase is either activated independently of its recruitment to the CD3⑀ ITAM or is not involved in the cytoskeletal responses in our assay. Since the PI-3 kinase inhibitor Wortmannin had no effect on T cell polarization in our hands, we favor the second interpretation. The discrepancy observed between the two systems could reflect either cell line-specific differences or the different modes used to induce polarization. To generate a strong enough signal, B cell-T cell conjugates may need the contribution of adhesion molecules that may signal via a PI-3 kinase-dependent mechanism. Furthermore, efficient signaling by peptide-MHC complexes requires the serial engagement of TCRs (Valitutti et al., 1995a). PI-3 kinase could be involved in the recruitment and concentration of TCR molecules at the B cell-T cell contact area.
ITAM phosphorylation is a key event in T cell activation. Our analysis of -chain chimeras and Jurkat mutants demonstrates that MTOC reorientation and actin polymerization require the presence of at least one intact ITAM and a functional ITAM-phosphorylating Src-family kinase. Similarly, Cox et al. (1996) showed recently that clustering of the ITAM-containing Fc receptor ␥ subunit can trigger submembranous actin assembly in the chicken B cell line DT40. Actin polymerization required ITAM phosphorylation and the presence of the PTK Syk. In T cells, the Syk/ZAP-70 family member ZAP-70 can bind to phosphorylated ITAMs of all TCR signaling subunits with very high affinity (Osman et al., 1996). Recruitment of ZAP-70 to the receptor results in ZAP-70 phosphorylation and activation. Overexpression of a dominant-negative mutant of ZAP-70, which blocks phosphorylated ITAMs, markedly reduced MTOC reorientation in our studies. This result suggests that ZAP-70 or another phospho-ITAM-binding molecule may be involved in the regulation of microtubule dynamics. In fact, both Zap-70 and Syk can associate and efficiently phosphorylate ␣-tubulin in vitro (Huby et al., 1995;Peters et al., 1996). Furthermore, two recent reports provide evidence that T cell activation leads to the rapid tyrosine phosphorylation of ␣-tubulin (Cardine et al., 1995;Ley et al., 1994). Importantly, phosphorylated ␣-tubulin was not incorporated into microtubules, but remained in the soluble, unpolymerized tubulin pool. Syk was found to phosphorylate a site in the COOH terminus of ␣-tubulin, which coincides with a region involved in MAP binding (Peters et al., 1996). Since MAPs are involved in regulating the polymerization status of tubulin, it is possible that TCR-induced activation of ZAP-70 leads to the phosphorylation of ␣-tubulin and the subsequent dissociation of MAPs. This in turn could destabilize existing microtubules and lower the pool of tubulin monomers available for polymerization. Such changes in microtubule dynamics would occur locally at the site of TCR engagement and might result in the repositioning of the MTOC. Although the model described is very attractive, the possibility remains that dominant-negative ZAP-70 impairs MTOC reorientation by inhibiting the binding of other SH2-containing effector molecules to ITAMs. Taking advantage of membrane-targeted ZAP-70/Syk chimeras, future experiments will assess whether independent activation of this PTK family is sufficient to induce T cell polarization.
Proteins involved in the regulation of microtubule dynamics are often good substrates for ERKs and protein kinase C (PKC) (Gelfand, 1991). Members of both kinase families are known to be activated upon T cell activation. Thus, apart from the aforementioned ZAP-70-mediated mechanism, changes in the microtubule cytoskeleton could be regulated by one of these pathways. Our experiments show that MTOC reorientation can occur in the absence of Ras activation. Neither overexpression of dominantnegative Ras, nor inhibition of MAPK/ERK significantly affected MTOC reorientation. These results suggest that Ras-independent pathways regulate microtubule rearrangements in response to TCR engagement. In earlier studies, MTOC reorientation was shown to be dependent on extracellular calcium (Kupfer et al., 1987). Incubation of B cell-T cell couples in the presence of EGTA reduced MTOC reorientation from Ͼ90% to 55 Ϯ 5%. Chelation of extracellular calcium showed similar effects in our con-jugate formation assay (data not shown). MTOC reorientation was significantly reduced, but not abolished (from 78% to 50 Ϯ 8%). This suggests that MTOC reorientation may in part be regulated by a calcium-dependent pathway. Indeed, repositioning of the MTOC could be a complex event involving the shortening of microtubules and the subsequent anchoring of the MTOC at the site of TCR engagement. Thus, impairment of one of these events could result in partial MTOC reorientation. A more simple explanation relates to the fact that intracellular calcium stores are not depleted under our assay conditions. Thus, release of intracellular calcium may be sufficient to partially activate calcium-dependent effector molecules. Interestingly, Ca 2ϩ /calmodulin-dependent kinase family members phosphorylate MAPs (Gelfand, 1991). Thus, several kinase families might participate in the regulation of MTOC reorientation.
The assay system described here should prove useful to further characterize the effector molecules involved in T cell cytoskeleton dynamics. Once identified, it will be interesting to assess the physiological role of these effectors in maintaining a polarized intercellular immune response.


extfieldsofstudy: Biology Medicine

text:
Differential Activation of p70 and p85 S6 Kinase Isoforms during Cardiac Hypertrophy in the Adult Mammal*

An adult feline right ventricular pressure overload (RVPO) model was used to examine the two S6 kinase (S6K) isoforms, p70S6K and p85S6K, that are involved in translational and transcriptional activation. Biochemical and confocal microscopy analyses at the level of the cardiocyte revealed that p70S6K is present predominantly in the cytosol, substantially activated in 1-h RVPO (>12 fold), and phosphorylated in the pseudosubstrate domain at the Ser-411, Thr-421, and Ser-424 sites. p85S6K, which was localized exclusively in the nucleus, showed activation subsequent to p70S6K, with a sustained increase in phosphorylation for up to 48 h of RVPO at equivalent sites of p70S6K, Thr-421 and Ser-424, but not at Ser-411. Neither isoform translocated between the cytosol and the nucleus. Further studies to determine potential upstream elements of S6K activation revealed: (i) similar time course of activation for protein kinase C isoforms (α, γ, and ε) and c-Raf, (ii) absence of accompanying phosphatidylinositol 3-kinase activation, (iii) activation of c-Src subsequent to p70S6K, and (iv) similar changes in adult cardiocytes after treatment with 12-O-tetradecanoylphorbol-13-acetate. Thus, these studies suggest that a protein kinase C-mediated pathway couples pressure overload to growth induction via differential activation of S6K isoforms in cardiac hypertrophy.

Hypertrophic cardiac growth occurs by an increase in cellular mass via a relatively coordinate increase in cellular protein content (1). In the terminally differentiated adult cardiac muscle cell, translational mechanisms have a key role in regulating the rate of protein synthesis during hypertrophic growth. Phosphorylation of the carboxyl terminus of 40 S ribosomal S6 protein (S6 protein) has been shown to regulate protein synthesis rate by mediating protein translational initiation and elongation (2), and a growing number of studies show strong correlation between translational efficiency and S6 protein phosphorylation (3)(4)(5). Two distinct families of S6 kinases, p90 Rsk (ribosomal S6 kinase) and p70/85 S6 kinase (S6K), 1 appear to be involved in phosphorylation of the S6 protein (2,6,7). However, S6K is suggested to be primarily responsible for the S6 protein phosphorylation upon stimulation of cells with insulin and a variety of mitogenic stimuli (8 -10).
S6K consists of two isoforms transcribed by a single gene due to alternative splicing: a 70-kDa cytoplasmic isoform (p70 S6K ) and a 85-kDa nuclear isoform (p85 S6K ) with an additional 23 residues at the amino terminus consisting of a putative nuclear targeting sequence (11). Both isoforms are known to phosphorylate the S6 protein and mediate the translation of polypyrimidine tract mRNA, including transcripts of ribosomal proteins and translational elongation factors (12). In addition to playing a role in protein translational control, nuclear p85 S6K has been shown to be important for transcriptional activation (13), increased DNA synthesis, and G 1 to S phase transition (14).
For activation, both isoforms of S6K undergo phosphorylation on multiple sites, which are largely classified into two groups (although the same numbering is used for both p70 S6K and p85 S6K , 23 residues should be added to convert to the numbering of p85 S6K ). The first set of rapamycin-sensitive phosphorylation sites consists of Thr-229 in the catalytic domain and Thr-389 and Ser-404 in the linker region and is implicated in the catalytic activity (15,16). The second set of phosphorylation sites consists of four Ser/Thr residues (Ser-411, Ser-418, Thr-421, and Ser-424) in the pseudosubstrate region of S6K (also known as autoinhibitory domain), and their phosphorylation, except Ser-411, is independent of the rapamycin-sensitive pathway (15). Phosphorylation of these residues in the pseudosubstrate domain has been shown to play a role in regulating kinase function in vivo, although it is not essential for catalytic activity (17,18). Recently, a third phosphorylation locus has been identified in which phosphorylation of the Ser-371 site is shown to be required for kinase activity (19). S6K activation has been shown to be mediated both by protein kinase C (PKC)-dependent and -independent mechanisms (6,20). The PKC-independent pathway has been attributed primarily to growth factor receptor/tyrosine kinase-mediated activation of phosphatidylinositol-3-OH kinase (PI 3-kinase) (21). In the PKC-dependent pathway, S6K activation has been shown to proceed in the absence of PI 3-kinase activation (21)(22)(23).
As S6K is important in the control of both translational and transcriptional activation, the key events mediating cardiac hypertrophy, we initiated a study to determine whether the isoforms of this kinase are activated in pressure-overloaded myocardium. In the present study, we show for the first time a substantial activation of S6K in pressure overloaded myocardium in vivo. This activation is accompanied by a distinct pattern and time course of phosphorylation of the cytoplasmic and the nuclear isoforms of S6K occurring at the level of the cardiocytes of pressure overloaded myocardium. Our studies also indicate that this mechanically induced signaling pathway is mediated by PKC and is independent of PI 3-kinase.

EXPERIMENTAL PROCEDURES
Experimental Animal Models-Right ventricular pressure overload (RVPO) hypertrophy was induced by partial occlusion of the pulmonary artery in adult cats of either sex, weighing 2.6 -3.9 kg, either by external banding (for 48-h and long term RVPO; Ref. 24) or via a balloontipped catheter (for 1-or 4-h RVPO; Ref. 25). In both models, pressure in the right ventricle (RV) was more than doubled, whereas systemic arterial pressure remained unaltered. Controls, as appropriately specified, consisted of sham-operated cats submitted to thoracotomy and pericardiotomy without hemodynamic intervention, or the normally loaded left ventricle (LV) from each cat. At least three animals were used per group for subsequent studies. After removing the heart from the surgically anesthetized cats, the aorta was cannulated, flushed retrograde with ice-cold phosphate-buffered saline, and the heart was either immediately processed or flash-frozen in liquid nitrogen for later use. Cardiocytes were isolated from normal adult cat hearts as described previously (26).
Preparation of Cell and Tissue Lysates-Triton X-100-lysed subfractions, namely Triton X-100-soluble high spin supernatant, Triton X-100-insoluble low spin pellet (cytoskeleton), and high spin pellet (membrane skeleton) from ventricular tissue samples were prepared as we and others have described previously (27,28).
Cytosolic and membrane fractions from ventricles were prepared as described previously with minor modifications (29). Briefly, 100 mg of tissue was homogenized in a Tekmar Tissuemizer (Tekmar Co., Cincinnati, OH) in 2 ml of homogenization buffer containing 30 mM Tris-HCl, pH 7.4, 1 mM DTT, 10 mM EGTA, protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, 2 g/ml pepstatin, 2 M E-64, and 200 g/ml p-aminobenzamidine) and phosphatase inhibitors (1 M okadaic acid, 10 mM ␤-glycerophosphate, and 1 mM sodium orthovanadate). The homogenate was transferred into a Dounce homogenizer (Wheaton Co., Millville, NJ) and further processed with 40 strokes on ice. To remove unbroken cells and debris, the homogenate was centrifuged at 3,000 ϫ g for 10 min at 4°C. The supernatant was transferred to a fresh set of tubes and further centrifuged at 30,000 ϫ g for 30 min. The pellet, which contains predominantly the membrane fraction, was resuspended in 1 ml of SDS sample buffer (30) and boiled for 5 min. The supernatant, referred to as the cytosolic fraction, was used either for kinase assays or for Western blot analysis after boiling with an equal volume of SDS sample buffer.
Immunoprecipitation and Kinase Assays-p70 S6K activity was measured after immunoprecipitation with C-18 antibody using cytosolic samples obtained from control and pressure-overloaded ventricles. For this, 100 l of the cytosolic fraction was added to 0.9 ml of dilution buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, and protease and phosphatase inhibitors as described above). The samples were then mixed with 1 g of antibody or non-immune IgG (controls), and incubated overnight at 4°C. Pre-washed protein A beads (10 l, Pierce) were then added, and the incubation was continued for an additional 1 h. The immune complexes were washed three times with dilution buffer and once with kinase assay buffer (30 mM Tris, pH 7.4, 10 mM ␤-glycerophosphate, 1 mM DTT, and 1 mM EGTA). The immune complexes were then used for measuring kinase activity as defined by Kuppuswamy et al. (29) in the presence or absence of 2 mM synthetic peptide substrate (31), homologous to a region in the S6 protein. In order to measure the p70 S6K activity in TPA-treated cells, adult cardiocytes (4 ϫ 10 6 ) were cultured on 100-mm laminin-coated plates. After 12 h, the cardiocytes were treated with 100 nM TPA for various time periods, washed with phosphate-buffered saline, and used for preparing cytosol as described for the tissue samples except that the homogenization was performed by sonication for 1 min on ice. p70 S6K was immunoprecipitated with C-18 antibody and assayed for kinase activity. For measuring and comparing p70 S6K activity with p85 S6K in pressure-overloaded myocardium, respective antibodies, C-18 and C-3 (specific for p85 S6K ), were conjugated with secondary antibody-coated magnetic beads (Dynabeads), as specified by the manufacturer and used for immunoprecipitation with total extracts obtained with 2% Triton buffer as described previously (27) and assayed for kinase activity.
Measurement of PI 3-Kinase Activity-PI 3-kinase activity was measured similar to the protocol described by Auger et al. (32). Briefly, 50 mg of tissue or 4 ϫ 10 6 cultured cardiocytes were homogenized in 1 ml of extraction buffer containing 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM DTT, and protease and phosphatase inhibitors as described before. The supernatant after centrifugation at 30,000 ϫ g was diluted to 1 ml with extraction buffer (final protein concentration at 0.5 mg/ml), mixed with 1 g of antibody raised against the p85 subunit of PI 3-kinase (Upstate Biotechnology, Inc.; polyclonal), and incubated overnight at 4°C. The immune complex was precipitated with protein-A agarose beads, and sequentially washed as follows: three times with the extraction buffer; three times with 0.1 M Tris, pH 7.4 containing 5 mM LiCl and 0.1 mM sodium orthovanadate; and twice with 10 mM Tris, pH 7.4 containing 150 mM NaCl and 0.1 mM sodium orthovanadate. The PI 3-kinase activity was measured using phosphatidylinositol (PI) as a substrate. The organic phase, containing the lipid products, was used for TLC separation in chloroform/methanol/ water/ammonia (60:47:11:3.2 (v/v)). Unlabeled phosphatidylinositol 3-phosphate was run in parallel to determine the position of phosphorylated PI.
Immunofluorescence Staining and Confocal Microscopy-Fresh frozen ventricular tissue sections (7 m) were fixed and immunolabeled as follows. Sections were cross-linked with 1 mM dithiobissuccinimidylpropionate in Hanks' balanced salt solution at 37°C, extracted with 0.5% Triton X-100 prepared in Hanks' balanced salt solution, and fixed in 4% paraformaldehyde (33). After blocking the sections with 10% goat serum in Tris-buffered saline (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 1% bovine serum albumin, they were incubated with primary antibodies overnight. After washing with Tris-buffered saline, the sections were further incubated with fluorescence-labeled secondary antibodies (Jackson Laboratories Inc., West Grove, PA), as well as TR-phalloidin and YoYo-1 (Molecular Probes Inc., Eugene, OR). Imaging was done on a Bio-Rad MRC-1000 laser scanning confocal microscope, and the pictures were processed using Adobe Photoshop 4.0 software (Adobe Systems Inc., Mountain View, CA).
Statistical Analysis-The ratios of the nuclear phosphorylation signal (P-Thr-421 and P-Ser-424) from RV and LV were calculated as described by Angst (34) and compared for differences between control, 4-h, and 48-h RVPO using an analysis of variance test followed by Fisher's protected least significant difference post hoc test (Super-Anova, Abacus Concepts Inc., Berkeley, CA). After Bonferroni's correction for significance levels, values with p Ͻ 0.016 were considered as statistically significant. Data were tested for normal distribution and homogeneity of variance prior to analysis and shown as means Ϯ S.E.

RESULTS
Pressure Overload Induces Phosphorylation and Activation of the Cytoplasmic p70 S6K -The focus of the present study was to determine whether pressure overload-induced hypertrophic cardiac growth is accompanied by the activation of one or both the isoforms of S6K. In order to determine the complete activation profile, both short term (1-4 h) and long term (48 h or longer) pressure overload feline models were used. Three dif-ferent approaches were utilized to study the changes associated with S6K isoforms: (i) Western blot analysis with either regular S6K antibody or with phosphospecific S6K antibodies for measuring phosphorylation, (ii) immune complex assays for kinase activity, and (iii) confocal immunostaining studies for determining phosphorylation occurring at the level of cardiocytes in the pseudosubstrate domain of cytoplasmic (p70 S6K ) as well as nuclear (p85 S6K ) isoforms of S6K.
To demonstrate whether S6K isoforms are activated in pressure-overloaded myocardium, pressure-overloaded right ventricles and normally loaded same-animal control left ventricles were used to obtain total lysates by extracting with Triton X-100 lysis buffer as described under "Experimental Procedures." After resolving the proteins on SDS-PAGE, Western blotting was performed first with the C-18 antibody that detects both the phosphorylated and unphosphorylated forms of S6K. A 70-kDa protein doublet (Fig. 1A) representing p70 S6K could be readily detected in the normally loaded control ventricles (LV or RV). Analysis of pressure-overloaded right ventricular samples revealed a shift in the mobility of p70 S6K (i.e. disappearance of the lower band with the appearance of a new band on top of the upper band), which is indicative of phosphorylated species of S6K. This change in SDS gel migration of p70 S6K was observed as early as 1 h RVPO, persisted up to 4 h, reverted partially at 48 h (i.e. reappearance of the bottom band), and returned to the control level in 1-week pressure-overloaded RV. The detection of p85 S6K (Fig. 1A), which also showed a doublet in control ventricles, revealed distinct differences in pressure-overloaded ventricles, when compared with p70 S6K . Although retarded mobility was similar at 1 and 4 h RVPO, p85 S6K did not decline appreciably at 48 h RVPO (i.e. the lower band for p85 S6K was still absent in contrast to the reappearance of the lower band for p70 S6K ) and was back to control levels only at the later time point of 1-week RVPO. Furthermore, the exposure time for p85 S6K during ECL development of the Western blots needed to be doubled. This relatively low level of detection could be due to either an isoformspecific change in the antibody binding epitope or low levels of the p85 S6K isoform in the heart. We confirmed these changes at each time point of pressure overloading with at least three independent experimental cats. In summary, the data clearly indicate that both isoforms of S6K exhibit mobility band shift on SDS-PAGE in response to pressure overloading with observable differences in the time course.
Although mobility change on SDS-PAGE has been reported to be indicative of kinase activation due to phosphorylation of the rapamycin-sensitive sites (35,36), it does not represent phosphorylation of the pseudosubstrate domain. To address the possibility that the pseudosubstrate domain of S6K also undergoes phosphorylation in response to pressure overloading, phosphospecific antibodies were employed in the Western blot experiments. Among the four known phosphorylation sites of the pseudosubstrate domain, only Ser-411 has been shown to be sensitive to inhibition by rapamycin (15). When P-Ser-411 antibody was used in the Western blot analysis, Ser-411 phosphorylated p70 S6K was detected exclusively in the pressureoverloaded myocardium, whereas it was almost completely absent in all the normally loaded ventricular samples (Fig. 1B). The time course of Ser-411 phosphorylation, which was seen maximally between 1 and 4 h of RVPO and declined sharply at 48 h, matches the time course of load-induced p70 S6K band shifting shown in the previous experiment (Fig. 1A). These experiments, therefore, suggest that p70 S6K is phosphorylated on the Ser-411 residue in addition to other sites of S6K (as multiple species were observed in Fig. 1A) in response to pressure overloading. Interestingly, the P-Ser-411 antibody did not detect the p85 S6K isoform either in control or pressure-overloaded ventricular samples, indicating that this isoform is not phosphorylated at the Ser-411 residue in response to pressure overload. This was true even after increasing either the sample volume for SDS-PAGE (twice the concentration, 40 g of protein) and/or a longer time period of ECL detection in the Western blot analysis (data not shown). All these studies indicate that the kinase(s) responsible for the phosphorylation of the Ser-411 site of p85 S6K is either absent or inactive in the nucleus, whereas it is activated in the cytoplasm in response to pressure overloading.
To determine whether the changes in the phosphorylation status of S6K are associated with kinase activation, we analyzed the activity of p70 S6K using a synthetic peptide substrate homologous to the S6 protein after immunoprecipitating the kinase from cytosolic samples of control and pressure-overloaded ventricles with the regular S6K antibody (C-18). Western blot analysis with C-18 antibody did not detect p85 S6K in the cytosolic samples (data not shown). Therefore, when the immune complexes are prepared with the cytosolic samples, their activities represent mostly that of p70 S6K . As shown in Fig. 2, a more than 12-fold activation of S6K (most of it due to p70 S6K ) was observed following 1-h pressure overloading, slightly decreased at the 4-h time point, markedly declined when RVPO was extended for 48 h, and reached the basal condition by 1 week of RVPO. We confirmed the time course of this kinase activation in two other independent experimental cats for each time point. Therefore, both Western blot experiments and immune complex kinase assays show that the cytosolic p70 S6K is activated substantially in pressure-overloaded myocardium as early as 1 h of RVPO.
Next, to analyze whether the activated p70 S6K translocates to the nucleus during pressure overloading, we used confocal microscopy of fresh-frozen tissue sections. Although P-Ser-411 antibody detected exclusively p70 S6K (Fig. 1B), this antibody was not effective for immunostaining. Therefore, we took advantage of the regular C-18 antibody, which was found to be strongly reactive with the p70 S6K (cytoplasmic) compared with the p85 S6K (nuclear) isoform of S6K (Fig. 1A). When this antibody was used for confocal microscopy, it detected only the cytoplasmic S6K (corresponding to the p70 S6K isoform) as anticipated, i.e. the signal is distributed evenly in the sarcoplasm (decorated in red) of both control and 4 h pressure-overloaded ventricular cardiocytes (Fig. 3). No S6K signal was found in the nuclei (labeled in green) when analyzed either in the normally loaded control ventricles or in the 4-h pressure-overloaded right ventricles. These data suggest that p70 S6K , following load-induced phosphorylation and activation, did not translocate to the nucleus.
p70 S6K and p85 S6K Undergo Differential Phosphorylation and Activation during RVPO-As phosphorylation of the Ser-411 residue of the pseudosubstrate domain was observed exclusively for the cytoplasmic isoform (p70 S6K ) during pressure overloading (Fig. 1B), we employed another phosphospecific antibody (P-Thr-421) to determine phosphorylation at other known sites in the pseudosubstrate domain of S6K isoforms. This antibody detects S6K when both Thr-421 and Ser-424 residues in the pseudosubstrate domain are phosphorylated simultaneously. Interestingly, Western blot analysis with this antibody revealed phosphorylation not only of p70 S6K but also of p85 S6K in response to pressure overload (Fig. 4A). Neither isoform was phosphorylated in normally loaded control ventricles (LV or RV). Phosphorylation of Thr-421/Ser-424 residues of both the isoforms could be detected as early as 1 h of RVPO; however, although phosphorylation of these residues for the p70 S6K isoform declined sharply after pressure overloading for 48 h, phosphorylation for the p85 S6K isoform was sustained in the 48-h pressure-overloaded myocardium and required a longer time period for the phosphorylation to decline, as the basal level was found to be reached only when pressure overloading was extended for 1 week. This time course of phosphorylation was confirmed for each time point with two other independent groups of experimental cat samples. These data imply that both nuclear and cytosolic isoforms undergo pressure overload-induced phosphorylation at Thr-421 and Ser-424 sites; however, the phosphorylation of p70 S6K is more transient than the phosphorylation of the nuclear p85 S6K . Overall, these experiments show that, in addition to a difference in phosphorylation at the Ser-411 site (Fig. 1B), these two S6K isoforms exhibit a difference in the time course of load-induced Thr-421/Ser-424 phosphorylation.
In order to confirm that the differential phosphorylation of S6K isoforms occurs at the level of individual cardiocytes, analysis of fresh-frozen tissue sections by confocal microscopy was performed with the P-Thr-421 antibody using the same ventricular tissue samples used for the Western blot analysis (Fig.  4A). In the confocal pictures shown in Fig. 4B, sarcomeric actin is decorated in blue (TR-phalloidin), nuclei in green (YoYo-1 dye), and the Thr-421/Ser-424 phosphorylated S6K in red. Control ventricular tissue sections (both LV and RV) show nuclear and sarcomeric staining, but show no signal for the phosphorylated S6K isoforms (Fig. 4B). Interestingly, upon pressure overloading for 1 or 4 h, diffuse red staining could be seen throughout the sarcoplasm, suggesting phosphorylation of the cytoplasmic p70 S6K . In the 1-h, 4-h, and 48-h pressure-overloaded RV, staining of the phosphorylated p85 S6K (nuclear isoform) could be seen in yellow/orange due to the merger of the p85 S6K (red) signal with the nuclear (green) signal. Therefore, phosphorylation of p85 S6K at Thr-421/Ser424 sites persists longer when compared with that of p70 S6K , and the data precisely match the Western blot data shown in Fig. 4A. The quantification of phosphorylation from the confocal microscopy pictures (Fig. 4C) confirms this finding that pressure overloading resulted in an increase in the level of phosphorylation of nuclear S6K (p85 S6K ), reaching a statistically significant maximal level after 48-h RVPO, when compared with 4-h RVPO and control. Furthermore, based on the results obtained with phospho-Ser-411 (Fig. 1B) and phospho-Thr-421 (Fig. 4, A and  B) antibodies, phosphorylation in the pseudosubstrate domain of p85 S6K appears to occur preferentially at Thr-421/Ser-424 sites when compared with Ser-411 phosphorylation, and the load-induced differential activation/deactivation of S6K isoforms occurs at the level of individual cardiocytes.
To further confirm that there is a different time course of p85 S6K activation relative to p70 S6K activation, as well as to demonstrate that the p85 S6K is indeed catalytically active in pressure-overloaded myocardium, we measured and compared the kinase activities between 1 and 4 h in pressure-overloaded cat ventricles (Fig. 5). For this, an antibody specific for the FIG. 2. Time course of p70 S6K activation during RVPO. Cytosolic fractions were prepared from a control feline heart (Cont) and from feline hearts after RVPO for 1 h, 4 h, 48 h, and 1 week. The cytosolic samples were used for immunoprecipitation with C-18 antibody, and the kinase activity was measured in duplicates with S6 peptide as substrate. Activities in terms of RV/LV ratio were calculated for each experimental feline heart and plotted against the time period of RVPO. In a typical 4-h RVPO experiment, the activity (in cpm) was 206,618 Ϯ 5,543 for RV and 29,529 Ϯ 5,414 for LV. hr, hour; wk, week.
FIG. 3. Immunofluorescence confocal microscopy showing no translocation of p70 S6K to the nucleus. LVs and RVs from control (Cont) and 4-h RVPO cats were used to prepare fresh-frozen tissue sections and processed for confocal microscopy as described under "Experimental Procedures." In the figure, S6K is decorated in red with C-18 antibody, the nucleus is decorated in green with YoYo-1 dye, and actin is decorated in blue with TR-phalloidin. Bar, 25 m. hr, hour. p85 S6K isoform (C-3) was used to immunoprecipitate the kinase from Triton X-100 tissue extracts and compared with the previously used C-18 antibody. As mentioned earlier, C-18 antibody detects mostly the p70 S6K isoform. Therefore, when the immune complex kinase assays were performed with this antibody, there was a remarkable kinase activation following 1 h pressure overloading (14-fold) relative to normally loaded LV control, which decreases substantially (7-fold) when pressure overloading is extended for 4 h. This activation profile is very similar to the activation profile of p70 S6K when the cytosolic samples were used (Fig. 2). Interestingly, when the assays were performed for p85 S6K , there was a clear difference in the activation profile, i.e. there was almost no activation of p85 S6K at 1 h of pressure overloading (1.5-fold), whereas there was a significant increase in the kinase activity after 4 h of RVPO (6.2-fold). These experiments reveal two major points. First, similar to p70 S6K , pressure overloading results in the catalytic activation of p85 S6K ; and second, p85 S6K activation occurs subsequent to the activation of p70 S6K .
Finally, as the P-Thr-421 antibody detected the phosphorylated nuclear isoform (p85 S6K ) in pressure-overloaded myocardium, we used this antibody to examine whether this activated kinase shifts to the cytoplasm during load-induced activation.
Total lysates prepared with Triton X-100 extraction buffer and samples prepared with detergent-free buffers (cytosol) were obtained from a 4-h RVPO cat ventricle and used for Western blotting with P-Thr-421 antibody. Interestingly, in the pressure-overloaded RV, whereas the Thr-421/Ser-424 phosphorylated p85 S6K could be readily detected in the Triton X-100solubilized total tissue lysates, it was absent in the cytosolic preparation, although phosphorylated p70 S6K could be detected in both types of tissue samples (Fig. 6). These studies clearly suggest that during pressure overloading the Thr-421/Ser-424phosphorylated p85 S6K did not translocate from the nucleus to the cytoplasm. S6K Activation Is Accompanied by PKC-mediated Signaling Independent of PI 3-Kinase-S6K has been shown to be activated by both PKC-dependent and -independent pathways (6,20). In the case of the PKC-independent pathway, tyrosine kinases are known to play an important role in S6K activation (21,37), with the possible involvement of PI 3-kinase (37). Therefore, to determine if PI 3-kinase was activated during pressure overloading, we immunoprecipitated PI 3-kinase from either 1-h or 4-h RVPO ventricular lysates and examined both for tyrosine phosphorylation and kinase activity (32, 38, 39). As a positive control, insulin-treated feline adult cardiocytes were used in these studies. Measurement of PI 3-kinase activity showed no appreciable load-induced increase in the basal activity, i.e. there were no observable differences between RV and LV of either control or 1-or 4-h RVPO cat samples (Fig. 7A). As shown previously, activation of both the isoforms of S6K was observed between 1 and 4 h of pressure-overloaded myocardium (Fig. 5). Furthermore, our observation showing absence of PI 3-kinase activation could not be due to any experimental difficulties, since, as observed in other cell types (21,23), a substantial amount of stimulated activity could be observed when cultured adult cardiocytes were treated with insulin for 30 min (Fig. 7A, right panel), and this activation was accompanied by a retarded mobility of both S6K isoforms on SDS-PAGE (data not shown). Therefore, these studies clearly indicate that load-induced activation of S6K is not accompanied by PI 3-kinase activation. As studies show PI 3-kinase activation via c-Src-mediated tyrosine kinase pathways (40), as a next step, c-Src activation was analyzed in pressure-overloaded ventricular samples. We have recently shown that pp60c-Src, a non-receptor tyrosine kinase, associates with the cytoskeleton of pressure-overloaded myocardium and becomes substantially tyrosine-phosphorylated for kinase activation (27). In order to determine whether c-Src plays an upstream role for S6K activation, we analyzed Triton X-100-lysed ventricular subfractions, namely Triton-soluble, cytoskeleton, and membrane skeleton (27). Similar to what we have shown before, c-Src movement to the cytoskeleton was present in significant amounts in the 48-h pressure-overloaded ventricles and was absent in all normally loaded ventricles (Fig. 7B). Such analysis in the 4-h pressure-overloaded ventricles showed very low levels of cytoskeleton-bound c-Src, and was completely absent in the 1-h pressure-overloaded ventricles. In addition, we did not observe any differences in the levels of tyrosine-phosphorylated proteins when 1-h RVPO samples were used for Western blots with anti-phosphotyrosine antibody, whereas in 48-h RVPO samples several cytoskeleton-bound proteins including c-Src became tyrosine-phosphorylated (Ref. 27 and data not shown). In contrast, the maximal activation of p70 S6K in these tissue samples was seen at 1 h of pressure overload (Fig. 2). Therefore, c-Src association and activation at the cytoskeleton occurs subsequent to p70 S6K activation. However, as shown in the previous experiments (Figs. 4 and 5), p85 S6K activation was observed at 4 h of RVPO, whereas phosphorylation at Thr-421/ Ser-424 sites occurred as early as 1 h of RVPO. Therefore, whereas these studies clearly ruled out the possible involvement of c-Src in p70 S6K activation, it is not clear whether c-Src is linked to p85 S6K activation.
As PKC is a well known activator of S6K (20,22), we analyzed whether members of the PKC family are activated with a time course similar to S6K activation. Our initial screening showed the presence of the following PKC isoforms in the feline heart sample: ␣, ␤, ␥, ⑀, , , , , and very low expression of the and ␦ isoforms. Of these isoforms, we were able to detect a FIG. 5. Activation of p85 S6K occurs subsequent to p70 S6K activation during pressure overloading. Triton X-100 extracts were prepared from 1-and 4-h pressure-overloaded RVs and normally loaded same animal control LVs and used for immunoprecipitation with either C-18 (precipitates predominantly p70 S6K ) or C-3 (specific for p85 S6K ) antibodies conjugated covalently to magnetic beads. The kinase activity associated with the immune complexes was measured in duplicate, and the results represent the mean Ϯ S.D. -Fold stimulation (RV/LV ratio) due to pressure overloading is shown in parentheses on top of corresponding bars.
FIG. 6. Nuclear p85 S6K does not translocate to the cytoplasm during activation. LVs and RVs from a 4-h RVPO feline heart were extracted either with Triton X-100-containing buffer (Triton-soluble) or with detergent-free buffer (cytosol) and used for Western blotting with P-Thr-421 antibody. Of note, load-induced phosphorylation of p85 S6K could be detected only in the detergent-extracted fraction, whereas the same for p70 S6K is seen with both types of sample preparations.

FIG. 7. Measurement of PI 3-kinase activity and cytoskeletal redistribution of c-Src.
A, PI 3-kinase (PI3K) activity. LVs and RVs from control cat and RVPO cat at 1 and 4 h and feline cardiocytes after treatment with Ϯ 200 nM insulin for 30 min were extracted with Triton X-100 buffer. Extracts (0.5 mg of protein) were immunoprecipitated with PI 3-kinase antibody to obtain immune complexes as described under "Experimental Procedures" and assayed for kinase activity. Insulin-treated cells and 4-h RV extracts processed in the absence of antibody (ϪAb) were used for background activity. The product of the kinase reaction, phosphatidylinositol 3-kinase (PIP), is shown in the figure. Each set of data for tissue or cardiocyte samples represents independent experiments, and the results were confirmed at least two times. B, cytoskeletal redistribution of c-Src. LVs and RVs from control (Cont) and RVPO cats at 1 h, 4 h, 48 h, and 1 week were processed to obtain Triton X-100-lysed subfractions, namely soluble (Sol), cytoskeleton (CSK), and membrane-skeleton (MSK) as described previously (27). The samples were processed for Western blotting with c-Src mAb.
shift in PKC ␣, PKC ␥, and PKC ⑀ to the membrane between 1 and 4 h of pressure overloading (Fig. 8A), which persisted thereafter for a long time period (1 week). Such membrane translocation of PKC has been shown to be indicative of kinase activation (41). Furthermore, we analyzed their immediate downstream effector kinase, c-Raf, for load-induced changes in the membrane preparations used for PKC analysis. As an unexpected finding, c-Raf was found to be membrane-associated in both normal and pressure-overloaded ventricles without any appreciable difference (data not shown). However, when we used Triton X-100-lysed subfractions (27), the following loadinduced changes in c-Raf, measured in terms of both translo-cation and mobility band shift on SDS-PAGE separation, were observed (Fig. 8B). First, c-Raf was present in all of the Triton X-100-lysed subfractions of 1-4-h pressure-overloaded ventricles and exhibited retarded mobility on SDS-PAGE separation, indicative of phosphorylation and activation as reported previously (42). Second, we show for the first time that pressure overload results in the association of substantial amounts of c-Raf with the cytoskeletal fraction, which was found to be completely absent in all of the normally loaded control ventricular fractions. Third, a significant amount of c-Raf was present in the Triton X-100-insoluble membrane skeletal fraction of both normal and pressure-overloaded ventricles, even after the detergent extraction. Most of these changes observed between 1 and 4 h of RVPO returned to the basal level after long term pressure overloading (1 week), except for the membrane skeleton-bound c-Raf, which was present at high levels even up to 1 week of RVPO, as was observed for PKC isoforms (Fig. 8A).
Load-induced Changes in S6K Activation Can Be Mimicked in TPA-stimulated Adult Feline Cardiocytes-To demonstrate that PKC stimulation could result in the activation of S6K in cardiocytes, we treated cultured adult cardiocytes for various time points with 100 nM TPA, and total extract was prepared. Extracts prepared from control and TPA-treated cells were Western blotted (Fig. 9A) with C-18 antibody (top panel), P-Ser-411 (middle panel), and P-Thr-421 antibody (lower panel). Treatment of adult cardiocytes with TPA for 1, 4, and 24 h resulted in a mobility shift of both p70 S6K as well as p85 S6K on SDS-PAGE separation (top panel), although the latter isoform required double the exposure time during ECL reaction similar to the previous observation with tissue samples (Fig. 1A). This mobility shift decreased to almost control levels after 48 h of treatment. Furthermore, Western blot experiments with P-Ser-411 and P-Thr-421 antibodies (Fig. 9A, middle and lower panels, respectively) using detergent extracts of TPA-treated cells revealed the following. First, Ser-411 phosphorylation of p70 S6K was seen as early as 1 h after TPA treatment, continued to be present up to 24 h, and then decreased to undetectable levels after 48 h of treatment; second, similar to the observation seen in pressure-overloaded ventricular samples, Ser-411phosphorylated p85 S6K was not present in detectable amounts in control and TPA-treated cell extracts; and third, both iso-FIG. 8. Redistribution of PKC isoforms and c-Raf in pressureoverloaded myocardium. A, translocation of PKC isoforms to the membrane. LVs and RVs from control (Cont) and RVPO cats at 1 h, 4 h, 48 h, and 1 week were processed to obtain cytosol and membrane fractions (Memb.) as described under "Experimental Procedures." The samples were processed for Western blotting with isoform-specific PKC antibodies. B, cytoskeletal association and activation of c-Raf. LVs and RVs from control (Cont) and RVPO cats at 1 h, 4 h, 48 h, and 1 week were processed to obtain Triton X-100-lysed subfractions, namely soluble (Sol), cytoskeleton (CSK), and membrane-skeleton (MSK) as described previously (27). The samples were processed for Western blotting with c-Raf monoclonal antibody. Data were confirmed in at least two independent experiments. hr, hour; wk, week. forms of S6K were found to be phosphorylated at Thr-421/Ser-424 sites in 1-24-h TPA-treated cells and returned to the control level after 48 h of TPA treatment. All of these observations obtained with TPA treatment reflect the changes seen in the pressure-overloaded myocardium. One noticeable difference was that the p85 S6K (nuclear isoform) phosphorylation at Thr-421/Ser-424 did not persist longer than that of p70 S6K as observed with pressure overloading experiments (compare experiments shown in Fig. 9A with Fig. 4A).
In order to determine whether the band shift and pseudosubstrate phosphorylation of p70 S6K in TPA-treated cells (Fig. 9A) represent enhanced kinase activity, cytosolic samples from control and TPA-treated cardiocytes were immunoprecipitated with the C-18 antibody and the associated kinase activity measured (as described in Figs. 2 and 5). TPA treatment for 1 h resulted in a 13.8-fold stimulation (82,469 Ϯ 5,920 cpm when compared with control cells 5,964 Ϯ 355 cpm). Kinase activity fell to 61% of the 1 h stimulated activity after 4 h of treatment (50,432 Ϯ 1,855 cpm), remained at 45% of the 1-h stimulated activity after 24 h of treatment (36,705 Ϯ 4,126 cpm), and returned to the basal condition after 48 h of TPA treatment. This profile of TPA-stimulated p70 S6K activation was confirmed in two other independent experiments. Therefore, both the band shifting and pseudosubstrate phosphorylation of the p70 S6K in TPA-treated cardiocytes correlate with its kinase activation, and the time course of this activation was similar to the in vivo observations with pressure-overloaded myocardium.
Next, as in pressure-overloaded myocardium many of the changes seen with S6K isoforms were accompanied by activation of c-Raf, we also examined whether such changes could be seen in TPA-treated adult cardiocytes. Therefore, control and TPA-treated adult cardiocytes were processed to obtain the Triton X-100-soluble fraction, as performed for tissue samples, and Western blotted with anti-c-Raf antibody. As shown in Fig.  9B, c-Raf exhibited a mobility band shift in 1-, 4-, and 24-h TPA-treated cells, which returned to the control status by 48 h of TPA treatment. The time course of these changes associated with c-Raf are very similar to the time course of changes seen with S6K isoforms (Fig. 9A). DISCUSSION Quantitative and qualitative changes, as a result of transcriptional and translational activation, are two major events associated with hypertrophic growth. The present study was designed to determine whether the two isoforms of S6K, p70 S6K and p85 S6K , which are known to regulate both transcriptional and translational activation (20,43), are activated in response to pressure overloading of the heart. Furthermore, in a finding having general rather than cardiac-restricted implications, this study distinguishes for the first time the activation profiles of the two isoforms of S6K. Previous studies have demonstrated p70 S6K activation in several cell types after either mitogenic stimulation (for review, see Ref. 20), mechanical stretch (44), or integrin receptor engagement (37). In cultured neonatal cardiocytes, treatment with angiotensin II (45,46) or phenylephrine (47) has been shown to result in increased p70 S6K activity, and blocking of this kinase activation leads to a significant reduction in protein synthesis (45)(46)(47) and total RNA (47). Therefore, p70 S6K could be a key intermediate for the loadinduced hypertrophic growth process.
In the present study, we show for the first time a remarkable stimulation of p70 S6K activity following 1 h of pressure overloading. This activation was confirmed via three independent approaches that show: (i) changes in the mobility on SDS-PAGE, which are indicative of distinct phosphorylation required for kinase activation (35, 36); (ii) phosphorylation at Ser-411, Thr-421, and Ser-424 sites of the pseudosubstrate domain, which is shown to be required for kinase function (17,18); and (iii) enhanced kinase activity as determined by the immune complex kinase assay, which is observed maximally at 1 h of RVPO, begins to decline at 4 h, and decreases sharply when the pressure overload is extended for 48 h (Fig. 2). p70 S6K is known to phosphorylate S6 protein, resulting in the translation of specific mRNAs with a polypyrimidine tract in the 5Ј-untranslated region (12). Therefore, activation of p70 S6K to a substantial level between 1 and 4 h of RVPO indicates that the kinase might play a pivotal role in the initial acute-phase increase in translational efficiency. By taking a similar approach for p85 S6K , our data also show for the first time activation of this isoform in pressure-overloaded myocardium based on the following facts. First, p85 S6K exhibits retarded mobility on SDS-PAGE (Fig. 1A); second, RVPO results in a sustained phosphorylation at Thr-421 and Ser-424 sites of the pseudosubstrate domain during 4 -48 h of pressure overloading; third, our immune complex kinase assay performed in 1-and 4-h pressure-overloaded myocardium clearly show activation at 4 h of RVPO, which occurs as an event subsequent to p70 S6K activation. One possible role of activated p85 S6K in the nucleus is to phosphorylate and activate transcriptional factors such as the cyclic AMP-responsive element-binding protein (CREB) and the cyclic AMP responsive element-binding protein modulator (CREM) (13). Activation of these factors has been shown to regulate the transcriptional activation of immediate early and delayed response genes (48 -50).
Both the pattern and time course of phosphorylation in the pseudosubstrate regions are significantly different for the two S6K isoforms, suggesting that distinct regulatory mechanisms control their phosphorylation during pressure overload. First, the phosphorylation of p70 S6K is more transient, peaks between 1 and 4 h of RVPO, and returns almost to the basal level at 48 h of RVPO, whereas p85 S6K phosphorylation persists longer by peaking between 4 and 48 h of pressure overload and returns to the basal condition at 1 week of RVPO. This suggests that the kinase(s) and/or phosphatase(s) that control phosphorylation of these residues for the two isoforms are differently regulated. Second, as mentioned earlier, whereas in pressureoverloaded myocardium p70 S6K is phosphorylated at Ser-411, Thr-421, and Ser-424 sites, phosphorylation of p85 S6K is only observed at Thr-421 and Ser-424 but not at the Ser-411 site. This was also true for the TPA-treated cardiocytes. All of these studies indicate that the kinase that phosphorylates Thr-421/ Ser-424 sites of both the S6K isoforms might be different from the one that phosphorylates Ser-411, although all of these phosphorylation sites in the pseudosubstrate domain are flanked by a COOH-terminal proline. Previous studies show that the phosphorylation of Ser-411, but not Thr-421 or Ser-424, could be prevented by treatment with rapamycin (15), suggesting that a rapamycin-sensitive pathway is responsible for Ser-411 phosphorylation. The mammalian target of rapamycin (mTOR) has been identified recently as a kinase responsible for the phosphorylation of PHAS-I (eIF-4E binding protein) at Ser/Thr residues flanked by a COOH-terminal proline (51)(52)(53). As Ser-411 phosphorylation is sensitive to rapamycin and is followed by a COOH-terminal proline, the same kinase that phosphorylates PHAS-I could be responsible for the phosphorylation of Ser-411. The kinase(s) responsible for the phosphorylation of Thr-421 and Ser-424 sites are largely unknown at the present time. We suspect cyclin-dependent kinases as possible candidates, as they phosphorylate not only proteins containing the "Ser/Thr-Pro" motif but also are present both in the nucleus and in the cytoplasm, and are activated in response to growth factors (29).
Our efforts to identify upstream pathways of S6K activation indicate that the PKC-independent PI 3-kinase pathway is not the primary mechanism for load-induced activation of both of the S6K isoforms for the following reasons: first, in 1-4-h pressure-overloaded myocardium where both isoforms of S6K are active, neither was the 85-kDa subunit of PI 3-kinase found to be tyrosine-phosphorylated (data not shown), indicative of kinase activation (38,39), nor was its overall kinase activity increased (Fig. 7A). Second, analysis of the phosphorylation status of Akt/protein kinase B, an intermediate kinase that functions downstream of PI 3-kinase (54), using phosphospecific antibody reveals the absence of phosphorylation at all time points of S6K activation (data not shown). Taken together, these data strongly suggest that a PI 3-kinase-mediated pathway is not the mechanism for load-induced activation of the S6K isoforms. However, our studies do not exclude the possibility of PI 3-kinase activation occurring prior to 1 h of RVPO. Unfortunately, such early time points are difficult to examine by surgical interventions in the acute pressure overload model. In addition, our studies indicate the absence of any changes in the pattern of tyrosine-phosphorylated proteins present in 1-h RVPO tissue samples (Ref. 27 and data not shown). Also, similar to our earlier observation (27), cytoskeletal association and activation of c-Src, which could potentially serve as an upstream activator of PI 3-kinase (40), was not detected during the early period of 1-h pressure overloading, although c-Src activation was observed at later time points of pressure overloading ( Fig. 7B and Ref. 27). Taken together, these data suggest that the cytoskeletal association and activation of c-Src that were observed between 4 and 48 h of RVPO and the resulting tyrosine phosphorylation may not contribute to the activation of p70 S6K that was maximally observed at 1 h of RVPO.
We favor the idea that a PKC-dependent pathway is responsible for load-induced S6K activation. PKC, which has been shown to activate c-Raf (55) as well as S6K (22,44), is known to undergo activation in pressure-overloaded myocardium (41,56). Our present studies also show membrane translocation of classical PKC isoforms ␣ and ␥ as well as the novel isoform PKC-⑀ in the same tissue samples wherein the S6K isoforms were found to be activated. Previous studies have shown PKCmediated S6K activation in the absence of PI 3-kinase activity (21,23), which supports the idea that load-induced S6K activation could be partly due to PKC-mediated signaling. Furthermore, our studies also demonstrate that c-Raf, which is immediately downstream of PKC, showed retarded mobility on SDS-PAGE, indicating kinase activation (42), as well as cytoskeletal association in response to RVPO. As a recent study (57) indicates that constitutively active c-Raf could activate S6K, it is possible that PKC-mediated c-Raf activation might contribute to the activation of S6K in the pressure-overloaded myocardium. The functional role of cytoskeleton-bound c-Raf is largely unknown at the present time, but might be linked to the subsequent recruitment of c-Src to the cytoskeleton for kinase activation. Importantly, most of the load-induced in vivo changes of the S6K isoforms can be mimicked in TPA-stimulated cardiocytes. Overall, these data from TPA-treated cardiocytes support our view that activation of S6K is mediated by a PKC-dependent pathway, and c-Raf could be an intermediate player. However, it is possible that additional pathways might regulate S6K phosphorylation in pressure-overloaded myocardium as opposed to the defined PKC-mediated pathway after TPA stimulation.
In summary, we have demonstrated for the first time that acute pressure overloading of the heart results in substantial activation of both isoforms of S6K, with changes that are distinctly different for the two isoforms. Our attempts to identify potential upstream pathways that are responsible for the phosphorylation and activation of both S6K isoforms suggest that a PKC-mediated pathway independent of PI 3-kinase is responsible for the load-induced kinase activation. In addition, our study extends the importance of S6K, wherein the activation of specific isoforms might regulate both translational and transcriptional events that are critical for the cardiac hypertrophic response.