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© The Rockefeller University Press,
0021-9525/1999//315 $5.00
The Journal of Cell Biology, Volume 144, Number 2,
, 1999 315-324
Regular Articles |
Dissociation of FAK/p130CAS/c-Src Complex during Mitosis: Role of Mitosis-specific Serine Phosphorylation of FAK
Department of Cancer Research, Parke-Davis Pharmaceutical Research Division, Ann Arbor, Michigan 48105; Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and || Cancer Institute of New Jersey, Busch Campus, Piscataway, New Jersey 08855
At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130CAS (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading.
We have found two significant alterations in FAK-mediated signal transduction during mitosis. First, the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16 and 13% of the interphase levels, respectively. Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.
Key Words: FAK • CAS • paxillin • mitosis • phosphorylation • c-Src
Abbreviations used in this paper: CAS, p130CAS; ECM, extracellular matrix; FAK, focal adhesion kinase; PP1, protein phosphatase 1.
DURING mitosis, normal adherent cells show massive changes in their adhesive interactions with the extracellular matrix (ECM).1 During prophase, cells round up and lose attachments to a substrate. After cytokinesis, cells start to reattach and spread. The attachment to the substrate during early G1 phase is essential for cells to pass through the restriction point at each cell cycle (O'Neill et al., 1986), indicating that cell adhesion transmits a signal for cell cycle progression. The cycle of attachment and detachment during the cell cycle is thus critical to proliferation of normal adherent cells.
Focal adhesions are responsible for the cell-substrate adhesion of fibroblasts and other adhesive animal cells (reviewed in Schaller and Parsons, 1994; Jockusch et al., 1995; Burridge and Chrzanowska-Wodnicka, 1996; Craig and Johnson, 1996). They are specialized sites that link the extracellular matrix and the actin cytoskeleton. Since bundles of actin filaments (stress fibers) are anchored to focal adhesions, focal adhesions regulate the organization of stress fibers, thereby controlling cell morphology. In addition to this structural role, focal adhesions are involved in integrin-mediated signal transduction. This is evident from the fact that focal adhesions contain a variety of enzymes involved in signal transduction including protein kinase C, tyrosine kinases, and tyrosine phosphatases (Rohrschneider, 1980; Hanks et al., 1992; Schaller et al., 1992; Liao and Jaken, 1993; Serra-Pages et al., 1995; Shen et al., 1998). Thus, the loss of adhesion during mitosis should not only lead to the disassembly of focal adhesions, but also cause inactivation of integrin-mediated signal transduction. While the importance of the modulation of focal adhesion activities in synchrony with the cell cycle is recognized, little is known regarding the mechanisms responsible for the alterations in these activities during mitosis and post-mitotic spreading. Although mitotic cells and trypsinized cells are both rounded with disassembled focal adhesions and stress fibers, they are clearly different in terms of their responsiveness to ECM. Trypsinized cells immediately begin to reattach and spread when replated on ECM-coated substrates, focal adhesions are reassembled, and certain signals are transduced to allow cell spreading and proliferation. In contrast, mitotic cells stay detached until the completion of cytokinesis, even if they are placed on ECM. There must be distinctive mechanisms in mitotic cells that suppress the formation of focal adhesions and inhibit signal transduction.
Focal adhesion kinase (FAK) appears to be a primary mediator of integrin-mediated signal transduction (reviewed in Parsons et al., 1994; Schaller and Parsons, 1994; Richardson and Parsons, 1995; Hanks and Polte, 1997). FAK is a tyrosine kinase prominently localized at focal adhesions (Hanks et al., 1992; Schaller et al., 1992), and tyrosine phosphorylated FAK binds to the SH2 domains of Src (Schaller et al., 1994; Xing et al., 1994; Eide et al., 1995; Schlaepfer et al., 1997) and Grb2 (Schlaepfer et al., 1994, 1997). In addition, FAK is known to bind to a variety of other signaling proteins, including paxillin (Turner and Miller, 1994; Tachibana et al., 1995), PI-3 kinase (Chen and Guan, 1994; Bachelot et al., 1996; Chen et al., 1996; Saito et al., 1996), p130CAS (CAS) (Polte and Hanks, 1995, 1997; Harte et al., 1996), talin (Chen et al., 1995), and the cytoplasmic domain of integrin beta subunit (Schaller et al., 1995). Among these FAK-associated proteins, both paxillin and CAS are considered as nonenzymatic "docking" proteins (Sakai et al., 1994; Turner, 1994; Turner and Miller, 1994; Burnham et al., 1996; Nojima et al., 1996; Vuori et al., 1996; Klemke et al., 1998). Paxillin is phosphorylated at tyrosine residues during integrin-mediated cell attachment and binds to vinculin, FAK, and Crk (Burridge et al., 1992; Birge et al., 1993; Turner, 1994; Turner and Miller, 1994; Wood et al., 1994; Hildebrand et al., 1995; Schaller and Parsons, 1995; Tachibana et al., 1995). CAS binds to FAK via its SH3 domain (Polte and Hanks, 1995, 1997; Harte et al., 1996), and is also known to associate with other signal transduction molecules, including Src, Crk (Sakai et al., 1994; Petch et al., 1995; Burnham et al., 1996; Nakamoto et al., 1996), and PTP1B (Liu et al., 1996). It is further suggested that the association of Src with either CAS or FAK leads to the activation of Src (Hanks and Polte, 1997). Thus, FAK, CAS, and paxillin are likely to play a central role in signal transduction mediated via focal adhesions.
In this report we investigated possible mechanisms underlying mitosis-induced focal adhesion disassembly and inactivation of integrin-mediated signaling. We found that FAK, paxillin, and CAS each become prominently phosphorylated on serine and/or threonine residues in a mitosis-specific fashion, coincident with their becoming dephosphorylated on tyrosine. We also found that the FAK/ CAS/c-Src signaling complex is dissociated during mitosis. While FAK tyrosine dephosphorylation appears to disrupt the FAK/c-Src interaction, we found that the FAK/CAS dissociation is due to mitosis-specific serine phosphorylation of FAK. We also observed that mitotic FAK shows decreased binding to the cytoplasmic domain of integrin beta subunit. Our findings suggest that mitosis-specific phosphorylation of FAK and other focal adhesion components contributes to the arrest of integrin-associated signaling activities during mitosis.
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Materials and Methods |
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Preparation of Cells and Synchronization for Cell Division
Mitotic cells were prepared as described in Yamashiro et al. (1990). Briefly, 6–10 large dishes (245 x 245 mm; Nalge Nunc International) of REF-2A cells (subconfluent) were treated for 3 h with 0.25 µg/ml nocodazole, and collected by the shake-off method. In some experiments, mitotic cells were collected by the shake-off method without treatment of nocodazole. Trypsinized cells were prepared from one large dish (245 x 245 mm) by incubation with trypsin (0.05% trypsin and 2 mM EDTA in PBS), followed by inactivation of trypsin with the addition of 3% BSA and 0.5 mg/ ml of soybean trypsin inhibitor. Trypsinized cells were collected and incubated for 30 min in DME containing 1% NCS before harvest. Interphase cells were also collected from one large dish (245 x 245 mm).
Cells at later stages of cell division (such as cytokinesis or post-mitotic cell spreading) were prepared as described in Hosoya et al. (1993). Briefly, cells were first treated for 3 h with 0.25 µg/ml nocodazole, and mitotic cells were collected (prometaphase). After being washed with cold DME to remove nocodazole, cells were plated into fresh culture dishes, and incubated at 37°C in DME containing 10% NCS to allow cell cycle progression. Cell morphologies at different mitotic stages were checked by phase-contrast light microscopy. As reported previously (Hosoya et al., 1993), these cells show synchronized cell division in a reasonably short time window. Mitotic cells recovered spindles after 10–20 min of incubation following release of nocodazole arrest, and underwent cytokinesis at 40–60 min. Post-mitotic cell spreading occurred in 80–180 min, at which time both stress fibers and focal adhesions were reassembled. Mitotic 3T3 and HeLa cells were also prepared as above.
In some experiments, REF-2A cells at mitosis were labeled with 32P- orthophosphoric acid as described previously (Yamashiro et al., 1990; Hosoya et al., 1993). Briefly, cells were incubated for 3 h in phosphate-free DME containing 10% dialyzed NCS, 0.25 µg/ml nocodazole, and 0.125 mCi/ml 32P-orthophosphoric acid and mitotic cells were collected. Interphase cells were labeled in the same way except that nocodazole was omitted.
Immunoprecipitation
Immunoprecipitation was performed under the following three conditions. We found that FAK immunoprecipitates prepared under condition II gave consistent results in examination of FAK-associated proteins including CAS, c-Src, paxillin, and talin.
Condition I.
Cells at different stages of mitosis were solubilized in buffer I (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 20 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.2 mM PMSF), and stored in a –80°C freezer. After being thawed quickly, the lysates were homogenized by several passes through a 25-gauge needle, and centrifuged at 100,000 g for 15 min. 2–3 µg of mouse mAb against FAK, paxillin (Transduction Laboratories), or rabbit polyclonal antibody (pAb) against FAK or CAS (Santa Cruz Biotechnology, Inc.) was added to the supernatants. After incubation at 4°C for 1.5 h, protein A–Sepharose-conjugated rabbit antibody against mouse IgG (for mAb) or protein A–Sepharose (for pAb) was added, and incubation continued for 1 h. After extensive washing with buffer I, the immunocomplexes were analyzed by SDS-PAGE, followed by immunoblotting.
Condition II.
Cells were lysed with buffer II (50 mM Hepes, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 25 mM NaF, 1 mM sodium vanadate, and 50 mM beta-glycerophosphate), and homogenized by a Dounce homogenizer. After centrifugation at 16,000 g for 20 min, the cell extracts (made to an equal protein concentration of 5–10 mg/ml) were incubated with rabbit pAb against the COOH-terminal peptides of FAK or CAS (Santa Cruz Biotechnology, Inc.) or mouse mAb against paxillin (Transduction Laboratories), followed by incubation with protein A–Sepharose beads (Pharmacia). The immunocomplexes were washed three times with buffer II, analyzed by SDS-PAGE, followed by immunoblotting. In some experiments, protein A–conjugated primary antibodies were used.
Condition III.
Cells were lysed in buffer III (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.05% SDS, 0.5% sodium deoxycholate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 25 mM NaF, 1 mM sodium vanadate, and 50 mM beta-glycerophosphate), and processed as described for condition II.
Immunoblotting
The following antibodies were used for immunoblotting: antibodies against FAK (mAb, Transduction Laboratories; pAb, Santa Cruz Biotechnology, Inc.), paxillin (Transduction Laboratories), phosphotyrosine (PY20; Transduction Laboratories), CAS (Transduction Laboratories), PI-3 kinase (Transduction Laboratories), c-Src (mAb; Upstate Biotechnology; pAb, Santa Cruz Biotechnology, Inc.), Crk (Santa Cruz Biotechnology, Inc.), cyclin B1 (Oncogene Science, Inc.), and talin (Sigma Chemical Co.). For preparation of total cell lysates, cells at different stages of mitosis were lysed in 2x SDS-PAGE sample buffer. The equivalent amounts of total proteins were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting with the indicated antibodies. FAK immunoprecipitates were separated by SDS-PAGE, and analyzed by immunoblotting with the antibodies against FAK, CAS, c-Src, and phosphotyrosine. The blots were quantitated by densitometry to determine levels of FAK, CAS, c-Src, and phosphotyrosine. The levels of CAS, c-Src, and phosphotyrosine were normalized by the levels of FAK immunoprecipitate. The association of CAS and c-Src, as well as FAK phosphotyrosine levels, from mitotic and trypsinized cells was expressed by ratios to those of interphase FAK.
To determine the level of phosphotyrosine, immunoprecipitates of FAK, paxillin, or CAS were separated on SDS gels, and each was transferred to PVDF membranes. The membranes were first immunoblotted with antiphosphotyrosine antibody (PY20; Transduction Laboratories). After removal of the PY20 antibody, the same membrane blots of paxillin, FAK, and CAS immunoprecipitates were stripped and reprobed with anti-FAK, antipaxillin, and anti-CAS antibodies, respectively. The blots were quantitated by densitometry to determine ratios of phosphotyrosine levels divided by the levels of FAK, paxillin, and CAS.
Protein Phosphatase Treatment
FAK, paxillin, and CAS were immunoprecipitated under condition I from mitotic and interphase cells using specific antibodies, and half of the immunoprecipitates were treated for 30 min at 30°C with one unit of recombinant serine/threonine phosphatase (PP1, 
-isoform from rabbit muscle; Calbiochem-Novabiochem Corp.) in 50 mM imidazole buffer, pH 7.0, containing 0.25 mM MnCl2, 5 mM DTT, 100 mM KCl, 2 mM MgCl2, and 0.2 mg/ml BSA. Under this condition, no tyrosine dephosphorylation was observed. Both treated and untreated samples were separated by SDS-PAGE, followed by immunoblotting with anti-FAK (Transduction Laboratories), antipaxillin (Transduction Laboratories), and anti-CAS (Transduction Laboratories) antibodies to examine changes in mobility shifts.
Reconstitution of a FAK/CAS Complex
The reconstitution of a FAK/CAS complex was performed by incubating FAK immunoprecipitates with interphase extracts. First, FAK was immunoprecipitated from mitotic cells under condition I, and the immunocomplex was divided into three equal aliquots. The first was treated with PP1 as described above, while the second two were untreated. All three samples were washed extensively with the immunoprecipitation buffer II and 1 µg of the antigenic peptide (CNLLDVIDQARLKMLG) was added in order to block the residual sites of FAK antibody. The PP1-treated sample and one untreated sample were incubated with an equivalent amount of interphase extracts, which had been prepared by homogenization of interphase cells in the immunoprecipitation buffer II, followed by centrifugation at 16,000 g for 20 min. The incubation with the interphase extracts continued for 30 min at 4°C. The other untreated sample was incubated with the immunoprecipitation buffer II alone. All three were again extensively washed with the same buffer, washed once with PBS, and analyzed by SDS-PAGE followed by Western blotting with antibodies against FAK, CAS, and c-Src.
Binding of FAK to a Cytoplasmic Peptide of Integrin Beta Subunit
Binding of FAK or paxillin to a peptide (called SP1; CKLLMIIHDRREFA) was performed as described by Schaller et al. (1995) as SP1 represents a FAK binding site within the cytoplasmic tail of the integrin beta subunit (Schaller et al., 1995). Briefly, varying concentrations (0.3–3 mg/ml) of the lysates of mitotic, interphase, or trypsinized cells were incubated for 60 min at 4°C with beads which had been conjugated with the SP1 peptide (Schaller et al., 1995). The beads were then washed five times with the lysis buffer, and the bound proteins were solubilized with SDS sample buffer, separated by SDS-PAGE, and analyzed by Western blotting using anti-FAK or antipaxillin antibody.
FAK Kinase Assay
The kinase activity of FAK immunoprecipitates was measured using a synthetic random co-polymer (GluTyr = 4:1, Sigma Chemical Co.) as a substrate. Immunoprecipitated FAK (under condition I or II) and the synthetic peptide were incubated at 30°C in the kinase assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM MnCl2, 0.1 mM ATP, and 2 µCi of gamma-[32P]ATP) for 20 min. The reaction mixtures were analyzed by SDS-PAGE followed by autoradiography. The amounts of FAK in immunoprecipitates were normalized by immunoblotting with anti-FAK antibody. The activities were determined by either densitometry of autoradiography or by measuring radioactivity of the phosphorylated co-polymer using a PhosphorImager (Molecular Dynamics Inc.).
Far Western Blot Analysis
The SH2 domain of chicken c-Src (amino acid residues 148–251) was expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli and affinity purified by glutathione–agarose adsorption essentially as described by Guan and Dixon (1991). The GST-SrcSH2 fusion protein was used at 10 µg/ml to probe immunoprecipitated FAK immobilized on nitrocellulose membrane as described by Hildebrand et al. (1995). GST-SrcSH2 bound to FAK was detected by antibody against GST (1 µg/ml; Sigma Chemical Co.) followed by ECL with HRP-conjugated protein A (1:1,000; Amersham Life Science). The membrane was then stripped and reprobed with anti-FAK antibody (0.2 µg/ml; Santa Cruz Biotechnology) for normalization.
Other Procedures
SDS-PAGE was performed as described by Blatter et al. (1972) using 12.5% polyacrylamide, except the buffer system of Laemmli (1970) was used. Phosphoamino-acid analysis was performed as described (Hunter and Sefton, 1980). Protein concentrations were determined by the method of Bradford (1976) using BSA as a standard.
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Dissociation of FAK/CAS/c-Src Complex during Mitosis
To explore the functional significance of mitosis-specific phosphorylation of FAK, we examined whether the association between FAK and FAK-associated proteins (including CAS, paxillin, c-Src, and talin) was altered. We found that the levels of CAS and c-Src associated with FAK immunoprecipitates were greatly reduced in mitotic cells (Fig. 3 a). In contrast, similar levels of paxillin or talin associated with FAK in mitotic or interphase cells were found (data not shown). Quantitative analyses (Fig. 3 b) reveal the levels of CAS or c-Src associated with mitotic FAK are 16 ± 9% and 13 ± 10% of those associated with interphase FAK. The difference is not due to the rounded morphology of mitotic cells because FAK immunoprecipitated from rounded trypsinized cells was associated with considerable amounts of CAS and c-Src. The levels of CAS and c-Src were 83 ± 23% and 70 ± 34% of the interphase levels, respectively. The dissociation of FAK/CAS/ c-Src complex in mitotic cells is not due to nocodazole treatment because similar dissociation was observed with mitotic cells prepared without nocodazole treatment.
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Mitotic Serine Phosphorylation Is Responsible for the Dissociation of FAK and CAS
To test whether mitosis-specific serine phosphorylation or tyrosine dephosphorylation causes the disruption of the FAK/CAS/c-Src complex, we dephosphorylated mitotic FAK by PP1, and examined whether serine dephosphorylated FAK was able to reassociate with CAS or c-Src. FAK was first immunoprecipitated from mitotic cells under condition I. One-third of the immunoprecipitate was used to confirm that neither CAS nor c-Src is associated with mitotic FAK (lane 1 of Fig. 4 a). The rest of the immunoprecipitate was divided in half; one (lane 2) was dephosphorylated by PP1, and the other (lane 3) was untreated. Fig. 4 a shows PP1 treatment resulted in the loss of mobility shift of FAK, confirming dephosphorylation of mitotic FAK. Both PP1 treated and untreated FAK were incubated with interphase extracts. After extensive washing, the binding of CAS or c-Src was examined by immunoblotting. Dephosphorylated FAK (Fig. 4 a, lane 2) was able to reassociate with CAS of interphase extracts (second panel). The level of CAS reassociation was comparable to interphase FAK immunoprecipitates (lane 4). In contrast, the phosphatase untreated mitotic FAK (lane 3) showed reduced binding to CAS. Quantitative analyses revealed that the binding of CAS to dephosphorylated FAK is more than three times higher than for untreated FAK (Fig. 4 b).
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Unlike the FAK/CAS binding, the association between FAK and c-Src was not reconstituted by serine dephosphorylation of FAK. The anti–c-Src antibody could not detect c-Src in any of the FAK preparations (data not shown). These results suggest that near complete tyrosine dephosphorylation observed during mitosis is likely to be involved in the FAK/c-Src dissociation, and that the low level tyrosine phosphorylation seen in trypsinized cells is responsible for the retained FAK/c-Src association.
Mitotic FAK Shows Reduced Binding to the Cytoplasmic Domain of Integrin Beta Subunit
Both FAK and paxillin have been reported to bind directly to a peptide (SP1) representing a membrane-proximal region of the cytoplasmic domain of the beta subunit of integrin (Schaller et al., 1995). We examined whether mitotic FAK or paxillin exhibited altered binding to the SP1 peptide. SP1 peptide–conjugated beads were incubated in varying concentrations of extracts prepared from mitotic, interphase, and trypsinized cells. After washing, levels of bound FAK and paxillin were detected by immunoblotting. As Fig. 5 a shows, mitotic FAK bound to SP1 peptide–conjugated beads less (lane M) than FAK from interphase (lane I), or trypsinized (lane T) cells. Quantitative analysis (Fig. 5 b) revealed the binding of mitotic FAK is 24–33% of the level shown by interphase FAK, while FAK from trypsinized cells showed 65–85% binding of the interphase level (Fig. 5 b). The binding of paxillin to SP1 beads, in contrast, revealed no difference between mitotic and interphase cells (data not shown).
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Mechanisms for Dissociation of a FAK/CAS/c-Src Complex during Mitosis
FAK binds directly to the SH3 domain of CAS via proline-rich domains of FAK (Polte and Hanks, 1995, 1997; Harte et al., 1996). It is likely the mitosis-specific serine phosphorylation alters such interactions. It is interesting to note that one of the proline-rich domains contains serine, and it may be possible that phosphorylation would occur at this serine, reducing the interaction between FAK and CAS.
The association of c-Src with FAK or CAS, on the other hand, has been reported to depend on the binding between phosphotyrosine of FAK or CAS and the SH2 domain of c-Src (Schaller et al., 1994; Xing et al., 1994; Eide et al., 1995; Nakamoto et al., 1996; Schlaepfer et al., 1997). However, FAK from trypsinized cells retains a high level of association of c-Src. It seems likely that the residual tyrosine phosphorylation of FAK in trypsinized cells may be able to preserve the association between FAK and c-Src. Furthermore, a recent report has shown, in addition to the SH2-binding site, FAK has a Src SH3-binding site (Thomas et al., 1998). This SH3-mediated interaction could reinforce the retained association between FAK and c-Src in trypsinized cells.
Reduced Binding of Mitotic FAK to the Cytoplasmic Domain of Integrin Beta Subunit
The NH2-terminal noncatalytic domain of FAK was reported to be responsible for its binding to SP1 beads (Schaller et al., 1995). It is worthy to note that this domain does not contain tyrosine phosphorylation sites. Because FAK from both trypsinized and mitotic cells showed low tyrosine phosphorylation, it seems likely that mitosis-specific phosphorylation of FAK is involved in the reduced binding to the cytoplasmic domain of integrin beta subunit. At present, we cannot directly examine the effects of serine dephosphorylation of mitotic FAK on the binding to SP1 beads because purification of mitotic FAK has been unsuccessful. Therefore, we cannot exclude a possibility that tyrosine dephosphorylation is involved in the loss of FAK to SP1 beads.
Mitosis-specific Phosphorylation of CAS and Paxillin
We have demonstrated that CAS and paxillin, like FAK, show mitosis-specific serine phosphorylation. Currently, we do not know the functional consequence of the serine/ threonine phosphorylation of CAS or paxillin. CAS, either from interphase or mitotic cells, does not bind to mitotic FAK (Figs. 3 and 4). This suggests that mitosis-specific phosphorylation of CAS does not seem to be essential for the dissociation between CAS and FAK, although it is possible that phosphorylation of CAS further decreases the binding between FAK and CAS. In addition, the association of paxillin with FAK is not disturbed during mitosis. Both paxillin and CAS function as docking proteins. It is possible that mitosis-specific phosphorylation would disrupt the association with other proteins, which may contribute to the phenotypic alterations seen during mitosis. One interesting possibility would be mitosis-specific phosphorylation of CAS inhibiting cell motility of mitotic cells.
Physiological Significance of the Disruption of FAK/CAS/c-Src Complexes
First, the disruption of FAK/CAS association during mitosis is likely to result in uncoupling of the FAK- and CAS-mediated signal transduction during mitosis. The reduced binding of mitotic FAK to the cytoplasmic domain of integrin beta subunit (Fig. 5) would amplify the uncoupling effects by altering the interactions between ECM and focal adhesions. Further, the dissociation of the FAK/CAS/c-Src complex could abolish FAK-mediated c-Src activation during mitosis because the association of CAS or FAK with c-Src has been suggested to activate Src by displacing the inhibitory tail domain of Src from its catalytic domain (Hanks and Polte, 1997). Src has been reported to be activated during mitosis by dephosphorylation at Tyr-527 of the tail domain (Bagrodia et al., 1991; Shalloway et al., 1992), which appeared to be inconsistent with the above notion. However, the mitotic activation of Src occurs independently of FAK since FAK is neither tyrosine phosphorylated nor associated with Src during mitosis. A substrate of mitotic Src, sam68, does not seem to be involved in FAK-mediated signal transduction (Fumagalli et al., 1994; Taylor and Shalloway, 1994; Taylor et al., 1995).
Second, the uncoupling probably represents an "off" state of the FAK-mediated signaling pathway of mitotic cells, and this off state would be required for mitotic cells to switch "on" the signal transduction pathway at later stages of cell cycle. For proliferation, normal adherent cells must attach to a substrate during early G1 (O'Neill et al., 1986; Lo and Chen, 1994), at which time FAK-mediated signaling is believed to occur. The mitotic inactivation of FAK would thus act to reestablish the requirement of adhesion for the next round of cell cycle progression.
Third, the inactivation of FAK may contribute to the inhibition of cell spreading and/or cell migration in mitosis. FAK has been implicated to play roles in cell spreading (Richardson and Parsons, 1996; Richardson et al., 1997) and in cell migration (Cary et al., 1996; Gilmore and Romer, 1996). The disruption of FAK/CAS/c-Src complex during mitosis could be a mechanism for the inhibition of cell migration during mitosis because the FAK/CAS or CAS/Crk association has been shown to be important for cell migration by FAK (Cary et al., 1998; Klemke et al., 1998).
Mitosis-specific phosphorylation of FAK could block RhoA-mediated activation of FAK during cytokinesis. It has been reported that the activation of RhoA is required for the completion of cytokinesis because botulinum C3 exoenzyme (which inhibits Rho specifically) blocks cytokinesis (Kishi et al., 1993; Mabuchi et al., 1993). Activation of RhoA, on the other hand, is also known to occur upon serum stimulation of 3T3 fibroblasts, which induces the formation of both stress fibers and focal adhesions (Ridley and Hall, 1992, 1994; Ridley, 1995) as well as tyrosine phosphorylation of FAK (Seufferlein and Rozengurt, 1994a,b, 1995). FAK phosphorylation is blocked by botulinum C3 exoenzyme (Rankin et al., 1994), placing FAK downstream of RhoA. Mitotic cells should have a mechanism that suppresses Rho's ability to activate FAK, and mitosis-specific phosphorylation of FAK may be involved in such a mechanism. FAK phosphorylation continues through cytokinesis, and the reversal of the modification occurs during post-mitotic cell spreading (Fig. 6). FAK phosphorylation may act as an internal clock by preventing premature activation of FAK during cytokinesis. The lack of such phosphorylation in trypsinized cells would explain the rapid adhesion shown when they are plated on ECM-coated substrates.
The mitosis-specific modification of FAK, CAS, and paxillin is apparently a general phenomenon. We have observed similar mobility shifts with other types of adherent cultured cells including 3T3 and HeLa cells. A similar mobility shift has been reported for paxillin (Yamaguchi et al., 1997). We are in the process of identifying kinase(s) and phosphatase(s) responsible for the alterations in the phosphorylation states of these proteins. Cdc2 kinase is not responsible for the mitosis-specific phosphorylation (our unpublished data), which is consistent with the time course of phosphorylation, i.e., phosphorylation continues past cytokinesis. The identification and characterization of kinase(s) and phosphatase(s) would help us understand the mechanisms that define the timing of mitotic events including cell rounding during prophase, cytokinesis, and post-mitotic cell spreading.
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Acknowledgments |
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This work is supported by grants R37-CA42742 (to F. Matsumura) and 2R01-GM49882 (to S.K. Hanks), from the National Institutes of Health.
Submitted: 7 August 1998
Revised: 14 December 1998
Address correspondence to Fumio Matsumura, Dept. of Molecular Biology and Biochemistry, Nelson Labs/Busch Campus, Rutgers University, Piscataway, NJ 08855. Tel.: (732) 445-2838. Fax.: (732) 445-4213. E-mail: matsumura{at}mbcl.rutgers.edu
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