FAK activation and focal adhesions formation are closely related events. Until recently, the exact order in which they occur following integrin stimulation was greatly debated. Recent experiments (Wilson et al., 1995
; Chrzanowska-Wodnicka and Burridge, 1996
) showed that FAK activation is a result of focal adhesion formation. Due to the physical tension caused by the stress fiber formation in a cell, under the control of the Rho GTPase, the associated integrins cluster and associate with other structural proteins like α-actinin and tensin (Burridge and Chrzanowska-Wodnicka, 1996
). FAK proteins are associated with integrins and can transphosphorylate in a manner similar to receptor tyrosine kinases following extracellular ligand binding. This phosphorylation activates FAK and provides docking sites for other signal transduction proteins that propagate integrin-triggered pathways.
However, the relationship between focal adhesion formation and FAK activation is apparently not strictly linear. Among the signals sent by FAK, some were shown to feed back and play a role in the turnover rate of focal adhesions. The existence of such signals was concluded when the FAK null cell lines showed an increase in focal adhesion size and number (Ilìc et al., 1995
). However, these results did not take into account the effects of other members of the FAK family that were later identified, such as Pyk2 (Sasaki et al., 1995
). Recently, FAK was shown to play a role in migration, an event that requires focal adhesion formation and breakdown. This increase in migration occurs via the tyrosine phosphorylation of an adapter protein, p130CAS
(Cary et al., 1998
). Also, gene disruption of a cytoplasmic tyrosine phosphatase with two SH2 domains, SHP-2, was shown to impair the ability of fibroblasts to spread and migrate on fibronectin (Yu et al., 1998
). This phenotype was associated with a decrease in FAK dephosphorylation following cell detachment and suspension. In this paper, we examined another cell line with a gene inactivated by homologous recombination, cytosolic PTP-PEST. A major substrate for this enzyme in both mice and humans was shown to be p130CAS
(Garton et al., 1996
; Côté et al., 1998
), and we show in Fig. that, in COS-1 cells, PTP-PEST can translocate to the membrane periphery following integrin activation, possibly in order to reach the p130CAS
substrate which also translocates to focal adhesions when FAK is activated (Nakamoto et al., 1997
The effects of PTP-PEST removal on cell migration and focal adhesion number were similar to the phenotype of the FAK mutant cells (Ilìc et al., 1995
). Both showed a decrease in fibroblast migration using fibronectin as an extracellular matrix, and the presence of numerous immature focal adhesions scattered on the ventral face of the cell (Figs. and ). Even though FAK and PTP-PEST have opposite catalytic activities on a common substrate, p130CAS
, PTP-PEST does not seem to antagonize the effects of FAK in cell migration, but rather to potentiate these effects. This suggests a mechanism where both a formation and a breakdown pathway are triggered at the same time to increase the turnover rate of the focal adhesion structures. Without the pathway required for focal adhesion breakdown, the adhesive contacts between the extracellular matrix and the cell are too strong, and the cell adheres and does not migrate (Schwarzbauer, 1997
). In FAK (−/−) cells, the actin stress fibers were dense around the cell periphery rather than in the center, where they can exert the transcellular tension necessary for cell migration (Lauffenburger and Horwitz, 1996
). In contrast, in the PTP-PEST (−/−) cells, central stress fibers are omnipresent, suggesting that the cells cannot turn them over. This state is also incompatible with cell migration. The absence of differences in the early stages of cell attachment to fibronectin (Fig. , a–d) suggests that PTP-PEST is not implicated in the initial formation of stress fibers or focal adhesions. However, at equilibrium, clear differences appear (Fig. , e–h). The focal adhesions in the PTP-PEST (−/−) cells are numerous and scattered throughout the ventral (i.e., substrate-attached) membrane of the cell, which can in part explain the absence of motility of the cells when tested in the wound healing assay. In contrast, the heterozygous cells became rounded, their focal adhesions were punctate and found only at the tips of their membranes, which is consistent with a migrating cell. This led us to believe that PTP-PEST is implicated in focal adhesion and stress fiber breakdown, a role that is the counterpart of FAK or Src but is required for the successful achievement of the same event, cell migration.
In the PTP-PEST mutant cell line, p130CAS
, paxillin, and FAK were found in a hyperphosphorylated state. The phosphorylation of paxillin was shown to be associated with cell spreading on an extracellular matrix (Richardson et al., 1997
), which is consistent with data reported here. Paxillin can associate with PTP-PEST (Shen et al., 1998
), but it does not constitute a direct physiological substrate for this PTP (Côté, J.-F., C.E. Turner, and M.L. Tremblay, manuscript submitted for publication) and the exact site that is hyperphosphorylated is still not clear.
SH2-domain affinity assays shown in Fig. strongly suggest the sites that are hyperphosphorylated on p130CAS
are within the SH2-binding domains of Src and Crk. One hypothesis is that the only tyrosine that is substrate for PTP-PEST is the 762YDYV765 Src SH2-binding region on p130CAS
, which binds Src in a tyrosine-dependant manner (Sakai et al., 1994
). In PTP-PEST (−/−) cells, this can cause Src to constitutively bind p130CAS
and hyperphosphorylate the Crk-binding motifs on p130CAS
. This would explain why p130CAS
is so hyperphosphorylated in the PTP-PEST (−/−) cells, and why the difference in affinity is greater with the Src SH2 domain compared with the Crk SH2 domain, even though there are 15 putative binding domains for Crk compared with only one for Src on p130CAS
. In the same line of thought, this could also be the cause for the small FAK hyperphosphorylation in the PTP-PEST (−/−) cells, since the SH3 domain of p130CAS
can bind a proline-rich region on FAK (Polte and Hanks, 1995
), and FAK is also a substrate for Src (Calalb et al., 1995
; Schlaepfer and Hunter, 1996
). This increased affinity between Crk and p130CAS
in the PTP-PEST (−/−) cells can be related to the phenotypes observed in Figs. and . The CAS/Crk coupling has been shown to play a role in the induction of cell migration (Klemke et al., 1998
). Thus, incorrect regulation of this molecular switch in the absence of PTP-PEST could lead to aberrant cell migration. These results provide a concrete, tyrosine phosphorylation–dependent way for PTP-PEST to regulate cell migration, and strongly suggest the phenotypes observed here are the result of the gene targeting. Still, reintroduction at physiological levels of different PTP-PEST constructs in the knockout cells to differentially rescue the phenotypes described in this paper is currently under way in our laboratory.
Studies using the PTP inhibitor phenylarsine oxide showed that treatment of cells with this compound was sufficient to induce the formation of stress fibers even after starving them for 16 h (Retta et al., 1996
). Phenylarsine oxide reacts with two thiol groups of closely spaced cysteine residues in the active site of the phosphatase. The PTP-PEST catalytic domain contains the sequence 231CSAGC235 (Charest et al., 1995
), the cysteine at position 231 being crucial for its catalytic activity. These studies also showed that focal adhesion disassembly results in stimulation of phosphatase activity, which could be assayed using FAK and paxillin as substrates. That, and the fact that paxillin is hyperphosphorylated in the PTP-PEST knockout cell line, suggest that PTP-PEST is a candidate PTP involved in the focal adhesion breakdown in the conditions studied in these experiments.
The role of a PTP in focal adhesion breakdown would suggest that overexpression of the PTP would also inhibit cell migration, by impairing the formation of the focal adhesions at the leading edge of the cell. Experiments involving another PTP that can dephosphorylate p130CAS
, PTP1B (Liu et al., 1996
), showed that overexpression of this PTP in rat fibroblasts decreased cell migration while increasing the time required for the cell to spread on fibronectin (Liu et al., 1998
). This was linked to a disordered formation of focal adhesions. In this article, we demonstrated that removal of a PTP that can dephosphorylate p130CAS
, PTP-PEST, increased the spreading speed of targeted cells on the same extracellular matrix protein (Fig. ). Interestingly, the PTP1B-overexpressing cells eventually formed numerous, large focal complexes scattered over their ventral surface, like ones found in the PTP-PEST (−/−) cells. These experiments and the ones presented in this paper suggest that an intermediate level of PTP activity towards p130CAS
is required for the formation of normal focal adhesions and for cell migration, which is consistent with a role in focal adhesion turnover.
PTP-PEST may also play a role in the regulation of the cell cytoskeleton, this time via the cleavage furrow–associated protein PSTPIP. PSTPIP was originally identified as a binding partner and a substrate for the phosphatase PTP-HSCF (Spencer et al., 1997
), a PEST tyrosine phosphatase. PTP-HSCF dephosphorylates tyrosine residues in PSTPIP that are modified either by coexpression of the v-Src tyrosine kinase or in the presence of the unspecific PTP inhibitor pervanadate. One of these sites, within the SH3 domain of PSTPIP, was shown to regulate binding with the proline-rich region found on WASP (Wu et al., 1998), and to control aspects of the actin cytoskeleton.
The possibility that tyrosine phosphorylation is involved in furrow development and the signaling events coordinating nuclear division was reported by Cool et al. (1992)
. Their data show that BHK cells overexpressing a truncated PTP, namely T cell PTP, become highly multinucleate, apparently through a failure in cytokinesis. This deletion mutant lacked the COOH-terminal extension responsible for its proper localization, and the redistribution of the enzyme to the soluble fraction caused both a furrowing defect and an asynchronous entry into S phase of two nuclei within the same syncytial cell.
It has not been shown whether PTP-PEST binds directly to PSTPIP, but peptides derived from the conserved proline-rich COOH terminus of PTP-PEST can compete the binding of PTP-HSCF to PSTPIP (Spencer et al., 1997
), consistent with the suggestion that PTP-PEST binds to PSTPIP in a manner similar to PTP-HSCF binding. In addition, while no previous data had shown that PTP-PEST can dephosphorylate PSTPIP, the fact that PSTPIP is hyperphosphorylated in the PTP-PEST (−/−) cells (Fig. ) strongly suggests that PTP-PEST plays a direct role in modulating the tyrosine phosphorylation level of PSTPIP. PTP-HSCF and another member of the PEST family, PTP-PEP, are generally not expressed in fibroblasts, and it is possible that PTP-PEST regulates PSTPIP in fibroblasts the way PTP-HSCF does in hematopoietic cells. The exact site that is hyperphosphorylated on PSTPIP in PTP-PEST (−/−) cells is not known, and the effects of this hyperphosphorylation on binding, for example, to WASP (Wu et al., 1998b
) are still under investigation. One of the observable effects appears to be an increase in the relative amount of cells found in the last stages of cytokinesis in a field of unsynchronized cells (Fig. ), possibly due to the role of PSTPIP in cleavage furrow assembly or disassembly. It is also possible that the increase of PSTPIP tyrosine phosphorylation is the result, and not the cause, of the increase of the number of cells in this phase.
The fact that PTP-PEST (−/−) cells still divide and grow at rates comparable to other cells suggests the presence of other mechanisms involved in cell division. Isolation and characterization of cytokinesis-deficient mutants in Dictyostelium discoideum
, a highly motile organism that undergoes cell cleavage much like higher eukaryotes, provided examples of cell division occurring with impaired cytokinesis (Vithalani et al., 1996
). In particular, a mutation in the myosin gene that prevents the protein assembly in thick filaments resulted in organisms clearly defective in the contractile events involved in cytokinesis (Fukui et al., 1990
). Another mutant, called 10BH2, also had a complete defect in cytokinesis (Vithalani et al., 1996
). However, both mutants, which showed cell division defects when grown in suspension, were able to divide when plated on solid substratum by pinching off a portion of their cytoplasm in a process known as traction-mediated cytofission (Fukui et al., 1990
). It is possible that the PTP-PEST (−/−) cells in part rely on this event to normally grow, even if the defect in chemokinesis observed in the wound-healing assay could impair the required migration. Interestingly, another D. discoideum
mutant, in the single gene encoding calmodulin, formed and constricted a contractile cleavage furrow ring, but the midbody linking the daughter cells failed to completely close (Liu et al., 1992
). The resultant population contained cells resembling cells observed in the PTP-PEST (−/−) population. This cytoplasmic bridge could be broken by shear forces when the cells were grown in suspension cultures and the cells could multiply normally. This suggests that cells in this state require little force to successfully complete division, and could explain the fact that the PTP-PEST (−/−) cells can still divide.
In this article, we reported two roles that a specific PTP, PTP-PEST, could play in regulating the cytoskeleton of fibroblasts. The first, possibly via its capacity to dephosphorylate p130CAS
, is to break down focal adhesions, an event which is required for cell migration on an extracellular matrix like fibronectin. PTP-PEST was shown to localize at the membrane periphery when COS-1 cells were plated on fibronectin and this confers to PTP-PEST a physiological role in cell migration that is not a secondary effect of overexpression. The fact that PTP-PEST is mostly found in a cytoplasmic pool (Charest et al., 1995
) and can be recruited to the plasma membrane after fibronectin-mediated attachment provides the cell a mechanism to increase its focal adhesions turnover rate proportional to the stimulus, even when FAK, p130CAS
, or v-Src is overexpressed (Akasaka et al., 1995
; Cary et al., 1998
; Fincham and Frame, 1998
). PTP-PEST also plays a role in modulating the phosphorylation level of PSTPIP, a protein that associates with the cytoskeleton (Spencer et al., 1997
). The role of this PTP-PEST activity is not known, but it may involve the binding of WASP to PSTPIP (Wu et al., 1998b
). Since WASP has been shown to regulate actin fiber assembly and cytokinesis in both yeast (Li, 1997
) and mammalian (Symons et al., 1996
) cells. These data suggest that this interaction may be somehow modulated in the PTP-PEST (−/−) cells.