Ras is either mutated or activated in many types of human cancer (6
). However, the mechanism by which Ras regulates focal contacts and consequently promotes tumor cell motility remains to be further elucidated. We have demonstrated here that activated Ras regulates the binding of PTP-PEST to FAK via ERK1/2-mediated phosphorylation and PIN1-dependent isomerization of PTP-PEST. The increased interaction between PTP-PEST and FAK leads to FAK dephosphorylation at Y397 in v-Ras-transformed cells and promotes cell migration, invasion, and metastasis.
PTP-PEST has been implicated in regulation of focal adhesion disassembly and cell migration (1
mouse fibroblasts have more focal adhesions, spread faster on fibronectin substrate, and have slower migration than PTP-PEST+/+
), all of which suggest that PTP-PEST promotes focal adhesion turnover and cell migration. Mutations of PTP-PEST that alter its catalytic activity have been found in both breast and squamous carcinoma cells (34
), which implies a role for misregulation of PTP-PEST in tumor cell motility. PTP-PEST can be posttranslationally modified, which affects its catalytic activity or its access to substrates. It has been shown that treatment of HeLa cells with 12-O
-tetradecanoylphorbol-13-acetate (TPA), forskolin, or 3-isobutyl-1-methylxanthine results in phosphorylation of the S39 and S435 residues of PTP-PEST, which is regulated by cyclic AMP-dependent protein kinase A (PKA) and protein kinase C (PKC) (13
). Phosphorylation of S39 in vitro
reduces PTP-PEST's affinity for its substrate. In addition, PTP-PEST immunoprecipitated from TPA-treated cells displays reduced phosphatase activity (13
). In line with the fact that PTP-PEST can be regulated by phosphorylation, we have shown here that activation of ERK1/2 by Ras results in phosphorylation of PTP-PEST at S571. Phosphorylation of S571, which by itself does not alter the catalytic activity of PTP-PEST toward phosphopeptide substrates, recruits PIN1 for the cis-trans
isomerization of PTP-PEST. The conformational changes of PTP-PEST induced by PIN1, which may alter the structure of the protein-interacting domains adjacent to S571 (10
), increased PTP-PEST's association with FAK and led to FAK dephosphorylation. The fact that mutation of S571 in PTP-PEST inhibits v-H-Ras-induced FAK Y397 dephosphorylation and cell migration, invasion, and metastasis highlights the significance of ERK1/2-mediated PTP-PEST phosphorylation in v-H-Ras-enhanced tumor cell motility.
PIN1 isomerizes the phosphorylated pS/TP bonds in its substrate proteins and catalytically induces their conformational change, which can be involved in regulating protein-protein interactions (18
). We previously showed that activation of Ras and ERK1/2 results in the colocalization and interaction of PIN1, PTP-PEST, and FAK at the lamellipodia, the front leading edge of cells (45
). ERK1/2 phosphorylate FAK S910 and recruit PIN1 for FAK cis-trans
isomerization, which leads to interaction between FAK and PTP-PEST (45
). However, Ras can still induce the interaction between PIN1 and ERK1/2-phosphorylated PTP-PEST in FAK−/−
cells. In addition, Ras promotes the association between PIN1 and ERK1/2-phosphorylated FAK in PTP-PEST−/−
cells. These results indicate that ERK1/2- and PIN1-mediated phosphorylation and the isomerization of both FAK and PTP-PEST occur simultaneously in a mutually independent manner. Nevertheless, both events are coordinated in the regulation of the interaction between FAK and PTP-PEST and subsequent FAK Y397 phosphorylation.
Increased FAK expression and activity, both of which promote tumor growth and metastasis, have been detected in several types of human cancer (17
). In contrast, paradoxical evidence shows that FAK expression was reduced in metastatic tumors compared to their corresponding primary tumors from patients with colorectal adenocarcinoma, cervical cancer, and intrahepatic cholangiocarcinoma (3
). Previously, we and other groups showed that activation of the ErbB family members EGFR, ErbB2, ErbB3, and ErbB4 and of the insulin-like growth factor 1 (IGF-1) receptor induced Tyr dephosphorylation of FAK, inactivation of FAK, and increased tumor cell motility, invasion, and metastasis (4
). In response to IGF-1 signaling, SHP2 is instrumental in tumor cell invasion (25
), and SHP2 activity is required for FAK Tyr dephosphorylation (25
). Notably, activation of EGFR and Ras results in a reduction of total focal adhesions, with limited focal contacts remaining at the leading-edge lamellipodia and tail regions (21
). Moreover, FAK phosphorylation can be dynamically regulated because EGFR- and Ras-induced FAK dephosphorylation is overridden by activated integrin signaling during cell attachment. However, integrin-induced FAK phosphorylation occurs only during the process of adhesion and is downregulated by EGFR and Ras signaling thereafter (21
). On the basis of all these findings, we proposed that overexpression of FAK in human tumor cells might contribute to malignancy by promoting survival through the activation of a positive FAK-c-Src feedback loop and subsequently of downstream tumor-promoting molecules, such as ERK and AKT (17
). However, some other tumors that have aberrant Ras activity, which does not require Src for its transforming ability (data not shown), can activate cell proliferation molecules such as ERK and AKT in a FAK-c-Src-independent manner. Activated Ras signaling inhibits FAK activity, which leads to reduced cell adhesion and increased cell migration. Importantly, FAK activity can be dynamically regulated by integrated Ras and integrin signaling, which may play an instrumental role in the regulation of lamellipodial dynamics and turnover of focal adhesions at the leading edge of the cells during migration (44
). Thus, FAK might have different roles in different tumors and at different stages of tumor progression.
In summary, we have uncovered an important mechanism of PTP-PEST-regulated FAK dephosphorylation in response to oncogenic Ras signaling (). Activation of Ras results in phosphorylation of FAK S910 and PTP-PEST S571 by ERK1/2, which leads to the recruitment of PIN1 and the cis-trans isomerization of both FAK and PTP-PEST. The conformational alterations of FAK and PTP-PEST enable them to bind each other to facilitate subsequent FAK Y397 dephosphorylation and enhanced tumor cell migration, invasion, and metastasis.
Fig. 8. A proposed mechanism for activated Ras-induced and ERK1/2- and PIN1-dependent FAK Y397 dephosphorylation by PTP-PEST. Activated Ras results in ERK1/2-dependent phosphorylation of PTP-PEST S571 and FAK S910 and subsequent recruitment of PIN1 for cis-trans (more ...)