Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration

We have previously shown that overexpression of focal adhesion kinase (FAK) in Chinese hamster ovary (CHO) cells promoted their migration on fibronectin. This effect was dependent on the phosphorylation of FAK at Tyr-397. This residue was known to serve as a binding site for both Src and phosphatidylinositol 3-kinase (PI3K), implying that either one or both are required for FAK to promote cell migration. In this study, we have examined the role of PI3K in FAK-promoted cell migration. We have demonstrated that the PI3K inhibitors, wortmannin and LY294002, were able to inhibit FAK-promoted migration in a dose-dependent manner. Furthermore, a FAK mutant capable of binding Src but not PI3K was generated by a substitution of Asp residue 395 with Ala. When overexpressed in CHO cells, this differential binding mutant failed to promote cell migration although its association with Src was retained. Together, these results strongly suggest that PI3K binding is required for FAK to promote cell migration and that the binding of Src and p130(Cas) to FAK may not be sufficient for this event.     

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.     

Analysis of FAK-associated signaling pathways in the regulation of cell cycle progression

Focal adhesion kinase (FAK) is an important mediator of signal transduction pathways initiated by integrins in cell migration, survival and cell cycle regulation. The ability of FAK to mediate integrin signaling in the regulation of cell cycle progression depends on the phosphorylation of Tyr397, which implies a functional significance for the formation of FAK signaling complexes with Src, phosphatidylinositol-3-kinase (PI3K) and Grb7. We have previously described a FAK mutant, D395A, that selectively disrupts FAK binding to PI3K, but allows FAK association with Src. Using this mutation in a mislocalized FAK mutant background, we show here that formation of a FAK/PI3K complex is not sufficient for cell cycle progression but the formation of a FAK/Src complex plays an essential role. We also show that mutation of D395 to A disrupted FAK association with Grb7. This suggests that a FAK/Grb7 complex is not involved in the cell cycle regulation either, which is supported by direct analysis of cells expressing a dominant negative Grb7 construct. Finally, we provide evidence that the Src-dependent association of FAK with Grb2 and p130(Cas) are both required for the regulation of cell cycle progression by FAK. Together, these studies identify important FAK downstream signaling pathways in cell cycle regulation.     

Differential Effect of the Focal Adhesion Kinase Y397F Mutant on v-Src-Stimulated Cell Invasion and Tumor Growth

Summary Upon cell adhesion to extracellular matrix proteins, focal adhesion kinase (FAK) rapidly undergoes autophosphorylation on its Tyr-397 which consequently serves as a binding site for the Src homology 2 domains of the Src family protein kinases and several other intracellular signaling molecules. In this study, we have attempted to examine the effect of the FAK Y397F mutant on v-Src-stimulated cell transformation by establishing an inducible expression of the Y397F mutant in v-Src-transformed FAK-null (FAK^−/−) mouse embryo fibroblasts. We found that the FAK Y397F mutant had both positive and negative effects on v-Src-stimulated cell transformation; it promoted v-Src-stimulated invasion, but on the other hand it inhibited the v-Src-stimulated anchorage-independent cell growth in vitro and tumor formation in vivo . The positive effect of the Y397F mutant on v-Src-stimulated invasion was correlated with an increased expression of matrix metalloproteinase-2, both of which were inhibited by the specific phosphatidylinositol 3-kinase inhibitor wortmannin or a dominant negative mutant of AKT, suggesting a critical role for the phosphatidylinositol 3-kinase/AKT pathway in both events. However, the expression of the Y397F mutant rendered v-Src-transformed FAK^−/− cells susceptible to anoikis, correlated with suppression on v-Src-stimulated activation of ERK and AKT. In addition, under anoikis stress, the induction of the Y397F mutant in v-Src-transformed FAK^−/− cells selectively led to a decrease in the level of p130^Cas, but not other focal adhesion proteins such as talin, vinculin, and paxillin. These results suggest that FAK may increase the susceptibility of v-Src-transformed cells to anoikis by modulating the level of p130^Cas.     

Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities

Elevated levels of p130^Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130^Cas promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130^Cas protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130^Cas-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130^Cas exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130^Cas on cell biology and therefore will be the target of future studies.     

JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cell migration

c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130(Cas)) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130(Cas), which required p130(Cas) hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130(Cas) pathway by expression of a dominant-negative form of p130(Cas) or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1.FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors.     

Suppression of ultraviolet irradiation-induced apoptosis by overexpression of focal adhesion kinase in Madin-Darby canine kidney cells.

Focal adhesion kinase (FAK) has been implicated to play a role in suppression of apoptosis. In this study, we have demonstrated that UV irradiation induced cleavage of FAK and two of its interacting proteins Src and p130(Cas) in Madin-Darby canine kidney cells, concomitant with an increase in cell death. The cleavage of these proteins upon UV irradiation was completely inhibited by ZVAD-FMK, a broad range inhibitor of caspases, and apparently delayed by Bcl2 overexpression. To examine if FAK plays a role in suppressing UV-induced apoptosis, stable Madin-Darby canine kidney cell lines overexpressing FAK were established. Our results showed that a marked (30-40%) increase in cell survival upon UV irradiation was achieved by this strategy. In our efforts to determine the mechanism by which FAK transduces survival signals to the downstream, we found that a FAK mutant deficient in binding to phosphatidylinositol 3-kinase failed to promote cell survival. Moreover, the expression of the Src homology 3 domain of p130(Cas), which competed with endogenous p130(Cas) for FAK binding, abrogated the FAK-promoted cell survival. Together, these results suggest that the integrity of FAK and its binding to phosphatidylinositol 3-kinase and p130(Cas) are required for FAK to exert its antiapoptotic function.     

Naphtho[1,2-b]furan-4,5-dione inhibits MDA-MB-231 cell migration and invasion by suppressing Src-mediated signaling pathways

Naphtho[1,2-b]furan-4,5-dione (NFD), a bioactive component of Avicennia marina, has been demonstrated to display anti-cancer activity. Breast cancer is a highly malignant carcinoma and most deaths of breast cancer are caused by metastasis. In this study, we showed that NFD blocked migration and invasion of MDA-MB-231 breast cancer cells without affecting apoptosis or growth arrest. NFD caused significant block of Src kinase activity in MDA-MB-231 cells. Moreover, NFD treatment was correlated with reduced phosphorylation of FAK at Tyr 576/577, 861 and 925 sites, p130^Cas at Tyr 410, and paxillin at Tyr 118. NFD also suppressed the activation of phosphatidylinositol 3-kinase/Akt. Consistent with inhibition of these signaling pathways and invasion, NFD reduced the expression of matrix metalloproteinase-9. Furthermore, Src antagonist PP2 caused a significant decrease in the phosphorylation of FAK, p130^Cas, paxillin, and PI3K/Akt. Our findings provide evidences that NFD inhibits Src-mediated signaling pathways involved in controlling breast cancer migration and invasion, suggesting that it has a therapeutic potential in breast cancer treatment.     

Association of focal adhesion kinase with Grb7 and its role in cell migration.

Focal adhesion kinase (FAK) has been implicated to play a key role in integrin-mediated signal transduction in cell migration. Grb7 is an Src homology (SH) 2-containing and pleckstrin homology domain-containing molecule, which shares significant homology with the Caenorhabditis elegans gene for Mig-10 involved in cell migration during embryogenesis. Here, we report that the SH2 domain of Grb7 can directly interact with FAK through Tyr-397, a major autophosphorylation site in vitro and in vivo. This interaction is cell adhesion-dependent, suggesting that the FAK-Grb7 complex is involved in integrin signaling. Using tetracycline-regulated expression system, we showed that overexpression of Grb7 enhanced cell migration toward fibronectin, whereas overexpression of its SH2 domain alone inhibited cell migration. In addition, we found that phosphorylation of FAK or p130(cas) was not affected by the expression of either Grb7 or its SH2 domain alone, suggesting that Grb7 is downstream of FAK and does not compete with Src for binding to FAK in vivo. Taken together, these results suggest that the FAK-Grb7 complex plays a role in cell migration stimulated by integrin signaling through FAK.     

Role of Grb7 targeting to focal contacts and its phosphorylation by focal adhesion kinase in regulation of cell migration

We have previously described Grb7 association with focal adhesion kinase (FAK) and its possible roles in cell migration. In this paper, we investigated the mechanisms by which Grb7 and its association with FAK regulate cell migration. We found that deletion of the Grb7 SH2 domain eliminated partial Grb7 localization to focal contacts and its ability to stimulate cell migration. Replacement of the SH2 domain with the focal adhesion targeting sequence from FAK resulted in the focal contacts localization of the chimeric molecule and restored its activity to stimulate cell migration. We also found that Grb7 could be phosphorylated by FAK, which was dependent on the FAK kinase activity but not the presence of the Src family kinases. Cell adhesion also enhanced Grb7 phosphorylation in FAK+/+ cells but not FAK-/- cells, suggesting that Grb7 is a physiological substrate of FAK. Furthermore, both Grb7 and the chimeric molecule did not increase migration of FAK-/- cells, although the chimeric molecule was targeted to the focal contacts. Last, we showed that other Grb7 family members could not stimulate cell migration under similar experimental conditions. Together, these results demonstrate a role for Grb7 targeting to focal contacts and its phosphorylation by FAK in the regulation of cell migration.     

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.     

Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration.

The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.     

Differential regulation of cell migration and cell cycle progression by FAK complexes with Src, PI3K, Grb7 and Grb2 in focal contacts.

Focal adhesion kinase (FAK) is a key mediator of integrin signaling, which has been implicated in the regulation of cell migration and cell cycle progression. Using chimeric molecules that fuse the focal adhesion targeting (FAT) sequence directly to several signaling molecules, we investigated the potential role of FAK recruitments of signaling molecules to focal contacts in the regulation of cell migration and cell cycle progression. We found that fusion of FAT to Src, the p85 subunit of phosphatidylinositol 3-kinase, Grb7 and Grb2 resulted in the efficient focal adhesion targeting of these signaling molecules. We showed that expression of Src-FAT, p85-FAT, or Grb7-FAT, but not Grb2-FAT, each stimulated cell migration. Interestingly, tyrosine phosphorylation of paxillin, but not p130cas, was induced by expression of Src-FAT, suggesting a potential role of paxillin in mediating stimulation of cell migration by the chimeric molecule. In contrast, targeting of Grb2, but not Src, p85, or Grb7, to focal contacts increased cell cycle progression. Biochemical analyses correlated Erk activation by Grb2-FAT with its stimulation of cell cycle progression. Together, these results suggest that at least part of the role of FAK interaction with these signaling molecules is to recruit them to focal contacts and that distinct FAK signaling complexes are involved in the regulation of cell migration vs. cell cycle progression.     

Transformation Suppression by Protein Tyrosine Phosphatase 1B Requires a Functional SH3 Ligand

We have recently shown that protein tyrosine phosphatase 1B (PTP1B) associates with the docking protein p130^Cas in 3Y1 rat fibroblasts. This interaction is mediated by a proline-rich sequence on PTP1B and the SH3 domain on p130^Cas. Expression of wild-type PTP1B (WT-PTP1B), but not a catalytically competent, proline-to-alanine point mutant that cannot bind p130^Cas (PA-PTP1B), causes substantial tyrosine dephosphorylation of p130^Cas (F. Liu, D. E. Hill, and J. Chernoff, J. Biol. Chem. 271:31290–31295, 1996). Here we demonstrate that WT-, but not PA-PTP1B, inhibits transformation of rat 3Y1 fibroblasts by v-crk, -src, and -ras, but not by v-raf. These effects on transformation correlate with the phosphorylation status of p130^Cas and two proteins that are associated with p130^Cas, Paxillin and Fak. Expression of WT-PTP1B reduces formation of p130^Cas-Crk complexes and inhibits mitogen-activated protein kinase activation by Src and Crk. These data show that transformation suppression by PTP1B requires a functional SH3 ligand and suggest that p130^Cas may represent an important physiological target of PTP1B in cells.     

Investigation of Binding Phenomenon of NSP3 and p130Cas Mutants and Their Effect on Cell Signalling

Members of the novel SH2-containing protein (NSP3) and Crk-associated substrate (p130Cas) protein families form a multi-domain signalling platforms that mediate cell signalling process. We analysed the damaging consequences of three mutations, each from NSP3 (NSP3^L469R, NSP3^L623E, NSP3^R627E) and p130Cas (p130Cas^F794R, p130Cas^L787E, p130Cas^D797R) protein with respect to their native biological partners. Mutations depicted notable loss in interaction affinity towards their corresponding biological partners. NSP3^L469R and p130Cas^D797R mutations were predicted as most prominent in docking analysis. Molecular dynamics (MD) studies were conducted to evaluate structural consequences of most prominent mutation in NSP3 and p130Cas obtained from the docking analysis. MD analysis confirmed that mutation in NSP3^L469R and p130Cas^D797R showed significant structural deviation, changes in conformations and increased flexibility, which in turn affected the binding affinity with their biological partners. Moreover, the root mean square fluctuation has indicated a rise in fluctuation of residues involved in moderate interaction acquired between the NSP3 and p130Cas. It has significantly affected the binding interaction in mutant complexes. The results obtained in this work present a detailed overview of molecular mechanisms involved in the loss of cell signalling associated with NSP3 and p130Cas protein.     

Regulation of focal adhesion and cell migration by ANKRD28-DOCK180 interaction

DOCK180 is an atypical guanine nucleotide exchange factor of Rac1 identified originally as one of the two major proteins bound to the SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130^Cas, and recruits the Crk-p130^Cas complex to focal adhesions. Recently, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and found that ANKRD28, a protein with twenty-six ankyrin domain-repeats, interacts with the SH3 domain of DOCK180. Knockdown of ANKRD28 reduced the migration velocity and altered the distribution of focal adhesion proteins such as Crk, paxillin, and p130^Cas. On the other hand, the expression of ANKRD28, p130^Cas, Crk, and DOCK180 induced hyper-phosphorylation of p130^Cas, which paralleled the induction of multiple long cellular processes. Depletion of ELMO, another protein bound to the SH3 domain of DOCK180, also retarded cell migration, but its expression together with p130^Cas, Crk, and DOCK180 induced extensive lamellipodial protrusion around the entire circumference without 130^Cas hyperphosphorylation. These data suggest the dual modes of DOCK180-Rac regulation for cell migration.     

Ankyrin repeat domain 28 (ANKRD28), a novel binding partner of DOCK180, promotes cell migration by regulating focal adhesion formation

DOCK180 is a guanine exchange factor of Rac1 originally identified as a protein bound to an SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130^Cas, and recruits the Crk-p130^Cas complex to focal adhesions. To understand the role of DOCK180 in cell adhesion and migration, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and identified ANKRD28, a protein that contains twenty-six ankyrin domain repeats. Knockdown of ANKRD28 by RNA interference reduced the velocity of migration of HeLa cells, suggesting that this protein plays a physiologic role in the DOCK180-Rac1 signaling pathway. Furthermore, knockdown of ANKRD28 was found to alter the distribution of focal adhesion proteins such as Crk, paxillin, and p130^Cas. On the other hand, expression of ANKRD28, p130^Cas, Crk, and DOCK180 induced hyper-phosphorylation of p130^Cas, and impaired detachment of the cell membrane during migration. Consequently, cells expressing ANKRD28 exhibited multiple long cellular processes. ANKRD28 associated with DOCK180 in an SH3-dependent manner and competed with ELMO, another protein bound to the SH3 domain of DOCK180. In striking contrast to ANKRD28, overexpression of ELMO induced extensive lamellipodial protrusion around the entire circumference. These data suggest that ANKRD28 specifies the localization and the activity of the DOCK180-Rac1 pathway.     

Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.     

Association of Grb7 with phosphoinositides and its role in the regulation of cell migration

Grb7 is the prototype of a family of adaptor molecules that also include Grb10 and Grb14 that share a conserved molecular architecture including Src homology 2 (SH2) and pleckstrin homology (PH) domains. Grb7 has been implicated as a downstream mediator of integrin-FAK signal pathways in the regulation of cell migration, although the molecular mechanisms are still not well understood. In this paper, we investigated the potential role and mechanisms of PH domain in Grb7 in the regulation of cell migration. We found that the PH domain mediated Grb7 binding to phospholipids both in vitro and in intact cells. Furthermore, both Grb7 and its PH domain preferentially interacted with phosphatidylinositol phosphates showing strongest affinity to the D3- and D5-phosphoinositides. The PH domain interaction with phosphoinositides was shown to play a role in the stimulation of cell migration by Grb7. It was also shown to be necessary for Grb7 phosphorylation by FAK, although it was not required for Grb7 interaction with FAK or recruitment to the focal contacts. Last, we found that PI 3-kinase activity played a role in both Grb7 association with phosphoinositides and its stimulation of cell migration. In addition, both FAK binding to PI 3-kinase via its autophosphorylated Tyr(397) and integrin-mediated cell adhesion increased Grb7 association with phosphoinositides. Together, these results identified the Grb7 PH domain interaction with phosphoinositides and suggested a potential mechanism by which several signaling molecules including Grb7, FAK, and PI 3-kinase and their interactions cooperate to mediate signal transduction pathways in integrin-mediated cell migration.