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Yoder, M., and J.D. Hildebrand (2007) Shroom4 (KIAA1202) is an actin-associated protein implicated in cytoskeletal organization. Cell Motil. Cytoskel. 64:49-63 All animal cells utilize a specialized set of cytoskeletal proteins to determine their overall shape and the organization of their intracellular compartments and organelles. During embryonic development, the dynamic nature of the actin cytoskeleton is critical for virtually all morphogenic events requiring changes in cell shape, migration, adhesion, and division. The behavior of the actin cytoskeleton is modulated by a myriad of accessory proteins. Shroom3 is an actin binding protein that regulates neural tube morphogenesis by eliciting changes in cell shape through a myosin II-dependent pathway. The Shroom-related gene SHROOM4 (formerly called KIAA1202) has also been implicated in neural development, as mutations in this gene are associated with human X-linked mental retardation. To better understand the function of Shrm4 in embryonic development, we have cloned mouse Shroom4 and characterized its protein product in vivo and in vitro. Shroom4 is expressed in a wide range of cell types during mouse development, including vascular endothelium and the polarized epithelium of the neural tube and kidney. In endothelial cells and embryo fibroblasts, endogenous Shroom4 co-distributes with myosin II to a distinct cytoplasmic population of F-actin and ectopic expression of Shroom4 in multiple cell types enhances or induces the formation of this actin-based structure. This localization is mediated, at least in part, by the direct interaction of Shroom4 and F-actin. Our results suggest that Shroom4 is a regulator of cytoskeletal architecture that may play an important role in vertebrate development. Cell Motil. Cytoskeleton 2006. (c) 2006 Wiley-Liss, Inc.
Hagens, O., A. Ballabio, V. Kalscheuer, J.P. Kraehenbuhl, M.V. Schiaffino, P. Smith, O. Staub, J. Hildebrand, and J.B. Wallingford (2006) A new standard nomenclature for proteins related to Apx and Shroom. BMC Cell Biol 7:18 Shroom is a recently-described regulator of cell shape changes in the developing nervous system. This protein is a member of a small family of related proteins that are defined by sequence similarity and in most cases by some link to the actin cytoskeleton. At present these proteins are named Shroom, APX, APXL, and KIAA1202. In light of the growing interest in this family of proteins, we propose here a new standard nomenclature.
Fairbank, P.D., C. Lee, A. Ellis, J.D. Hildebrand, J.M. Gross, and J.B. Wallingford (2006) Shroom2 (APXL) regulates melanosome biogenesis and localization in the retinal pigment epithelium. Development 133:4109-4118 Shroom family proteins have been implicated in the control of the actin cytoskeleton, but so far only a single family member has been studied in the context of developing embryos. Here, we show that the Shroom-family protein, Shroom2 (previously known as APXL) is both necessary and sufficient to govern the localization of pigment granules at the apical surface of epithelial cells. In Xenopus embryos that lack Shroom2 function, we observed defects in pigmentation of the eye that stem from failure of melanosomes to mature and to associate with the apical cell surface. Ectopic expression of Shroom2 in naive epithelial cells facilitates apical pigment accumulation, and this activity specifically requires the Rab27a GTPase. Most interestingly, we find that Shroom2, like Shroom3 (previously called Shroom), is sufficient to induce a dramatic apical accumulation of the microtubule-nucleating protein gamma-tubulin at the apical surfaces of naive epithelial cells. Together, our data identify Shroom2 as a central regulator of RPE pigmentation, and suggest that, despite their diverse biological roles, Shroom family proteins share a common activity. Finally, because the locus encoding human SHROOM2 lies within the critical region for two distinct forms of ocular albinism, it is possible that SHROOM2 mutations may be a contributing factor in these human visual system disorders.
Dietz, M.L., T.M. Bernaciak, F. Vendetti, and J.D. Hildebrand (2006) Differential actin-dependent localization modultes the evolutionarily conserved activity of shroom-family proteins. J. Biol. Chem. 281:20542-20554 Shroom is an actin-associated determinant of cell morphology that is required for neural tube closure in both mice and frogs. Shroom regulates this process by causing apical constriction of epithelial cells via a pathway involving myosin II. Here we report on characterization of the Shroom-related proteins Apxl and KIAA1202 and their role in cell architecture. Shroom, Apxl, and KIAA1202 exhibit differing abilities to interact with the actin cytoskeleton. In fibroblasts, Shroom readily associates with actin stress fibers and induces bundling, Apxl is found on cortical actin, and KIAA1202 is localized to a cytoplasmic population of F-actin. In epithelial cells, Apxl and KIAA1202 do not induce apical constriction as Shroom does, but have the capacity to do so if targeted to the Apical Junctional Complex. To determine if the activity of Shroom-like proteins is conserved in invertebrates, we have tested the ability of the lone Shroom-related protein in Drosophila, CG8603, to activate the constriction pathway. A chimeric protein consisting of the Shroom targeting domain and the Drosophila protein elicits constriction. Finally, we show that Apxl is involved in regulating the cytoskeletal organization and architecture of endothelial cells. We predict that the ability of Shroom-like proteins to regulate cellular morphology is conserved in evolution and is regulated in part by subcellular localization.
Hildebrand, J.D. (2005) CtBP proteins in vertebrate development. Pp in CtBP Family Proteins, Chinnadurai, G., Ed. Landes Bioscience Hildebrand, J.D. (2005) Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network. J. Cell Sci. 118:5191-5203 The actin-binding protein Shroom is essential for neural tube morphogenesis in multiple vertebrate organisms, indicating its function is evolutionarily conserved. Shroom facilitates neurulation by regulating the morphology of neurepithelial cells. Shroom localizes to the apical tip of adherens junctions of neural ectoderm cells in vivo and to the apical junctional complex (AJC) in MDCK cells. Induced expression of Shroom in polarized epithelia elicits apical constriction and dramatic reorganization of the apical arrangement and packing of cells without altering apical-basal polarity. These events likely mimic the cell shape changes and cellular movements required for neurulation in vivo. The observed phenotypes depend on the ability of Shroom to alter F-actin distribution and regulate the formation of a previously uncharacterized contractile actomyosin network associated with the AJC. Targeting the C-terminal domain of Shroom to the apical plasma membrane elicits constriction and reorganization of the actomyosin network, indicting that this domain mediates Shroom's activity. In vivo, Shroom-mutant neural epithelia show a marked reduction in apically positioned myosin. Thus, Shroom likely facilitates neural tube closure by regulating cell shape changes via the apical positioning of an actomyosin network in the neurepithelium.
Haigo, S.L., J.D. Hildebrand, R.M. Harland, and J.B. Wallingford (2003) Shroom induces apical constriction and is required for hingepoint formation during neural tube closure. Curr. Biol. 13:2125-2137 BACKGROUND: The morphogenetic events of early vertebrate development generally involve the combined actions of several populations of cells, each engaged in a distinct behavior. Neural tube closure, for instance, involves apicobasal cell heightening, apical constriction at hingepoints, convergent extension of the midline, and pushing by the epidermis. Although a large number of genes are known to be required for neural tube closure, in only a very few cases has the affected cell behavior been identified. For example, neural tube closure requires the actin binding protein Shroom, but the cellular basis of Shroom function and how it influences neural tube closure remain to be elucidated. RESULTS: We show here that expression of Shroom is sufficient to organize apical constriction in transcriptionally quiescent, naive epithelial cells but not in non-polarized cells. Shroom-induced apical constriction was associated with enrichment of apically localized actin filaments and required the small GTPase Rap1 but not Rho. Endogenous Xenopus shroom was found to be expressed in cells engaged in apical constriction. Consistent with a role for Shroom in organizing apical constriction, disrupting Shroom function resulted in a specific failure of hingepoint formation, defective neuroepithelial sheet-bending, and failure of neural tube closure. CONCLUSIONS: These data demonstrate that Shroom is an essential regulator of apical constriction during neurulation. The finding that a single protein can initiate this process in epithelial cells establishes that bending of epithelial sheets may be patterned during development by the regulation of expression of single genes.
Zhang, Q., Y. Yoshimatsu, J. Hildebrand, S.M. Frisch, and R.H. Goodman (2003) Homeodomain Interacting Protein Kinase 2 Promotes Apoptosis by Downregulating the Transcriptional Corepressor CtBP. Cell 115:177-186 Genetic knockout of the transcriptional corepressor CtBP in mouse embryo fibroblasts upregulates several genes involved in apoptosis. We predicted, therefore, that a propensity toward apoptosis might be regulated through changes in cellular CtBP. To identify pathways involved in this regulation, we screened a mouse embryo cDNA library with an E1A-CtBP complex and identified the homeodomain interacting protein kinase 2 (HIPK2), which had previously been linked to UV-directed apoptosis through its ability to phosphorylate p53. Expression of HIPK2 or exposure to UV irradiation reduced CtBP levels via a proteosome-mediated pathway. The UV effect was prevented by coexpression of kinase-inactive HIPK2 or reduction in HIPK2 levels via siRNA. Mutation of the residue phosphorylated by HIPK2 prevented UV- and HIPK2-directed CtBP clearance. Finally, reduction in CtBP levels, either by genetic knockout or siRNA, promoted apoptosis in p53-deficient cells. These findings provide a pathway for UV-induced apoptosis in cells lacking p53.
Lin, X., B. Sun, M. Liang, Y.Y. Liang, A. Gast, J. Hildebrand, F.C. Brunicardi, F. Melchior, and X.H. Feng (2003) Opposed regulation of corepressor CtBP by SUMOylation and PDZ binding. Mol. Cell 11:1389-1396 The transcription corepressor CtBP is often recruited to the target promoter via interaction with a conserved PxDLS motif in the interacting repressor. In this study, we demonstrate that CtBP1 was SUMOylated and that its SUMOylation profoundly affected its subcellular localization. SUMOylation occurred at a single Lys residue, Lys428, of CtBP1. CtBP2, a close homolog of CtBP1, lacked the SUMOylation site and was not modified by SUMO-1. Mutation of Lys428 into Arg (K428R) shifted CtBP1 from the nucleus to the cytoplasm, while it had little effect on its interaction with the PxDLS motif. Consistent with a change in localization, the K428R mutation abolished the ability of CtBP1 to repress the E-cadherin promoter activity. Notably, SUMOylation of CtBP1 was inhibited by the PDZ domain of nNOS, correlating with the known inhibitory effect of nNOS on the nuclear accumulation of CtBP1. This study identifies SUMOylation as a regulatory mechanism underlying CtBP1-dependent transcriptional repression.
Grooteclaes, M., Q. Deveraux, J. Hildebrand, Q. Zhang, R.H. Goodman, and S.M. Frisch (2003) C-terminal-binding protein corepresses epithelial and proapoptotic gene expression programs. Proc. Natl. Acad. Sci., USA 100:4568-4573 The genesis of carcinoma cells often involves epithelial-to-mesenchymal transitions and the acquisition of apoptosis resistance, but it is unclear whether these alterations are controlled coordinately or independently. Our previously reported effects of adenovirus E1a in human tumor cells raised the possibility that the E1a-interacting corepressor protein C-terminal-binding protein (CtBP) might selectively repress epithelial cell adhesion and proapoptotic genes. Here, we report that CtBP-knockout cells were hypersensitive to apoptosis. Correspondingly, microarray analysis of CtBP-knockout vs. CtBP-rescued mouse embryo fibroblasts revealed that many epithelial-specific and proapoptotic genes were indeed regulated by CtBP. Neither the apoptosis nor the repression activities of CtBP required histidine-315, suggesting that the proposed dehydrogenase activity is not essential for CtBP function. The results presented herein establish two functional roles of CtBP: to corepress epithelial genes, thus permitting epithelial-to-mesenchymal transitions, and to modulate the cellular threshold for apoptotic responses.
Hildebrand, J.D., and P. Soriano (2002) Overlapping and unique roles for C-terminal binding protein 1 (CtBP1) and CtBP2 during mouse development. Mol. Cell. Biol. 22:5296-5307 The C-terminal binding protein (CtBP) family of proteins has been linked to multiple biological processes through their association with numerous transcription factors. We generated mice harboring mutations in both Ctbp1 and Ctbp2 to address the in vivo function of CtBPs during vertebrate development. Ctbp1 mutant mice are small but viable and fertile, whereas Ctbp2-null mice show defects in axial patterning and die by E10.5 due to aberrant extraembryonic development. Mice harboring various combinations of Ctbp1 and Ctbp2 mutant alleles exhibit dosage-sensitive defects in a wide range of developmental processes. The strong genetic interaction, as well as transcription assays with CtBP-deficient cells, indicates that CtBPs have overlapping roles in regulating gene expression. We suggest that the observed phenotypes reflect the large number of transcription factors whose activities are compromised in the absence of CtBP.
Howell, B.W., T.M. Herrick, J.D. Hildebrand, Y. Zhang, and J.A. Cooper (2000) Dab1 tyrosine phosphorylation site relay positional signals during mouse brain development. Curr. Biol. 10:877-885
Background: The extracellular protein Reln controls neuronal migrations in parts of the cortex, hippocampus and cerebellum. In vivo, absence of Reln correlates with up-regulation of the docking protein Dab1 and decreased Dab1 tyrosine phosphorylation. Loss of the Reln receptor proteins, apolipoprotein receptor 2 and very low density lipoprotein receptor, results in a Reln-like phenotype accompanied by increased Dab1 protein expression. Complete loss of Dab1, however, recapitulates the Reln phenotype.
Schaller, M.D., J.D. Hildebrand, and J.T. Parsons (1999) Complex formation with focal adhesion kinase: A mechanism to regulate activity and subcellular localization of Src kinases. Mol. Biol. Cell 10:3489-3505 Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60(c-src) or p59(fyn) results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60(c-src) or p59(fyn) to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60(c-src) is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60(c-src) from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60(c-src) to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.
Hildebrand, J.D., and P. Soriano (1999) Shroom, a PDZ domain containing actin binding protein, is required for neural tube morphogenesis in mice. Cell 99:485-497 Formation of the vertebrate neural tube is a dynamic morphogenetic process requiring coordinated differentiation, proliferation, cell adhesion, and cell migration. Using gene trap mutagenesis, we have identified a recessive lethal mutation in mice that causes exencephaly, acrania, facial clefting, and spina bifida, all of which can be attributed to failed neural tube closure. This mutation has been designated shroom (shrm) because the neural folds do not move towards the dorsal midline and subsequently "mushroom" outward. The mutated gene is predicted to encode a novel PDZ-domain protein. Several lines of evidence suggest that Shrm may regulate aspects of cellular cytoarchitecture. First, endogenous Shrm is localized to adherens junctions and the actin cytoskeleton. Second, ectopically expressed Shrm is capable of influencing the subcellular distribution of F-actin. Third, Shrm directly binds F-actin. Finally, cytoskeletal organization within the neuroepithelium is perturbed in mutant embryos. In concert, these data suggest that Shrm is a critical determinant of cellular architecture that is required for proper neurulation.
Taylor, J.M., J.D. Hildebrand, C.P. Mack, M.E. Cox, and J.T. Parsons (1998) Characterization of Graf, the GTPase-activating protein for rho associated with focal adhesion kinase. Phosphorylation and possible regulation by mitogen-activated protein kinase. J. Biol. Chem. 273:8063-8070 Graf is a GTPase-activating protein for Rho that interacts with focal adhesion kinase and co-localizes with the actin cytoskeleton (Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (1996) Mol. Cell. Biol. 16, 3169-3178). We examined the expression and regulation of Graf as a prelude to understanding the role of Graf in mediating signal transduction in vivo. We demonstrated that Graf is a ubiquitously expressed 95-kDa protein with high levels observed in heart and brain and cells derived from these tissues. Stimulation of PC12 cells with epidermal growth factor or nerve growth factor induced a phosphatase-reversible mobility shift upon gel electrophoresis, indicative of phosphorylation. In vitro, purified mitogen-activated protein (MAP) kinase catalyzed the phosphorylation of Graf on serine 510, suggesting that Graf phosphorylation may be mediated through MAP kinase signaling. In addition, the mutation of serine 510 to alanine inhibited the epidermal growth factor-induced mobility shift of mutant Graf protein in vivo, consistent with serine 510 being the site of in vivo phosphorylation. Based on these data we suggest that phosphorylation of Graf by MAP kinase or related kinases may be a mechanism by which growth factor signaling modulates Rho-mediated cytoskeletal changes in PC12 and perhaps other cells.
Richarson, A., R.K. Malik, J.D. Hildebrand, and J.T. Parsons (1997) Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation. Mol. Cell. Biol. 17:6906-6914 pp125FAK is a tyrosine kinase that appears to regulate the assembly of focal adhesions and thereby promotes cell spreading on the extracellular matrix. In some cells, the C terminus of pp125FAK is expressed as a separate protein, pp41/43FRNK. We have previously shown that overexpression of pp41/43FRNK inhibits tyrosine phosphorylation of pp125FAK and paxillin and, in addition, delays cell spreading and focal adhesion assembly. Thus, pp41/43FRNK functions as a negative inhibitor of adhesion signaling and provides a tool to dissect the mechanism by which pp125FAK promotes cell spreading. We report here that the inhibitory effects of pp41/43FRNK expression can be rescued by the co-overexpression of wild-type pp125FAK and partially rescued by catalytically inactive variants of pp125FAK. However, coexpression of an autophosphorylation site mutant of pp125FAK, which fails to bind the SH2 domain of pp60c-Src, or a mutant that fails to bind paxillin did not promote cell spreading. In contrast, expression of pp41/43FRNK and pp60c-Src reconstituted cell spreading and tyrosine phosphorylation of paxillin but did so without inducing tyrosine phosphorylation of pp125FAK. These data provide additional support for a model whereby pp125FAK acts as a "switchable adaptor" that recruits pp60c-Src to phosphorylate paxillin, promoting cell spreading. In addition, these data point to tyrosine phosphorylation of paxillin as being a critical step in focal adhesion assembly.
Friedrich, G.A., J.D. Hildebrand, and P. Soriano (1997) The secretory protein Sec8 is required for gastrulation in the mouse. Dev. Biol. 192:364-374 The sec8 gene, isolated in a gene trap screen in embryonic stem cells, is required for paraxial mesoderm formation in the mouse. Homozygous sec8 mutant embryos initiate gastrulation but are unable to progress beyond the primitive streak stage and die shortly afterward. The genomic locus and cDNA of the sec8 gene have been cloned. An open reading frame in the cDNA encodes a 971-amino-acid leucine-rich protein similar to rat rSec8. A description of the mutant phenotype and the cloning of the gene is presented here and the results are considered in light of the possibility that the Sec8 protein is involved in secretion.
Hildebrand, J.D., J.M. Taylor, and J.T. Parsons (1996) An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase. Mol. Cell. Biol. 16:3169-3178 The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.
Harte, M.T., J.D. Hildebrand, M.R. Burnham, A.H. Bouton, and J.T. Parsons (1996) p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase. J. Biol. Chem. 271:13649-13655 p130(Cas) (crk associated substrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130(Cas) suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. In this report we show that p130(Cas) is localized to focal adhesions. We demonstrate that p130(Cas) associates both in vitro and in vivo with pp125(FAK) (focal adhesion kinase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130(Cas) also associates with pp41/43(FRNK) (pp125(FAK)-related, non-kinase), an autonomously expressed form of pp125(FAK) composed of only the C-terminal noncatalytic domain. We show that the association of p130(Cas) with pp125(Fak) and pp41/43(FRNK) is direct, and is mediated by the binding of the SH3 domain of p130(Cas) to a proline-rich sequence present in both the C terminus of pp125(FAK) and in pp41/43(FRNK). In agreement with recent studies we show that p130(Cas) is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130(Cas) with pp125(FAK), a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.
Hildebrand, J.D., M.D. Schaller, and J.T. Parsons (1995) Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase. Mol. Biol. Cell 6:637-647 Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions. pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals. Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk. In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin. The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK. The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.
Chen, H.C., P.A. Appeddu, J.T. Parsons, J.D. Hildebrand, M.D. Schaller, and J.L. Guan (1995) Interaction of focal adhesion kinase with cytoskeletal protein talin. J. Biol. Chem. 270:16995-16999 The interaction of cells with extracellular matrix proteins plays a critical role in a variety of biological processes. Recent studies suggest that cell-matrix interactions mediated by integrins can transduce biochemical signals to the cell interior that regulate cell proliferation and differentiation. These studies have placed the focal adhesion kinase (FAK), an intracellular protein tyrosine kinase, in a central position in integrin-initiated signal transduction pathways (Zachary, I., and Rozengurt, E. (1992) Cell 71, 891-894; Schaller, M., and Parsons, J. T. (1993) Trends Cell Biol. 3, 258-262). Here, we report data suggesting a possible association of FAK with the cytoskeletal protein talin in NIH 3T3 cells. We have identified a 48-amino acid sequence in the carboxyl-terminal domain of FAK necessary for talin binding in vitro. Furthermore, we have correlated the ability of integrin to induce FAK phosphorylation with its ability to bind talin using a mutant integrin lacking the carboxyl-terminal 13 amino acids. These studies suggest talin may be a mediator for FAK activation in signaling initiated by integrins and may provide an explanation for the dependence on the integrity of actin-cytoskeleton of multiple intracellular signaling pathways converging to FAK activation and autophosphorylation.
Schaller, M.D., C.A. Otey, J.D. Hildebrand, and J.T. Parsons (1995) Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains. J. Cell Biol. 130:1181-1187 The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.
Parsons, J.T., M.D. Schaller, J.D. Hildeband, T.-H. Leu, A. Richardson, and C.A. Otey (1994) Focal adhesion kinase: structure and signaling. J. Cell. Sci. 18:109-113 Schaller, M.D., J.D. Hildebrand, J.D. Shannon, J.W. Fox, R.R. Vines, and J.T. Parsons (1994) Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol. Cell. Biol. 14:1680-1688 The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.
Hildebrand, J.D., M.D. Schaller, and J.T. Parsons (1993) Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions. J. Cell Biol. 123:993-1005 The integrin family of heterodimeric cell surface receptors play critical roles in multiple biological processes by mediating cellular adhesion to the extracellular matrix (ECM). Adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation and elevation of [Ca2+]i. The Focal Adhesion Kinase (FAK or pp125FAK), a protein tyrosine kinase that colocalizes with integrins in cellular focal adhesions, is a prime candidate for a mediator of integrin signaling events. Here we report an analysis of the domain structure of FAK in which we have identified a contiguous stretch of 159 amino acids within the COOH terminus essential for correct subcellular localization. When placed in the context of an unrelated cytosolic protein, this Focal Adhesion Targeting (FAT) sequence functions to efficiently mediate the focal adhesion localization of this fusion protein. Furthermore, this analysis suggests that pp125FAK cannot be activated oncogenically by mutation. This result could be explained if pp125FK either exhibits a narrow substrate specificity or is diametrically opposed by cellular phosphatases or other cellular processes.
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