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1.
Article in English | MEDLINE | ID: mdl-22105806

ABSTRACT

Attachment to extracellular matrix (ECM) is required for the survival and proliferation of normal epithelial cells. Epithelial tumor cells, however, often acquire "anchorage independence," a property that may contribute to their ability to invade and grow in foreign environments. Although apoptosis is the most rapid and effective mechanism that causes the death of matrix-detached cells, it has become apparent that detachment from matrix alters other aspects of cell physiology prior to commitment to cell death and that some of these alterations can lead to cell death under conditions where apoptosis is suppressed. This report provides an overview of death processes that contribute to the death of matrix-detached normal cells and describes mechanisms that confer anchorage independence, with a focus on ECM regulation of cell metabolism. Loss of matrix attachment leads to metabolic stress characterized by reduced nutrient uptake, decreased ATP production, and increased levels of reactive oxygen species (ROS). The decrease in ATP levels is prevented by either constitutive activation of the PI3K/Akt pathway or exogenous antioxidants. Additionally, decreased Erk signaling in matrix-detached cells causes a disproportionate decrease in flux through pyruvate dehydrogenase (PDH), leading to decreased entry of glucose carbons into the citric acid cycle. Interestingly, forced overexpression of a PDH inhibitor suppresses de novo lipogenesis and proliferation, highlighting the importance of mitochondrial metabolism in supplying intermediates for biosynthetic processes required for proliferation. Thus, ECM attachment is a key regulator of cellular metabolism, and alterations in metabolism owing to changes or loss of ECM engagement during tumorigenesis may serve important tumor-suppressive functions.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Extracellular Matrix/metabolism , Animals , Autophagy , Cell Adhesion , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Reactive Oxygen Species/metabolism
2.
Curr Biol ; 11(22): 1799-804, 2001 Nov 13.
Article in English | MEDLINE | ID: mdl-11719224

ABSTRACT

Syk protein tyrosine kinase is essential for immune system development and function [1]and for the maintenance of vascular integrity [2,3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs)[1]. Syk can also be activated by integrin adhesion receptors [4,5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreased Syk binding and disrupted its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase.


Subject(s)
Antigens, CD/metabolism , Cell Cycle Proteins , Enzyme Precursors/metabolism , Integrins/metabolism , Platelet Membrane Glycoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Amino Acid Sequence , Animals , Antigens, CD/genetics , CHO Cells , Cricetinae , Cytoplasm/metabolism , Enzyme Activation , Focal Adhesion Protein-Tyrosine Kinases , Integrin beta3 , Integrins/genetics , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Phosphorylation , Platelet Membrane Glycoproteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-vav , Syk Kinase , ZAP-70 Protein-Tyrosine Kinase , src Homology Domains
3.
Nat Cell Biol ; 3(9): 785-92, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11533657

ABSTRACT

Both ErbB1 and ErbB2 are overexpressed or amplified in breast tumours. To examine the effects of activating ErbB receptors in a context that mimics polarized epithelial cells in vivo, we activated ErbB1 and ErbB2 homodimers in preformed, growth-arrested mammary acini cultured in three-dimensional basement membrane gels. Activation of ErbB2, but not that of ErbB1, led to a reinitiation of cell proliferation and altered the properties of mammary acinar structures. These altered structures share several properties with early-stage tumours, including a loss of proliferative suppression, an absence of lumen, retention of the basement membrane and a lack of invasive properties. ErbB2 activation also disrupted tight junctions and the cell polarity of polarized epithelia, whereas ErbB1 activation did not have any effect. Our results indicate that ErbB receptors differ in their ability to induce early stages of mammary carcinogenesis in vitro and this three-dimensional model system can reveal biological activities of oncogenes that cannot be examined in vitro in standard transformation assays.


Subject(s)
Epithelial Cells/cytology , ErbB Receptors/metabolism , Receptor, ErbB-2/metabolism , Animals , Breast , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Division/drug effects , Cell Division/physiology , Cell Line , Cell Polarity , Dimerization , Dogs , Epidermal Growth Factor/pharmacology , Epithelial Cells/physiology , Epithelial Cells/ultrastructure , Female , Humans , Kidney , Recombinant Fusion Proteins/metabolism
4.
Mol Cell Biol ; 20(17): 6364-73, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10938113

ABSTRACT

Vav proteins are guanine nucleotide exchange factors for Rho family GTPases which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Vav proteins contain several protein binding domains which can link cell surface receptors to downstream signaling proteins. Vav1 is expressed exclusively in hematopoietic cells and tyrosine phosphorylated in response to activation of multiple cell surface receptors. However, it is not known whether the recently identified isoforms Vav2 and Vav3, which are broadly expressed, can couple with similar classes of receptors, nor is it known whether all Vav isoforms possess identical functional activities. We expressed Vav1, Vav2, and Vav3 at equivalent levels to directly compare the responses of the Vav proteins to receptor activation. Although each Vav isoform was tyrosine phosphorylated upon activation of representative receptor tyrosine kinases, integrin, and lymphocyte antigen receptors, we found unique aspects of Vav protein coupling in each receptor pathway. Each Vav protein coprecipitated with activated epidermal growth factor and platelet-derived growth factor (PDGF) receptors, and multiple phosphorylated tyrosine residues on the PDGF receptor were able to mediate Vav2 tyrosine phosphorylation. Integrin-induced tyrosine phosphorylation of Vav proteins was not detected in nonhematopoietic cells unless the protein tyrosine kinase Syk was also expressed, suggesting that integrin activation of Vav proteins may be restricted to cell types that express particular tyrosine kinases. In addition, we found that Vav1, but not Vav2 or Vav3, can efficiently cooperate with T-cell receptor signaling to enhance NFAT-dependent transcription, while Vav1 and Vav3, but not Vav2, can enhance NFkappaB-dependent transcription. Thus, although each Vav isoform can respond to similar cell surface receptors, there are isoform-specific differences in their activation of downstream signaling pathways.


Subject(s)
Cell Cycle Proteins , Oncogene Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction , Amino Acid Sequence , Animals , CHO Cells , COS Cells , Cell Line , Cricetinae , DNA, Complementary/metabolism , Epidermal Growth Factor/pharmacology , Guanine Nucleotide Exchange Factors , Humans , Integrins/metabolism , Jurkat Cells , Mice , Molecular Sequence Data , Oncogene Proteins/chemistry , Phosphorylation , Platelet-Derived Growth Factor/pharmacology , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-vav , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, T-Cell/metabolism , Sequence Homology, Amino Acid , Tyrosine/metabolism
5.
Curr Biol ; 10(9): 551-4, 2000 May 04.
Article in English | MEDLINE | ID: mdl-10801448

ABSTRACT

We have previously shown that inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and that Raf is the critical target of PI 3-kinase regulation [1]. To investigate how PI 3-kinase regulates Raf, we examined sites on Raf1 required for regulation by PI 3-kinase and explored the mechanisms involved in this regulation. Serine 338 (Ser338), which was critical for fibronectin stimulation of Raf1, was phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) was independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, was also dependent on PI 3-kinase activity and expression of a kinase-inactive Pak-1 mutant blocked phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins.


Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Serine/metabolism , Animals , COS Cells , Integrins/metabolism , Mutagenesis , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-raf/genetics , Serine/genetics , p21-Activated Kinases
6.
J Biol Chem ; 274(53): 37893-900, 1999 Dec 31.
Article in English | MEDLINE | ID: mdl-10608855

ABSTRACT

The phosphatidylinositol 3-kinase (PI 3'-K) family of lipid kinases play a critical role in cell proliferation, survival, vesicle trafficking, motility, cytoskeletal rearrangements, and oncogenesis. To identify downstream effectors of PI 3'-K, we developed a novel screen to isolate proteins that bind to the major products of PI 3'-K: phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P(2)) and PtdIns-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)). This screen uses synthetic biotinylated analogs of these lipids in conjunction with libraries of radiolabeled proteins that are produced by coupled in vitro transcription/translation reactions. The feasibility of the screen was initially demonstrated using avidin-coated beads prebound to biotinylated PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3) to specifically isolate the pleckstrin homology domain of the serine/threonine kinase Akt. We then demonstrated the utility of this technique in isolating novel 3'-phosphorylated phosphatidylinositol (3'-PPI)-binding proteins through the preliminary screening of in vitro transcribed/translated cDNAs from a small pool expression library derived from mouse spleen. Three proteins were isolated that bound specifically to 3'PPIs. Two of these proteins have been previously characterized as PIP3BP/p42(IP4) and the PtdIns-3,4,5-P(3)-dependent serine/threonine kinase phosphoinositide-dependent kinase 1. The third protein is a novel protein that contains only a Src homology 2 domain and a pleckstrin homology domain; this protein has a higher specificity for both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) than for PtdIns-4, 5-bisphosphate. Transcripts of this novel gene are present in every tissue analyzed but are most prominently expressed in spleen. We have renamed this new protein PHISH for 3'-phosphoinositide-interacting Src homology-containing protein. This report demonstrates the utility of this technique for isolating and characterizing 3'-PPI-binding proteins and has broad applicability for the isolation of binding domains for other lipid products.


Subject(s)
Adaptor Proteins, Signal Transducing , Lipoproteins/metabolism , Phosphatidylinositol Phosphates/metabolism , Protein Isoforms/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Lipoproteins/genetics , Mice , Molecular Sequence Data , Phosphorylation , Protein Biosynthesis , Protein Isoforms/genetics , src Homology Domains
7.
Mol Cell Biol ; 19(10): 6845-57, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10490623

ABSTRACT

The four members of the ErbB family of receptor tyrosine kinases are involved in a complex array of combinatorial interactions involving homo- and heterodimers. Since most cell types express more than one member of the ErbB family, it is difficult to distinguish the biological activities of different homo- and heterodimers. Here we describe a method for inducing homo- or heterodimerization of ErbB receptors by using synthetic ligands without interference from the endogenous receptors. ErbB receptor chimeras containing synthetic ligand binding domains (FK506-binding protein [FKBP] or FKBP-rapamycin-binding domain [FRB]) were homodimerized with the bivalent FKBP ligand AP1510 and heterodimerized with the bifunctional FKBP-FRB ligand rapamycin. AP1510 treatment induced tyrosine phosphorylation of ErbB1 and ErbB2 homodimers and recruitment of Src homology 2 domain-containing proteins (Shc and Grb2). In addition, ErbB1 and ErbB2 homodimers activated downstream signaling pathways leading to Erk2 and Akt phosphorylation. However, only ErbB1 homodimers were internalized upon AP1510 stimulation, and only ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to form foci; however, cells expressing ErbB2 homodimers displayed a five- to sevenfold higher focus-forming ability. Using rapamycin-inducible heterodimerization we show that c-Cbl is unable to associate with ErbB1 in a ErbB1-ErbB2 heterodimer most likely because ErbB2 is unable to phosphorylate the c-Cbl binding site on ErbB1. Thus, we demonstrate that ErbB1 and ErbB2 homodimers differ in their abilities to transform fibroblasts and provide evidence for differential signaling by ErbB homodimers and heterodimers. These observations also validate the use of synthetic ligands to study the signaling and biological specificity of selected ErbB dimers in any cell type.


Subject(s)
ErbB Receptors/metabolism , Receptor, ErbB-2/metabolism , Ubiquitin-Protein Ligases , Animals , Cell Cycle , Cell Transformation, Neoplastic , Dimerization , Dose-Response Relationship, Drug , Endocytosis , Epidermal Growth Factor/metabolism , Ligands , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-cbl , Rats , Recombinant Fusion Proteins , Signal Transduction , Tacrolimus/analogs & derivatives , Tacrolimus/metabolism
8.
J Biol Chem ; 274(15): 10571-81, 1999 Apr 09.
Article in English | MEDLINE | ID: mdl-10187852

ABSTRACT

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.


Subject(s)
Adaptor Proteins, Signal Transducing , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases , Protein Kinase C/metabolism , Signal Transduction , src Homology Domains , Animals , COS Cells , Cell Adhesion , Cells, Cultured , Enzyme Activation , Fibronectins/pharmacology , GRB2 Adaptor Protein , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase 1 , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proteins/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Tetradecanoylphorbol Acetate/pharmacology , ras Proteins/metabolism
9.
J Biol Chem ; 274(11): 7341-50, 1999 Mar 12.
Article in English | MEDLINE | ID: mdl-10066798

ABSTRACT

The cyclin D1 gene is overexpressed in breast tumors and encodes a regulatory subunit of cyclin-dependent kinases that phosphorylate the retinoblastoma protein. pp60(c-src) activity is frequently increased in breast tumors; however, the mechanisms governing pp60(c-src) regulation of the cell cycle in breast epithelium are poorly understood. In these studies, pp60(v-src) induced cyclin D1 protein levels and promoter activity (48-fold) in MCF7 cells. Cyclin D1-associated kinase activity and protein levels were increased in mammary tumors from murine mammary tumor virus-pp60(c-src527F) transgenic mice. Optimal induction of cyclin D1 by pp60(v-src) involved the extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase members of the mitogen-activated protein kinase family. Cyclin D1 promoter activation by pp60(v-src) involved a cAMP response element-binding protein (CREB)/activating transcription factor 2 (ATF-2) binding site. Dominant negative mutants of CREB and ATF-2 but not c-Jun inhibited pp60(v-src) induction of cyclin D1. pp60(v-src) induction of CREB was blocked by the p38 inhibitor SB203580 or by mutation of CREB at Ser133. pp60(v-src) induction of ATF-2 was abolished by the c-Jun N-terminal kinase inhibitor JNK-interacting protein-1 or by mutation of ATF-2 at Thr69 and Thr71. CREB and ATF-2, which bind to a common pp60(v-src) response element, are transcriptionally activated by distinct mitogen-activated protein kinases. Induction of cyclin D1 activity by pp60(v-src) may contribute to breast tumorigenesis through phosphorylation and inactivation of the retinoblastoma protein.


Subject(s)
Breast Neoplasms/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cyclin D1/biosynthesis , Mitogen-Activated Protein Kinases , Oncogene Protein pp60(v-src)/metabolism , Signal Transduction , Activating Transcription Factor 2 , Animals , Breast Neoplasms/pathology , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclin D1/genetics , Gene Expression Regulation, Neoplastic , JNK Mitogen-Activated Protein Kinases , Mice , Mice, Transgenic , Plasmids , Promoter Regions, Genetic , Protein Binding , Transcription Factors/metabolism , Tumor Cells, Cultured , p38 Mitogen-Activated Protein Kinases
10.
Curr Biol ; 8(24): 1289-99, 1998 Dec 03.
Article in English | MEDLINE | ID: mdl-9843681

ABSTRACT

BACKGROUND: . Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK) occurs late in integrin signaling, downstream from actin polymerization. Our previous studies indicated that integrin-mediated activation of the non-receptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggested the existence of a distinct signaling pathway regulated by Syk. RESULTS: . Multiple proteins were found to be activated by Syk, downstream of engagement of the platelet/megakaryocyte-specific integrin alphaIIbbeta3. The guanine nucleotide exchange factor Vav1 was inducibly phosphorylated in a Syk-dependent manner in cells following their attachment to fibrinogen. Together, Syk and Vav1 triggered lamellipodia formation in fibrinogen-adherent cells and both Syk and Vav1 colocalized with alphaIIbbeta3 in lamellipodia but not in focal adhesions. Additionally, Syk and Vav1 cooperatively induced activation of Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase 2 (ERK2) and the kinase Akt, and phosphorylation of the oncoprotein Cbl in fibrinogen-adherent cells. Activation of all of these proteins by Syk and Vav1 was not dependent on actin polymerization. CONCLUSIONS: . Syk and Vav1 regulate a unique integrin signaling pathway that differs from the FAK pathway in its proximity to the integrin itself, its localization to lamellipodia, and its activation, which is independent of actin polymerization. This pathway may regulate multiple downstream events in hematopoietic cells, including Rac-induced lamellipodia formation, tyrosine phosphorylation of Cbl, and activation of JNK, ERK2 and the phosphatidylinositol 3'-kinase-regulated kinase Akt.


Subject(s)
Cell Cycle Proteins , Enzyme Precursors/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Ubiquitin-Protein Ligases , Actins/metabolism , Animals , CHO Cells , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cricetinae , Enzyme Precursors/genetics , GTPase-Activating Proteins , Intracellular Signaling Peptides and Proteins , Mitogen-Activated Protein Kinase 1 , Models, Biological , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Protein-Tyrosine Kinases/genetics , Proteins/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-cbl , Proto-Oncogene Proteins c-vav , Signal Transduction , Syk Kinase , Transfection , Tyrosine/metabolism
12.
J Cell Biol ; 142(2): 573-86, 1998 Jul 27.
Article in English | MEDLINE | ID: mdl-9679153

ABSTRACT

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal-regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.


Subject(s)
GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , GTPase-Activating Proteins , Integrins/metabolism , Actins/metabolism , Animals , Cell Adhesion , Cell Cycle Proteins/metabolism , Cell Line , Cytoskeleton/metabolism , Fibronectins/metabolism , GTP Phosphohydrolases/genetics , GTP-Binding Proteins/genetics , Mutation , Rats , Signal Transduction , cdc42 GTP-Binding Protein , rhoA GTP-Binding Protein
13.
EMBO J ; 16(21): 6414-25, 1997 Nov 03.
Article in English | MEDLINE | ID: mdl-9351824

ABSTRACT

pp72syk is essential for development and function of several hematopoietic cells, and it becomes activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to study Syk activation by the platelet integrin, alpha IIb beta 3. As in platelets, Syk underwent tyrosine phosphorylation and activation during CHO cell adhesion to alpha IIb beta 3 ligands, including fibrinogen. This involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited two novel features. Firstly, unlike alpha IIb beta 3-mediated activation of pp125FAK, Syk activation could be triggered by the binding of soluble fibrinogen and abolished by truncation of the alpha IIb or beta 3 cytoplasmic tail, and it was resistant to inhibition by cytochalasin D. Secondly, it did not require phosphorylated ITAMs since it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains. These studies demonstrate that Syk is a proximal component in alpha IIb beta 3 signaling and is regulated as a consequence of intimate functional relationships with the alpha IIb beta 3 cytoplasmic tails and with Src or a closely related kinase. Furthermore, there are fundamental differences in the activation of Syk by alpha IIb beta 3 and immune response receptors, suggesting a unique role for integrins in Syk function.


Subject(s)
Blood Platelets/metabolism , Enzyme Precursors/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , Amino Acid Sequence , Animals , CHO Cells , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cricetinae , Cricetulus , Enzyme Activation , Fibrinogen/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Point Mutation , Protein Binding , Protein Processing, Post-Translational , Receptors, Immunologic/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Syk Kinase
14.
Mol Cell Biol ; 17(8): 4406-18, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9234699

ABSTRACT

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Integrins/physiology , Mitogen-Activated Protein Kinase Kinases , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction/physiology , Androstadienes/pharmacology , Animals , COS Cells , Cell Adhesion , Cell Adhesion Molecules/metabolism , Chromones/pharmacology , Cytoskeletal Proteins/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Focal Adhesion Protein-Tyrosine Kinases , Guanosine Triphosphate/metabolism , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase 1 , Morpholines/pharmacology , Paxillin , Phosphatidylinositol 3-Kinases , Phosphatidylinositol Phosphates/metabolism , Phosphoproteins/metabolism , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Polylysine/metabolism , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-raf , Wortmannin , ras Proteins/metabolism
15.
J Immunol ; 158(4): 1650-9, 1997 Feb 15.
Article in English | MEDLINE | ID: mdl-9029101

ABSTRACT

Engagement of immunoreceptors in hemopoietic cells leads to activation of Src family tyrosine kinases as well as Syk or ZAP-70. Current models propose that Src family kinases are critical in immune response signal transduction through their role in phosphorylation of tyrosine residues within immunoreceptor tyrosine activation motifs (ITAMs; which recruit the SH2 domains of Syk or ZAP-70) and by direct phosphorylation of Syk and ZAP-70. Several lines of evidence suggest that Syk may not show the same dependence on activation by Src family kinases as ZAP-70. In this report, we used COS cells transiently transfected with components of the Fc epsilon RI complex (Lyn, Syk, and a chimeric CD8 receptor containing the cytoplasmic domain of the gamma subunit of Fc epsilon RI (CD8-gamma)) to examine the regulation of Syk activity. Syk was activated and phosphorylated in COS cells cotransfected with Lyn; however, in cells expressing CD8-gamma, activation of Syk and phosphorylation of CD8-gamma did not require coexpression of Lyn. Additional experiments indicate that gamma phosphorylation is dependent on Syk kinase activity and is independent of endogenous COS cell kinases. In parallel experiments, ZAP-70 was not activated by cotransfection with CD8-gamma, nor was CD8-gamma phosphorylated when coexpressed with ZAP-70 alone. Taken together, these studies indicate that Syk can be distinguished from ZAP-70 in its ability to be activated by coexpression with an ITAM-containing receptor without coexpression of a Src family kinase, and that Syk is capable of phosphorylating ITAM tyrosines under certain experimental conditions.


Subject(s)
Enzyme Precursors/immunology , Protein-Tyrosine Kinases/immunology , Receptors, IgE/immunology , Signal Transduction/immunology , src-Family Kinases/immunology , src-Family Kinases/physiology , Animals , COS Cells , Enzyme Activation/genetics , Enzyme Activation/immunology , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Phosphorylation , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Receptors, IgE/genetics , Receptors, IgE/metabolism , Signal Transduction/genetics , Syk Kinase , Transfection/immunology , ZAP-70 Protein-Tyrosine Kinase
16.
Annu Rev Immunol ; 15: 371-404, 1997.
Article in English | MEDLINE | ID: mdl-9143693

ABSTRACT

Intracellular signal transduction following the extracellular ligation of a wide variety of different types of surface molecules on leukocytes involves the activation of protein tyrosine kinases. The dependence of successful intracellular signaling on the functions of the nontransmembrane class of protein tyrosine kinases coupled with the cell type-specific expression patterns for several of these enzymes makes them appealing targets for therapeutic intervention. Development of drugs that can interfere with the catalytic functions of the nontransmembrane protein tyrosine kinases or that can disrupt critical interactions with regulatory molecules and/or substrates should find clinical applications in the treatment of allergic diseases, autoimmunity, transplantation rejection, and cancer.


Subject(s)
Drug Design , Leukocytes/enzymology , Protein-Tyrosine Kinases/metabolism , Amino Acid Sequence , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Models, Biological , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Signal Transduction
17.
Annu Rev Cell Dev Biol ; 13: 513-609, 1997.
Article in English | MEDLINE | ID: mdl-9442882

ABSTRACT

Src family protein tyrosine kinases are activated following engagement of many different classes of cellular receptors and participate in signaling pathways that control a diverse spectrum of receptor-induced biological activities. While several of these kinases have evolved to play distinct roles in specific receptor pathways, there is considerable redundancy in the functions of these kinases, both with respect to the receptor pathways that activate these kinases and the downstream effectors that mediate their biological activities. This chapter reviews the evidence implicating Src family kinases in specific receptor pathways and describes the mechanisms leading to their activation, the targets that interact with these kinases, and the biological events that they regulate.


Subject(s)
Cell Physiological Phenomena , src-Family Kinases/physiology , Animals , Humans , src-Family Kinases/genetics
18.
J Biol Chem ; 271(13): 7544-50, 1996 Mar 29.
Article in English | MEDLINE | ID: mdl-8631786

ABSTRACT

The vav proto-oncogene product, p95vav or Vav, is primarily expressed in hematopoietic cells and has been shown to be a substrate for tyrosine kinases. Although its function is unknown, Vav shares a region of homology with DBL, an exchange factor for the Rho family of GTP-binding proteins. The presence of this domain and the observation that cells transformed with Vav display prominent stress fibers and focal adhesions similar to those that are observed in RhoA transformed cells suggests that Vav may play a role in regulating the actin cytoskeleton. We have, therefore, examined Vav phosphorylation in platelets, which undergo dramatic cytoskeletal reorganization in response to agonists. Two potent platelet agonists, thrombin (via its G protein-coupled receptor) and collagen (via its interaction with the alpha2beta1 integrin), caused Vav to become phosphorylated on tyrosine. Weaker platelet agonists, including ADP, epinephrine and the thromboxane A2 analog, U46619, did not. The phosphorylation of Vav in response to thrombin was maximal within 15 s and was unaffected by aspirin, inhibitors of aggregation, or the presence of the ADP scavenger, apyrase. Vav phosphorylation was also observed when platelets became adherent to immobilized collagen (via integrin alpha2beta1), fibronectin (via integrin alpha5beta1), and fibrinogen (via integrin alphaIIbbeta3). These results show that Vav phosphorylation by tyrosine kinases 1) occurs during platelet activation by potent agonists, 2) also occurs when platelets adhere to biologically relevant matrix proteins, 3) requires neither platelet aggregation nor the release of secondary agonists such as ADP and TxA2, and 4) can be initiated by at least some members of two additional classes of receptors, G protein-coupled receptors and integrins, providing further evidence that both of these can couple to tyrosine kinases.


Subject(s)
Blood Platelets/metabolism , Cell Cycle Proteins , Integrins/physiology , Platelet Activation , Proto-Oncogene Proteins/blood , Receptors, Thrombin/physiology , Amino Acid Sequence , Antibodies/pharmacology , Aspirin/pharmacology , Blood Platelets/drug effects , Blood Platelets/physiology , Collagen/pharmacology , Extracellular Matrix Proteins , GTP-Binding Proteins/metabolism , Humans , Immunoblotting , Kinetics , Molecular Sequence Data , Peptide Fragments/pharmacology , Phosphorylation , Phosphotyrosine/analysis , Phosphotyrosine/metabolism , Platelet Adhesiveness , Proto-Oncogene Mas , Proto-Oncogene Proteins/isolation & purification , Proto-Oncogene Proteins c-vav , Thrombin/pharmacology , src Homology Domains
19.
Neuron ; 15(6): 1415-25, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8845164

ABSTRACT

We find that calcium influx through voltage-dependent calcium channels causes extensive neurite outgrowth in PC12 cells. The calcium signal transduction pathway promoting neurite outgrowth causes the rapid activation of protein tyrosine kinases, which include Src. Protein tyrosine phosphorylation results in the formation of an Shc/Grb2 complex, leading to Ras activation, MAP kinase activation, and the subsequent induction of the immediate early gene NGFI-A. Protein tyrosine phosphorylation, gene induction, and neurite outgrowth are inhibited by the expression of dominant negative forms of both Src and Ras, indicating a requirement for both proto-oncoproteins in calcium signaling. Our results suggest that a signaling cassette which includes Src and Ras is likely to underlie a broad range of calcium of actions in the nervous system.


Subject(s)
Adaptor Proteins, Signal Transducing , Calcium/metabolism , Genes, ras , Genes, src , Immediate-Early Proteins , Neurites/physiology , Signal Transduction , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Membrane/physiology , DNA-Binding Proteins/genetics , Early Growth Response Protein 1 , Electrophysiology , GRB2 Adaptor Protein , Gene Expression Regulation , PC12 Cells , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Proteins/metabolism , Rats , Transcription Factors/genetics , Transcriptional Activation , Tyrosine/metabolism , src Homology Domains/physiology
20.
Proc Natl Acad Sci U S A ; 92(24): 10909-13, 1995 Nov 21.
Article in English | MEDLINE | ID: mdl-7479908

ABSTRACT

The Src homology 3 (SH3) domain is a 50-aa modular unit present in many cellular proteins involved in intracellular signal transduction. It functions to direct protein-protein interactions through the recognition of proline-rich motifs on associated proteins. SH3 domains are important regulatory elements that have been demonstrated to specify distinct regulatory pathways important for cell growth, migration, differentiation, and responses to the external milieu. By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequence and to bind to SH3 domains in one of two pseudosymmetrical orientations, class I and class II. The class I sites have the consensus sequence ZP(L/P)PP psi P whereas the class II consensus is PP psi PPZ (where psi is a hydrophobic residue and Z is a SH3 domain-specific residue). We previously showed by M13 phage display that the Src, Fyn, Lyn, and phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPP psi P. These results failed to explain the specificity for cellular proteins displayed by SH3 domains in cells. In the current study, class I and class II core ligand sequences were displayed on the surface of bacteriophage M13 with five random residues placed either N- or C-terminal of core ligand residues. These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and PI3K SH3 domains. By this approach, additional ligand residue preferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides. The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and PI3K SH3 domains showed distinct binding specificities. These results indicate that residues that flank the core binding sequences shared by many SH3 domains are important determinants of SH3 binding affinity and selectivity.


Subject(s)
src Homology Domains , Amino Acid Sequence , Bacteriophage M13 , Gene Library , Ligands , Molecular Sequence Data , Peptides/chemistry , Protein Binding , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Structure-Activity Relationship
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