scholarly journals v-Crk Activates the Phosphoinositide 3-Kinase/AKT Pathway by Utilizing Focal Adhesion Kinase and H-Ras

2002 ◽  
Vol 22 (20) ◽  
pp. 7015-7023 ◽  
Author(s):  
Tsuyoshi Akagi ◽  
Kazutaka Murata ◽  
Tomoyuki Shishido ◽  
Hidesaburo Hanafusa
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Critical Role ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Regulatory Subunit ◽  
Akt Pathway ◽  
Dominant Negative Mutant ◽  
Phosphoinositide 3 Kinase

ABSTRACT v-Crk, an oncogene product of avian sarcoma virus CT10, efficiently transforms chicken embryo fibroblasts (CEF). We have recently reported that constitutive activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway plays a critical role in the v-Crk-induced transformation of CEF. In the present study we investigated the molecular mechanism by which v-Crk activates the PI3K/AKT pathway. First, we found that v-Crk promotes the association of the p85 regulatory subunit of PI3K with focal adhesion kinase (FAK) by inducing the phosphorylation of the Y397 residue in FAK. This FAK phosphorylation needs activation of the Src family tyrosine kinase(s) for which the v-Crk SH2 domain is responsible. v-Crk was unable to activate the PI3K/AKT pathway in FAK-null cells, indicating the functional importance of FAK. In addition, we found that H-Ras is also required for the activation of the PI3K/AKT pathway. The v-Crk-induced activation of AKT was greatly enhanced by the overexpression of H-Ras or its guanine nucleotide exchange factor mSOS, which binds to the v-Crk SH3 domain, whereas a dominant-negative mutant of H-Ras almost completely suppressed this activation. Furthermore, we showed that v-Crk stimulates the interaction of H-Ras with the Ras binding domain in the PI3K p110 catalytic subunit. Our data indicated that the v-Crk-induced activation of PI3K/AKT pathway was cooperatively achieved by two distinct interactions. One is the interaction of p85 with tyrosine-phosphorylated FAK promoted by the v-Crk SH2 domain, and another is the interaction of p110 with H-Ras dictated by the v-Crk SH3 domain.

scholarly journals Regulation of the RAP1/RAF-1/Extracellularly Regulated Kinase-1/2 Cascade and Prolactin Release by the Phosphoinositide 3-Kinase/AKT Pathway in Pituitary Cells

Endocrinology ◽  
2006 ◽  
Vol 147 (12) ◽  
pp. 6036-6045 ◽  
Author(s):  
David Romano ◽  
Morgane Pertuit ◽  
Ramahefarizo Rasolonjanahary ◽  
Jean-Vianney Barnier ◽  
Karine Magalon ◽  
...  
Keyword(s):  
Active Form ◽  
Dominant Negative ◽  
Akt Pathway ◽  
Dominant Negative Mutant ◽  
Pituitary Cells ◽  
Negative Effects ◽  
Igf I ◽  
Regulatory Effects ◽  
Phosphoinositide 3 Kinase ◽  
Rap1 Activation

In pituitary cells, prolactin (PRL) synthesis and release are controlled by multiple transduction pathways. In the GH4C1 somatolactotroph cell line, we previously reported that MAPK ERK-1/2 are a point of convergence between the pathways involved in the PRL gene regulation. In the present study, we focused on the involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the MAPK ERK-1/2 regulation and PRL secretion in pituitary cells. Either specific pharmacological PI3K and Akt inhibitors (LY294002, Akt I, and phosphoinositide analog-6) or Akt dominant-negative mutant (K179M) enhanced ERK-1/2 phosphorylation in unstimulated GH4C1 cells. Under the same conditions, PI3K and Akt inhibition also both increased Raf-1 kinase activity and the levels of GTP-bound (active form) monomeric G protein Rap1, which suggests that a down-regulation of the ERK-1/2 cascade is induced by the PI3K/Akt signaling pathway in unstimulated cells. On the contrary, ERK-1/2 phosphorylation, Raf-1 activity, and Rap1 activation were almost completely blocked in IGF-I-stimulated cells previously subjected to PI3K or Akt inhibition. Although the PRL promoter was not affected by either PI3K/Akt inhibition or activation, PRL release increased in response to the pharmacological PI3K/Akt inhibitors in unstimulated GH4C1 and rat pituitary primary cells. The IGF-I-stimulated PRL secretion was diminished, on the contrary, by the pharmacological PI3K/Akt inhibitors. Taken together, these findings indicate that the PI3K/Akt pathway exerts dual regulatory effects on both the Rap1/Raf-1/ERK-1/2 cascade and PRL release in pituitary cells, i.e. negative effects in unstimulated cells and positive ones in IGF-I-stimulated cells.

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-κB signaling in intestinal epithelial cells

2006 ◽  
Vol 290 (5) ◽  
pp. C1310-C1320 ◽  
Author(s):  
Huifang M. Zhang ◽  
Kaspar M. Keledjian ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Intestinal Epithelial Cells ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Induced Apoptosis

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-κB signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-κB activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF-α plus cycloheximide (TNF-α/CHX). Specific inhibition of NF-κB by the recombinant adenoviral vector containing the IκBα superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-κB activity and increased the sensitivity to TNF-α/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-κB activity and resulted in increased resistance to TNF-α/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-α/CHX-induced apoptosis, at least partially, through the activation of NF-κB signaling in IECs.

scholarly journals Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase.

1995 ◽  
Vol 129 (3) ◽  
pp. 831-842 ◽  
Author(s):  
C Guinebault ◽  
B Payrastre ◽  
C Racaud-Sultan ◽  
H Mazarguil ◽  
M Breton ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Subcellular Fraction ◽  
Critical Role ◽  
Physical Interaction ◽  
Activated Platelets ◽  
Phosphoinositide 3 Kinase

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.

scholarly journals Insulin-induced tyrosine dephosphorylation of paxillin and focal adhesion kinase requires active phosphotyrosine phosphatase 1D

1996 ◽  
Vol 318 (2) ◽  
pp. 609-614 ◽  
Author(s):  
D. Margriet OUWENS ◽  
Harald M. M. MIKKERS ◽  
Gerard C. M. van der ZON ◽  
Matthias STEIN-GERLACH ◽  
Axel ULLRICH ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Insulin Receptor ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Dominant Negative ◽  
Matrix Proteins ◽  
Dominant Negative Mutant ◽  
Phosphotyrosine Phosphatase ◽  
Insulin Receptor Substrates

Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor β-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(l-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D.

scholarly journals Cancer-associated mutations in the p85α N-terminal SH2 domain activate a spectrum of receptor tyrosine kinases

2021 ◽  
Vol 118 (37) ◽  
pp. e2101751118
Author(s):  
Xinran Li ◽  
Amy Y. T. Lau ◽  
Angel S. N. Ng ◽  
Abdullah Aldehaiman ◽  
Yuan Zhou ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Driver Mutations ◽  
Akt Pathway ◽  
Independent Manner ◽  
Inhibitory Interaction ◽  
Her Family ◽  
Protein Levels ◽  
Phosphoinositide 3 Kinase

The phosphoinositide 3-kinase regulatory subunit p85α is a key regulator of kinase signaling and is frequently mutated in cancers. In the present study, we showed that in addition to weakening the inhibitory interaction between p85α and p110α, a group of driver mutations in the p85α N-terminal SH2 domain activated EGFR, HER2, HER3, c-Met, and IGF-1R in a p110α-independent manner. Cancer cells expressing these mutations exhibited the activation of p110α and the AKT pathway. Interestingly, the activation of EGFR, HER2, and c-Met was attributed to the ability of driver mutations to inhibit HER3 ubiquitination and degradation. The resulting increase in HER3 protein levels promoted its heterodimerization with EGFR, HER2, and c-Met, as well as the allosteric activation of these dimerized partners; however, HER3 silencing abolished this transactivation. Accordingly, inhibitors of either AKT or the HER family reduced the oncogenicity of driver mutations. The combination of these inhibitors resulted in marked synergy. Taken together, our findings provide mechanistic insights and suggest therapeutic strategies targeting a class of recurrent p85α mutations.

scholarly journals Focal Adhesion Kinase Controls Cellular Levels of p27/Kip1 and p21/Cip1 through Skp2-Dependent and -Independent Mechanisms

2006 ◽  
Vol 26 (11) ◽  
pp. 4201-4213 ◽  
Author(s):  
Patrick Bryant ◽  
Qingxia Zheng ◽  
Kevin Pumiglia
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cell ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Kinase Inhibitors ◽  
Critical Role ◽  
Dominant Negative ◽  
Regulatory Control ◽  
Endothelial Cell Proliferation ◽  
F Box Protein

ABSTRACT Endothelial cell proliferation is a critical step in angiogenesis and requires a coordinated response to soluble growth factors and the extracellular matrix. As focal adhesion kinase (FAK) integrates signals from both adhesion events and growth factor stimulation, we investigated its role in endothelial cell proliferation. Expression of a dominant-negative FAK protein, FAK-related nonkinase (FRNK), impaired phosphorylation of FAK and blocked DNA synthesis in response to multiple angiogenic stimuli. These results coincided with elevated cyclin-dependent kinase inhibitors (CDKIs) p21/Cip and p27/Kip, as a consequence of impaired degradation. FRNK inhibited the expression of Skp2, an F-box protein that targets CDKIs, by inhibiting mitogen-induced mRNA. The FAK-regulated degradation of p27/Kip was Skp2 dependent, while levels of p21/Cip were regulated independent of Skp2. Skp2 is required for endothelial cell proliferation as a consequence of degrading p27. Finally, knockdown of both p21 and p27 in FRNK-expressing cells completely restored mitogen-induced endothelial cell proliferation. These data demonstrate a critical role for FAK in the regulation of CDKIs through two independent mechanisms: Skp2 dependent and Skp2 independent. They also provide important insights into the requirement of focal adhesion kinase for normal vascular development and reveal novel regulatory control points for angiogenesis.

scholarly journals Identification of p130Cas as a Mediator of Focal Adhesion Kinase–promoted Cell Migration

1998 ◽  
Vol 140 (1) ◽  
pp. 211-221 ◽  
Author(s):  
Leslie A. Cary ◽  
Dong Cho Han ◽  
Thomas R. Polte ◽  
Steven K. Hanks ◽  
Jun-Lin Guan
Keyword(s):  
Cell Migration ◽  
Binding Site ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Mek Inhibitor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Sh2 Domains ◽  
Promote Cell Migration

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130Cas and its subsequent binding to several SH2 domains, which depended on both the p130Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130Cas further increased cell migration on FN and coexpression of the p130Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.

scholarly journals Atypical Protein Kinase C Is Involved in the Evolutionarily Conserved Par Protein Complex and Plays a Critical Role in Establishing Epithelia-Specific Junctional Structures

2001 ◽  
Vol 152 (6) ◽  
pp. 1183-1196 ◽  
Author(s):  
Atsushi Suzuki ◽  
Tomoyuki Yamanaka ◽  
Tomonori Hirose ◽  
Naoyuki Manabe ◽  
Keiko Mizuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Protein Complex ◽  
Mdck Cells ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Surface Polarity ◽  
C Elegans ◽  
Evolutionarily Conserved

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

Prevention of TNF-α-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

2004 ◽  
Vol 286 (3) ◽  
pp. G479-G490 ◽  
Author(s):  
Sujoy Bhattacharya ◽  
Ramesh M. Ray ◽  
Leonard R. Johnson
Keyword(s):  
Epithelial Cells ◽  
Cytochrome C ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cytochrome C Release ◽  
Erk Phosphorylation ◽  
Tnf Α ◽  
Induced Apoptosis ◽  
Jnk Activation

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-α/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by α-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-α/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-α/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-α/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-α/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-α/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-α/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-α/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.

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