PubMed Health⌕ Search

Biomedical subjects

J K Westwick

Publications and source records attributed to J K Westwick.

At least 19 recordsLinked to original sources
PubMed1999

Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway.

The small GTP-binding protein Rac1, a member of the Ras superfamily, plays a fundamental role in cytoskeleton reorganization, cellular transformation, the induction of DNA synthesis, and superoxide production. Cyclin D1 abundance is rate-limiting in normal G(1) phase progression, and the abundance of cyclin D1 is induced by activating mutations of both Ras and Rac1. Nuclear factor-kappaB (NF-kappaB) proteins consist of cytoplasmic hetero- or homodimeric Rel-related proteins complexed to a member of the IkappaB family of inhibitor proteins. In the current studies, activating mutants of Rac1 (Rac(Leu-61), Rac(Val-12)) induced cyclin D1 expression and the cyclin D1 promoter in NIH 3T3 cells. Induction of cyclin D1 by Rac1 required both an NF-kappaB and an ATF-2 binding site. Inhibiting NF-kappaB by overexpression of an NF-kappaB trans-dominant inhibitor (nonphosphorylatable IkappaBalpha) reduced cyclin D1 promoter activation by the Rac1 mutants, placing NF-kappaB in a pathway of Rac1 activation of cyclin D1. Specific amino acid mutations in the amino-terminal effector domain of Rac(Leu-61) had comparable effects on NF-kappaB transcriptional activity and activation of the cyclin D1 promoter. The NF-kappaB factors Rel A (p65) and NF-kappaB(1) (p50) induced the cyclin D1 promoter, requiring both the NF-kappaB binding site and the ATF-2 site. Stable overexpression of Rac(Leu-61) increased binding of Rel A and NF-kappaB(1) to the cyclin D1 promoter NF-kappaB site. Activation of Rac1 in NIH 3T3 cells induces both NF-kappaB binding and activity and enhances expression of cyclin D1 through an NF-kappaB and ATF-2 site in the proximal promoter, suggesting a critical role for NF-kappaB in cell cycle regulation through cyclin D1 and Rac1.

3T3 Cells↗
PubMed1998

Transforming potential of Dbl family proteins correlates with transcription from the cyclin D1 promoter but not with activation of Jun NH2-terminal kinase, p38/Mpk2, serum response factor, or c-Jun.

The dbl family of oncogenes encodes a large, structurally related, family of growth-regulatory molecules that possess guanine nucleotide exchange factor activity for specific members of the Rho family of Ras-related GTPases. We have evaluated matched sets of weakly and strongly transforming versions of five Dbl family proteins (Lfc, Lsc, Ect2, Dbl, and Dbs) to determine their ability to stimulate signaling pathways that are activated by Rho family proteins. We found that the transforming potential of this panel did not correlate directly with their ability to activate Jun NH2-terminal kinase, p38/Mpk2, serum response factor, or c-Jun. In contrast, transient stimulation of transcription from the cyclin D1 promoter provided a strong correlation with transforming potential, and we found constitutive up-regulation of cyclin D1 protein in Dbl family protein-transformed cells. In addition, we observed that at least two Dbl family members (Lfc and Ect2) induced changes in the actin cytoskeleton and exhibited nuclear signaling profiles that are consistent with a broader range of in vivo substrate utilization than is predicted from their in vitro exchange specificities. In summary, although Dbl family proteins exhibit signaling profiles that are consistent with their in vivo activation of Rho proteins, stimulation of cyclin D1 transcription is the only activity that correlates with transforming potential, thus suggesting that deregulated cell cycle progression may be important for Dbl family protein transformation.

3T3 Cells↗
PubMed1998

Oncogenic ras induces gastrin gene expression in colon cancer.

BACKGROUND & AIMS: The expression of gastrin, as a tumor growth factor, is significantly increased in some colon cancers compared with the low levels found in normal mucosa. The aim of this study was to elucidate the transcriptional mechanisms of gastrin induction in colon cancer. METHODS: Gastrin messenger (mRNA) levels and K-ras genotype were determined in colon cancer cell lines and surgical specimens. Colon cancer cells were transfected with oncogenic ras expression vectors, and transcriptional activity was assayed with gastrin-luciferase reporter genes. RESULTS: Colon cancer cell lines and tissues with K-ras mutations all had significantly higher gastrin mRNA levels than those that were ras wild type. Treatment of several ras mutant cell lines with PD98059, an inhibitor of mitogen-activated protein kinase kinase, resulted in a decrease in endogenous gastrin mRNA levels. The effects of ras on gastrin expression appeared to be mediated through the gastrin promoter because transfection of oncogenic ras and activated raf expression vectors both induced gastrin-promoter, luciferase-reporter genes. The inductive effects of oncogenic ras could be blocked by the coexpression of dominant negative forms of raf and extracellular regulated kinase. CONCLUSIONS: Oncogenic ras induces gastrin gene expression through activation of the Raf-MEK-ERK signal transduction pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

Mas oncogene signaling and transformation require the small GTP-binding protein Rac.

The Mas oncogene encodes a novel G-protein-coupled receptor that was identified originally as a transforming protein when overexpressed in NIH 3T3 cells. The mechanism and signaling pathways that mediate Mas transformation have not been determined. We observed that the foci of transformed NIH 3T3 cells caused by Mas were similar to those caused by activated Rho and Rac proteins. Therefore, we determined if Mas signaling and transformation are mediated through activation of a specific Rho family protein. First, we observed that, like activated Rac1, Mas cooperated with activated Raf and caused synergistic transformation of NIH 3T3 cells. Second, both Mas- and Rac1-transformed NIH 3T3 cells retained actin stress fibers and showed enhanced membrane ruffling. Third, like Rac, Mas induced lamellipodium formation in porcine aortic endothelial cells. Fourth, Mas and Rac1 strongly activated the JNK and p38, but not ERK, mitogen-activated protein kinases. Fifth, Mas and Rac1 stimulated transcription from common DNA promoter elements: NF-kappaB, serum response factor (SRF), Jun/ATF-2, and the cyclin D1 promoter. Finally, Mas transformation and some of Mas signaling (SRF and cyclin D1 but not NF-kappaB activation) were blocked by dominant negative Rac1. Taken together, these observations suggest that Mas transformation is mediated in part by activation of Rac-dependent signaling pathways. Thus, Rho family proteins are common mediators of transformation by a diverse variety of oncogene proteins that include Ras, Dbl family, and G-protein-coupled oncogene proteins.

3T3 Cells↗
PubMed1997

Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K.

Transformation of mammary epithelial cells into invasive carcinoma results in alterations in their integrin-mediated responses to the extracellular matrix, including a loss of normal epithelial polarization and differentiation, and a switch to a more motile, invasive phenotype. Changes in the actin cytoskeleton associated with this switch suggest that the small GTPases Cdc42 and Rac, which regulate actin organization, might modulate motility and invasion. However, the role of Cdc42 and Rac1 in epithelial cells, especially with respect to integrin-mediated events, has not been well characterized. Here we show that activation of Cdc42 and Rac1 disrupts the normal polarization of mammary epithelial cells in a collagenous matrix, and promotes motility and invasion. This motility does not require the activation of PAK, JNK, p70 S6 kinase, or Rho, but instead requires phosphatidylinositol-3-OH kinase (PI(3)K). Further, direct PI(3)K activation is sufficient to disrupt epithelial polarization and induce cell motility and invasion. PI(3)K inhibition also disrupts actin structures, suggesting that activation of PI(3)K by Cdc42 and Rac1 alters actin organization, leading to increased motility and invasiveness.

Actins↗
PubMed1997

Oncogenic Ha-Ras-induced signaling activates NF-kappaB transcriptional activity, which is required for cellular transformation.

Ras proteins function in stimulating cell proliferation and differentiation through the activation of Raf-dependent and Raf-independent signal transduction pathways and the subsequent activation of specific transcription factors. The transcription factor NF-kappaB has been widely studied as a regulator of genes involved in immune and inflammatory responses. A variety of stimuli activate NF-kappaB through the induced phosphorylation and degradation of the inhibitor IkappaB followed by nuclear translocation of NF-kappaB. We show here that oncogenic forms of Ha-Ras activate NF-kappaB, not through induced nuclear translocation, but rather through the activation of the transcriptional function of the NF-kappaB RelA/p65 subunit. Importantly, RelA/p65 -/- cells are inefficient in the activation of kappaB-dependent gene expression in response to oncogenic Ras expression. Furthermore, IkappaBalpha expression blocks focus formation in NIH3T3 cells induced by oncogenic Ras. These results demonstrate that NF-kappaB is a critical downstream mediator of Ha-Ras signaling and oncogenic potential.

3T3 Cells↗
PubMed1997

The coupling of alpha6beta4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation.

The signaling pathways linking integrins to nuclear events are incompletely understood. We have examined intracellular signaling by the alpha6beta4 integrin, a laminin receptor expressed in basal keratinocytes and other cells. Ligation of alpha6beta4 in primary human keratinocytes caused tyrosine phosphorylation of Shc, recruitment of Grb2, activation of Ras and stimulation of the MAP kinases Erk and Jnk. In contrast, ligation of the laminin- and collagen-binding integrins alpha3beta1 and alpha2beta1 did not cause these events. While the stimulation of Erk by alpha6beta4 was suppressed by dominant-negative Shc, Ras and RhoA, the activation of Jnk was inhibited by dominant-negative Ras and Rac1 and by the phosphoinositide 3-kinase inhibitor Wortmannin. Adhesion mediated by alpha6beta4 induced transcription from the Fos serum response element and promoted cell cycle progression in response to mitogens. In contrast, alpha3beta1- and alpha2beta1-dependent adhesion did not induce these events. These findings suggest that the coupling of alpha6beta4 integrin to the control of cell cycle progression mediated by Shc regulates the proliferation of basal keratinocytes and possibly other cells which are in contact with the basement membrane in vivo.

3T3 Cells↗
PubMed1997

p120 GAP modulates Ras activation of Jun kinases and transformation.

Although recent evidence demonstrates that Ras causes transformation by activation of multiple downstream pathways, the specific role of non-Raf effector pathways is presently unknown. Although Ras causes activation of the Jun NH2-terminal kinases (JNKs) via a Raf-independent pathway, the contribution of JNK activation to Ras transformation and the effector that mediates JNK activation have not been established. We observed that a dominant negative mutant of SEK1/JNKK, an activator of JNKs, selectively inhibited oncogenic Ras activation of JNK and Ras transformation, but not Ras activation of the p42 mitogen-activated protein kinase. In contrast, overexpression of wild type SEK1 enhanced Ras activation of JNK and transforming activity. Thus, JNK activation promotes Ras transformation. Furthermore, a dominant negative mutant of p120 GAP (designated N-GAP), a candidate Ras effector, blocked Ras, but not Raf, transformation and blocked Ras, but not Rac, activation of JNK. Since N-GAP overexpression reduced the association of p190 Rac/Rho GAP with endogenous p120 GAP, N-GAP may form nonproductive complexes with components critical for p120 GAP function. In summary, p120 GAP may function as an effector for Ras activation of JNK and Ras transformation.

3T3 Cells↗
PubMed1997

Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways.

Rac1 and RhoA are members of the Rho family of Ras-related proteins and function as regulators of actin cytoskeletal organization, gene expression, and cell cycle progression. Constitutive activation of Rac1 and RhoA causes tumorigenic transformation of NIH 3T3 cells, and their functions may be required for full Ras transformation. The effectors by which Rac1 and RhoA mediate these diverse activities, as well as the interrelationship between these events, remain poorly understood. Rac1 is distinct from RhoA in its ability to bind and activate the p65 PAK serine/threonine kinase, to induce lamellipodia and membrane ruffling, and to activate the c-Jun NH2-terminal kinase (JNK). To assess the role of PAK in Rac1 function, we identified effector domain mutants of Rac1 and Rac1-RhoA chimeric proteins that no longer bound PAK. Surprisingly, PAK binding was dispensable for Rac1-induced transformation and lamellipodium formation, as well as activation of JNK, p38, and serum response factor (SRF). However, the ability of Rac1 to bind to and activate PAK correlated with its ability to stimulate transcription from the cyclin D1 promoter. Furthermore, Rac1 activation of JNK or SRF, or induction of lamellipodia, was neither necessary nor sufficient for Rac1 transforming activity. Finally, the signaling pathways that mediate Rac1 activation of SRF or JNK were distinct from those that mediate Rac1 induction of lamellipodia. Taken together, these observations suggest that Rac1 regulates at least four distinct effector-mediated functions and that multiple pathways may contribute to Rac1-induced cellular transformation.

3T3 Cells↗
PubMed1997

Lck regulates Vav activation of members of the Rho family of GTPases.

Vav is a member of a family of oncogene proteins that share an approximately 250-amino-acid motif called a Dbl homology domain. Paradoxically, Dbl itself and other proteins containing a Dbl domain catalyze GTP-GDP exchange for Rho family proteins, whereas Vav has been reported to catalyze GTP-GDP exchange for Ras proteins. We present Saccharomyces cerevisiae genetic data, in vitro biochemical data, and animal cell biological data indicating that Vav is a guanine nucleotide exchange factor for Rho-related proteins, but in similar genetic and biochemical experiments we fail to find evidence that Vav is a guanine nucleotide exchange factor for Ras. Further, we present data indicating that the Lck kinase activates the guanine nucleotide exchange factor and transforming activity of Vav.

Animals↗
PubMed1997

L-glutamine stimulates intestinal cell proliferation and activates mitogen-activated protein kinases.

We studied the mechanisms by which L-glutamine (Gln), a major fuel for enterocytes, signals proliferation in intestinal epithelial cell lines. Gln was additive to epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in stimulating DNA synthesis, as assessed by [3H]thymidine incorporation. Extracellular signal-regulated kinases (ERKs) p42mapk and p44mapk and Jun nuclear kinases (JNKs) phosphorylate and activate nuclear transcription factors. Proteins of the c-Jun, ATF-2, and c-Fos families aggregate to form DNA-binding homodimers or heterodimers called activating protein 1 (AP-1). In vitro assays and functional assays of phosphorylation demonstrated that Gln activates both ERKs and JNKs, resulting in a fourfold increase in AP-1-dependent gene transcription. Gln was required for EGF signaling through ERKs. Maximal stimulation of proliferation required approximately 2.5 mM Gln. c-Jun mRNA levels responded to Gln in "Gln-starved" porcine IPEC-J2 cells and in rat IEC-6 cells. Although Gln metabolism is required for the proliferative response, several Gln by-products did not stimulate [3H]thymidine incorporation, with the exception of arginine. Gln may be a unique nutrient for enterocytes, capable of dual signaling and augmenting the effects of growth factors that govern cellular proliferation and repair.

Animals↗
PubMed1996

Isolation of a NCK-associated kinase, PRK2, an SH3-binding protein and potential effector of Rho protein signaling.

The NCK adapter protein is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK we screened a bacterial expression library to isolate proteins that bind its SH3 domains. Two molecules were isolated, the Wiskott-Aldrich syndrome protein (WASP, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis the Prk2 gene was mapped to mouse chromosome 3. Unlike WASP, which bound the SH3 domains of several signaling proteins, PRK2 specifically bound to the middle SH3 domain of NCK and (weakly) that of phospholipase Cgamma. PRK2 also specifically bound to Rho in a GTP-dependent manner and cooperated with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling.

3T3 Cells↗
PubMed1996

Oncogenic Neu/ErbB-2 increases ets, AP-1, and NF-kappaB-dependent gene expression, and inhibiting ets activation blocks Neu-mediated cellular transformation.

Overexpression of Neu (ErbB-2/HER2) is found in approximately 20% of breast tumors. Activation of Neu by a point mutation (NeuT) causes constitutive tyrosine kinase activity of this transmembrane receptor and transforming activity in fibroblasts. To identify downstream targets of Neu, we have analyzed the ability of Neu to activate gene expression. Expression of NeuT, but not normal Neu, caused transcriptional activation of Ets, AP-1, or NF-kappaB-dependent reporter genes. Dominant inhibitory Ras or Raf mutants blocked the Neu-mediated transcriptional activation, confirming that Ras signaling pathways were required for this activation. Analysis with Ets2 mutants indicated that activation of Ets2 transcriptional activity mediated by NeuT or oncogenic Ras required phosphorylation of the same Ets2 residue, threonine 72. Cotransfection of dominant inhibitory Ets2 mutants specifically blocked NeuT-mediated activation of Ets-dependent reporter genes. Furthermore, in focus formation assays using NIH 3T3 cells, the transforming activity of NeuT was inhibited 5-fold when NeuT was cotransfected with a dominant negative Ets2 mutant. However, parallel colony formation assays showed that the Ets2 dominant negative mutant did not inhibit the growth of normal cells. Together, these data show that NeuT activates a variety of transcription factor families via the Ras signaling pathway and that Ets activation is required for NeuT-mediated cellular transformation. Thus, downstream targets of Neu, including Ets transcription factors, may be useful points for therapeutic intervention in Neu/ErbB-2-associated cancers.

3T3 Cells↗
PubMed1996

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.

3T3 Cells↗
PubMed1996

Differential regulation of hepatocyte DNA synthesis by cAMP in vitro in vivo.

Adenosine 3',5'-cyclic monophosphate (cAMP) prevents epidermal growth factor (EGF)-induced DNA synthesis in many types of cultured cells, including hepatocytes, but its effects on cellular proliferation in vivo are unknown. This study compares the effects of supplemental cAMP on hepatocyte proliferation induced in vivo by 70% partial hepatectomy (PH) and in vitro by EGF and determines the effects of cAMP on AP-1, a family of growth-regulatory transcription factors, and the kinase cascades that normally activate AP-1. Although injection of dibutyryladenosine 3',5'-cyclic monophosphate (30 mg/kgip) at the time of PH increased liver cAMP concentrations at least 100-fold for several hours, it did not inhibit hepatic incorporation of [3H]thymidine or proliferating cell nuclear antigen expression 24 h after PH. cAMP treatment led to a complete inhibition of extracellular signal-related kinase (ERK) activity and transiently reduced NH2-terminal Jun nuclear kinase (JNK) activity after PH but did not decrease the expression of c-jun mRNA or protein. Consistent with the known cAMP stimulation of jun-B in cultured cells, cAMP treatment increased jun-B mRNA, protein, and DNA binding activity post-PH. Surprisingly, cAMP treatment enhanced Raf kinase activity after PH in rats. In primary hepatocyte cultures, supplemental cAMP inhibited JNK and ERK activity, total AP-1 and c-Jun transcriptional activities, and DNA synthesis. Thus elevated cAMP inhibited ERK and JNK activity in culture and in vivo and inhibited hepatocyte proliferation in culture but not in vivo. This suggests that in vivo mechanisms compensate for cAMP inhibition of certain growth-related signaling cascades and emphasizes potential risks of extrapolating from simple cell culture systems to explain physiology in intact animals.

Animals↗
PubMed1995

Ceramide activates the stress-activated protein kinases.

Tumor necrosis factor alpha (TNF alpha) activates the stress-activated protein kinases (SAPKs, also known as Jun nuclear kinases or JNKs) resulting in the stimulation of AP-1-dependent gene transcription and induces the translocation of NF kappa B to the nucleus resulting in the stimulation of NF kappa B-dependent gene transcription. A potential second messenger for these signaling pathways is ceramide, which is generated when TNF alpha activates sphingomyelinases. We show that treatment of HL-60 human promyelocytic cells with exogenous sphingomyelinase leads to rapid stimulation of JNK/SAPK activity, an effect not mimicked by treatment with phospholipase A2, C, or D. Further, JNK/SAPK activity is stimulated 2.7- and 2.8-fold, respectively, in cells exposed to C2-ceramide (5 microM) or TNF alpha (10 ng/ml). The prolonged stimulation of this kinase activity by C2-ceramide is similar to that previously reported for TNF alpha. In contrast, the related mitogen-activated protein kinases ERK1 and ERK2 are weakly stimulated following TNF alpha treatment (1.5-fold) and are inhibited by C2-ceramide treatment. TNF alpha also potently stimulates NF-kappa B DNA binding activity and transcriptional activity, but these effects are not mimicked by addition of C2-ceramide or sphingomyelinase to intact cells. Furthermore, TNF alpha, sphingomyelinase, and C2-ceramide induce c-jun, a gene that is stimulated by the ATF-2 and c-Jun transcription factors. These data suggest that ceramide may act as a second messenger for a subset of TNF alpha's biochemical and biological effects.

Animals↗