Search PubMed⌕ Search

Biomedical subjects

G J Clark

Publications and source records attributed to G J Clark.

At least 37 records · Page 2Linked to original sources
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

Laparoscopic distal pancreatectomy procedures in a rural hospital.

Laparoscopic surgical procedures are replacing traditional, more invasive surgical procedures--even in small, rural hospitals. During a period of 12 months, the authors performed laparoscopic distal pancreatectomy procedures on two patients at a 20-bed hospital in North Dakota. Although surgeons in other countries have reported performing laparoscopic distal pancreatectomy procedures, this is the first published report of a US surgical team performing this procedure. Laparoscopic dissection of the distal pancreas allows preservation of patients' spleens, decreases postoperative pain and length of hospitalization, and permits patients to return to activities of daily living more quickly than with traditional open pancreatectomy procedures.

Adult↗
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↗
PubMed1996

Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.

3T3 Cells↗
PubMed1996

Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.

3T3 Cells↗
PubMed1996

Overexpression of the Ras-related TC21/R-Ras2 protein may contribute to the development of human breast cancers.

Although experimental studies suggest that aberrant Ras function can promote the malignant progression of human breast epithelial cells, the occurrence of mutated ras genes in breast tumors is infrequent. One possible explanation for this apparent paradox is that aberrant function of the Ras-related protein TC21/R-Ras2, which causes malignant transformation of NIH 3T3 cells via upregulation of the Ras signal transduction pathway, may contribute to breast tumor development in the absence of Ras mutations. To address this possibility, we utilized two complementary approaches. First, we determined that aberrant TC21 function caused transformation of the MCF-10A human breast epithelial cell line. TC21-transformed MCF-10A cells exhibited altered cellular morphology associated with a disruption of cell-cell adherens junctions, formed colonies in soft agar, and showed enhanced motility in vitro. These alterations were similar to, but more dramatic than, those observed with oncogenic Ras-transformed MCF-10A cells. Furthermore, overexpression of normal TC21, but not Ras, also caused transformation of these cells. Second, we observed that TC21 protein expression was greatly elevated in 7 of 9 breast tumor lines when compared to untransformed MCF-10A cells. Taken together, these results support the possibility that overexpression of TC21 may contribute to aberrant growth properties of breast carcinoma cells.

Breast Neoplasms↗
PubMed1996

TC21 causes transformation by Raf-independent signaling pathways.

Although the Ras-related protein TC21/R-Ras2 has only 55% amino acid identity with Ras proteins, mutated forms of TC21 exhibit the same potent transforming activity as constitutively activated forms of Ras. Therefore, like Ras, TC21 may activate signaling pathways that control normal cell growth and differentiation. To address this possibility, we determined if regulators and effectors of Ras are also important for controlling TC21 activity. First, we determined that Ras guanine nucleotide exchange factors (SOS1 and RasGRF/CDC25) synergistically enhanced wild-type TC21 activity in vivo and that Ras GTPase-activating proteins (GAPs; p120-GAP and NF1-GAP) stimulated wild-type TC21 GTP hydrolysis in vitro. Thus, extracellular signals that activate Ras via SOS1 activation may cause coordinate activation of Ras and TC21. Second, we determined if Raf kinases were effectors for TC21 transformation. Unexpectedly, yeast two-hybrid binding analyses showed that although both Ras and TC21 could interact with the isolated Ras-binding domain of Raf-1, only Ras interacted with full-length Raf-1, A-Raf, or B-Raf. Consistent with this observation, we found that Ras- but not TC21-transformed NIH 3T3 cells possessed constitutively elevated Raf-1 and B-Raf kinase activity. Thus, Raf kinases are effectors for Ras, but not TC21, signaling and transformation. We conclude that common upstream signals cause activation of Ras and TC21, but activated TC21 controls cell growth via distinct Raf-independent downstream signaling pathways.

3T3 Cells↗
PubMed1995

Cellular distribution and costimulatory function of rat CD28. Regulated expression during thymocyte maturation and induction of cyclosporin A sensitivity of costimulated T cell responses by phorbol ester.

CD28 has been identified in man and mouse as a potent costimulatory receptor on T cells. We have generated a mAb, called JJ319, to rat CD28 and show that it is expressed on virtually all peripheral rat alpha beta and on most gamma delta T cells, and on about half of NK cells. In contrast to the mouse but as in humans, most immature CD4+8+TCRlow thymocytes express little or no CD28, whereas CD28 expression is high on TCRintermediate and TCRhigh cells. mAb JJ319 very effectively costimulates T cell proliferation and IL-2 secretion by resting rat T cells. In contrast to results obtained in mice and humans, phorbol ester did not synergize in T cell activation with CD28-specific mAb but even induced sensitivity to cyclosporin A in T cell cultures that were optimally costimulated by mAbs to the TCR and to CD28. This result points to a novel effect of protein kinase activation by phorbol ester on signal transduction by TCR plus CD28 costimulation which only becomes apparent if, as in the rat, the TCR-mediated signal cannot be replaced by phorbol ester.

Animals↗
PubMed1995

Aberrant function of the Ras signal transduction pathway in human breast cancer.

Although ras mutations are infrequent (approximately 5%) in breast cancers, there is considerable evidence that suggests that the pathways which Ras services may still be deregulated in breast cancer cells. The recent identification of many of the components of the Ras signal transduction pathway has defined a network of proto-oncogene proteins controlling diverse signaling events that regulate cell growth and differentiation. Consequently, mutations that perturb the function of any one component of this signal pathway may trigger the same oncogenic events as mutation of ras itself. Moreover, several Ras-related proteins have recently been demonstrated to possess the ability to trigger malignant transformation via signaling pathways shared with Ras proteins. Thus, it is possible that the aberrant function of Ras-related proteins may contribute to breast cancer development. Consequently, it is important not to dismiss the Ras pathway in the development of breast cancer merely because of the infrequent detection of mutations in ras itself, but rather to consider the influence of aberrations upstream or downstream of Ras and of certain Ras-related proteins in the development of breast cancer. Finally, the critical importance of components upstream and downstream of Ras provides additional targets for rational drug design approaches to block the aberrant function of Ras signaling in human tumors.

Apoptosis↗
PubMed1995

Biological assays for Ras transformation.

The rodent fibroblast systems described above have provided sensitive and rapid biological assays to characterize the properties of normal and mutated Ras proteins. Furthermore, these assays have provided in vitro systems to measure the ability of other cellular components to modulate Ras signal transduction and transformation. However, while these assays provide an excellent measure of Ras-transforming activity, the fact that these cells are of fibroblastic origin, and can be transformed by a single hit, indicates that caution should be used in extrapolating observations from NIH 3T3 transformation assays to the situation in human tumors. Therefore, using human epithelial cell-based assays that more closely approximate the cell types where mutated ras alleles are most frequently detected may provide more realistic assays for examining the biochemical and biological consequences of aberrant Ras function in human tumors. Nevertheless, despite these cautions, these rodent transformation assays will continue to be the best and most widely applied assays for Ras biological activity.

3T3 Cells↗
PubMed1995

Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia.

Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-cell and cell-substrate interactions. Here we have identified changes in the adhesions and cytoskeletal interactions of transformed epithelial cells that contribute to their altered morphology. Using MCF-10A human breast epithelial cells as a model system, we have found that transformation by an activated form of ras is characterized by less developed adherens-type junctions between cells but increased focal adhesions. Contributing to the modified adherens junctions of the transformed cells are decreased interactions among beta-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including beta-catenin and p120 Cas. Whereas in the normal cells beta-catenin is found in association with E-cadherin, p120 Cas is not. In the ras-transformed cells, the situation is reversed; tyrosine-phosphorylated p120 Cas, but not tyrosine-phosphorylated beta-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated beta-catenin also shows increased detergent solubility, suggesting a decreased association with the actin cytoskeleton. p120 Cas, whether tyrosine phosphorylated or not, partitions into the detergent soluble fraction, suggesting that it is not tightly bound to the actin cytoskeleton in either the normal or ras-transformed cells. Inhibitors of tyrosine kinases decrease the level of tyrosine phosphorylation and restore a normal epithelial morphology to the ras-transformed cells. In particular, decreased tyrosine phosphorylation of beta-catenin is accompanied by increased interaction with both E-cadherin and the detergent insoluble cytoskeletal fraction. These results suggest that elevated tyrosine phosphorylation of proteins such as beta-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia.

Breast↗
PubMed1995

Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation.

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.

3T3 Cells↗
PubMed1994

A root moisture sensor for plants in microgravity.

Development of components for bioregenerative life-support systems is a vital step toward long-term space exploration. The culturing of plants in a microgravity environment may be optimized by the use of appropriate sensors and controllers. This paper describes a sensor developed for determining the amount of fluid (nutrient solution) available on the surface of a porous ceramic nutrient delivery substrate to the roots of conventional crop plants. The sensor is based on the change in thermal capacitance and thermal conductance near the surface as the moisture content changes. The sensor could be employed as a data acquisition and control sensor to support the automated monitoring of plants grown in a microgravity environment.

Ceramics↗
PubMed1993

Differential antagonism of Ras biological activity by catalytic and Src homology domains of Ras GTPase activation protein.

Ras p120 GTPase activation protein (GAP), a cytosolic protein, is a negative mediator and potential downstream effector of Ras function. Since membrane association is critical for Ras function, we introduced the Ras membrane-targeting signal (a 19-residue peptide ending in CAAX, where C = cysteine, A = aliphatic amino acid, and X = any amino acid) onto the GAP N-terminal Src homology 2 and 3 and the C-terminal catalytic domains (designated nGAP/CAAX and cGAP/CAAX, respectively) to determine the role of membrane association in GAP function. cGAP/CAAX and full-length GAP/CAAX, but not GAP or nGAP/CAAX, exhibited potent growth inhibitory activity. Whereas both oncogenic and normal Ras activity were inhibited by cGAP/CAAX, nGAP/CAAX, despite lacking the Ras binding domain, inhibited the activity of oncogenic Ras without affecting the action of normal Ras. Altogether, these results demonstrate that membrane association potentiates GAP catalytic activity, support an effector function for GAP, and suggest that normal and oncogenic Ras possess different downstream interactions.

3T3 Cells↗
PubMed1992

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.

3T3 Cells↗