Biomedical subjects
C J Der
Publications and source records attributed to C J Der.
Modulation of HIV-1 replication by a novel RhoA effector activity.
The RhoA GTPase is involved in regulating actin cytoskeletal organization, gene expression, cell proliferation, and survival. We report here that p115-RhoGEF, a specific guanine nucleotide exchange factor (GEF) and activator of RhoA, modulates HIV-1 replication. Ectopic expression of p115-RhoGEF or Galpha13, which activates p115-RhoGEF activity, leads to inhibition of HIV-1 replication. RhoA activation is required and the inhibition affects HIV-1 gene expression. The RhoA effector activity in inhibiting HIV-1 replication is genetically separable from its activities in transformation of NIH3T3 cells, activation of serum response factor, and actin stress fiber formation. These findings reveal that the RhoA signal transduction pathway regulates HIV-1 replication and suggest that RhoA inhibits HIV-1 replication via a novel effector activity.
Vav2 is an activator of Cdc42, Rac1, and RhoA.
Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.
Analyses of transforming activity of Rho family activators.
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Analysis of function and regulation of proteins that mediate signal transduction by use of lipid-modified plasma membrane-targeting sequences.
It is now established that the function of many signaling molecules is controlled, in part, by regulation of subcellular localization. For example, the dynamic recruitment of normally cytosolic proteins to the plasma membrane, by activated Ras or activated receptor tyrosine kinases, facilitates their interaction with other membrane-associated components that participate in their full activation (e.g., Raf-1). Therefore, the creation of chimeric proteins that contain lipid-modified signaling sequences that direct membrane localization allows the generation of constitutively activated variants of such proteins. The amino-terminal myristoylation signal sequence of Src family proteins and the carboxy-terminal prenylation signal sequence of Ras proteins have been widely used to achieve this goal. Such membrane-targeted variants have proved to be valuable reagents in the study of the biochemical and biological properties of many signaling molecules.
The Ras branch of small GTPases: Ras family members don't fall far from the tree.
The Ras branch of the Ras superfamily consists of small GTPases most closely related to Ras and include the R-Ras, Rap, Ral, Rheb, Rin and Rit proteins. Although our understanding of Ras signaling and biology is now considerable, recent observations suggest that Ras function is more complex than previously believed. First, the three Ras proteins may not be functionally identical. Second, Ras function involves functional cross-talk with their close relatives.
Understanding Ras: 'it ain't over 'til it's over'.
Since 1982, Ras has been the subject of intense research scrutiny, focused on determining the role of aberrant Ras function in human cancers and defining the mechanism by which Ras mediates its actions in normal and neoplastic cells. The long-term goal has been to develop antagonists of Ras as novel approaches for cancer treatment. Although impressive strides have been made in these endeavours, and our knowledge of Ras is quite extensive, it appears that we are at the beginning, rather than at the end, of fully understanding Ras function. This review highlights new issues that have further complicated our efforts to understand Ras.
Identification and characterization of an activating TrkA deletion mutation in acute myeloid leukemia.
In this study, we utilized retroviral transfer of cDNA libraries in order to identify oncogenes that are expressed in acute myeloid leukemia (AML). From screens using two different cell types as targets for cellular transformation, a single cDNA encoding a variant of the TrkA protooncogene was isolated. The protein product of this protooncogene, TrkA, is a receptor tyrosine kinase for nerve growth factor. The isolated transforming cDNA encoded a TrkA protein that contains a 75-amino-acid deletion in the extracellular domain of the receptor and was named DeltaTrkA. DeltaTrkA readily transformed fibroblast and epithelial cell lines. The deletion resulted in activation of the tyrosine kinase domain leading to constitutive tyrosine phosphorylation of the protein. Expression of DeltaTrkA in cells led to the constitutive activation of intracellular signaling pathways that include Ras, extracellular signal-regulated kinase/mitogen-activated protein kinase, and Akt. Importantly, DeltaTrkA altered the apoptotic and growth properties of 32D myeloid progenitor cells, suggesting DeltaTrkA may have contributed to the development and/or maintenance of the myeloid leukemia from which it was isolated. Unlike Bcr-Abl, expression of DeltaTrkA did not activate Stat5 in these cells. We have detected expression of DeltaTrkA in the original AML sample by reverse transcriptase PCR and by Western blot analysis. While previous TrkA mutations identified from human tumors involved fusion to other proteins, this report is the initial demonstration that deletions within TrkA may play a role in human cancers. Finally, this report is the first to indicate mutations in TrkA may contribute to leukemogenesis.
Dissection of Ras-dependent signaling pathways controlling aggressive tumor growth of human fibrosarcoma cells: evidence for a potential novel pathway.
Activation of multiple signaling pathways is required to trigger the full spectrum of in vitro and in vivo phenotypic traits associated with neoplastic transformation by oncogenic Ras. To determine which of these pathways are important for N-ras tumorigenesis in human cancer cells and also to investigate the possibility of cross talk among the pathways, we have utilized a human fibrosarcoma cell line (HT1080), which contains an endogenous mutated allele of the N-ras gene, and its derivative (MCH603c8), which lacks the mutant N-ras allele. We have stably transfected MCH603c8 and HT1080 cells with activating or dominant-negative mutant cDNAs, respectively, of various components of the Raf, Rac, and RhoA pathways. In previous studies with these cell lines we showed that loss of mutant Ras function results in dramatic changes in the in vitro phenotypic traits and conversion to a weakly tumorigenic phenotype in vivo. We report here that only overexpression of activated MEK contributed significantly to the conversion of MCH603c8 cells to an aggressive tumorigenic phenotype. Furthermore, we have demonstrated that blocking the constitutive activation of the Raf-MEK, Rac, or RhoA pathway alone is not sufficient to block the aggressive tumorigenic phenotype of HT1080, despite affecting a number of in vitro-transformed phenotypic traits. We have also demonstrated the possibility of bidirectional cross talk between the Raf-MEK-ERK pathway and the Rac-JNK or RhoA pathway. Finally, overexpression of activated MEK in MCH603c8 cells appears to result in the activation of an as-yet-unidentified target(s) that is critical for the aggressive tumorigenic phenotype.
Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF.
The long cytoplasmic tail of the human immunodeficiency virus (HIV)-1 transmembrane protein gp41 (gp41C) is implicated in the replication and cytopathicity of HIV-1 [1]. Little is known about the specific functions of gp41C, however. HIV-1 or simian immunodeficiency virus (SIV) mutants with defective gp41C have cell-type- or species-dependent phenotypes [2] [3] [4] [5] [6]. Thus, host factors are implicated in mediating the functions of gp41C. We report here that gp41C interacted with the carboxy-terminal regulatory domain of p115-RhoGEF [7], a specific guanine nucleotide exchange factor (GEF) and activator of the RhoA GTPase, which regulates actin stress fiber formation, activation of serum response factor (SRF) and cell proliferation [8] [9]. We demonstrate that gp41C inhibited p115-mediated actin stress fiber formation and activation of SRF. An amphipathic helix region with a leucine-zipper motif in gp41C is involved in its interaction with p115. Mutations in gp41C leading to loss of interaction with p115 impaired HIV-1 replication in human T cells. These findings suggest that an important function of gp41C is to modulate the activity of p115-RhoGEF and they thus reveal a new potential anti-HIV-1 target.
Involvement of NH(2)-terminal sequences in the negative regulation of Vav signaling and transforming activity.
Deletion of the NH(2)-terminal 65 amino acids of proto-Vav (to form onco-Vav) activates its transforming activity, suggesting that these sequences serve a negative regulatory role in Vav function. However, the precise role of these NH(2)-terminal sequences and whether additional NH(2)-terminal sequences are also involved in negative regulation have not been determined. Therefore, we generated additional NH(2)-terminal deletion mutants of proto-Vav that lack the NH(2)-terminal 127, 168, or 186 amino acids, and assessed their abilities to cause focus formation in NIH 3T3 cells and to activate different signaling pathways. Since Vav mutants lacking 168 or 186 NH(2)-terminal residues showed a several 100-fold greater focus forming activity than that seen with deletion of 65 residues, residues spanning 66 to 187 also contribute significantly to negative regulation of Vav transforming activity. The increase in Vav transforming activity correlated with the activation of the c-Jun, Elk-1, and NF-kappaB transcription factors, as well as increased transcription from the cyclin D1 promoter. Tyrosine 174 is a key site of phosphorylation by Lck in vitro and Lck-mediated phosphorylation has been shown to be essential for proto-Vav GEF function in vitro. However, we found that an NH(2)-terminal Vav deletion mutant lacking this tyrosine residue (DeltaN-186 Vav) retained the ability to be phosphorylated by Lck in vivo and Lck still caused enhancement of DeltaN-186 Vav signaling and transforming activity. Thus, Lck can stimulate Vav via a mechanism that does not involve Tyr(174) or removal of NH(2)-terminal regulatory activity. Finally, we found that NH(2)-terminal deletion enhanced the degree of Vav association with the membrane-containing particulate fraction and that an isolated NH(2)-terminal fragment (residues 1-186) could impair DeltaN-186 Vav signaling. Taken together, these observations suggest that the NH(2) terminus may serve as a negative regulator of Vav by intramolecular interaction with COOH-terminal sequences to modulate efficient membrane association.
Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals.
Mitogen-activated protein kinase (MAPK) cascades are activated by diverse extracellular signals and participate in the regulation of an array of cellular programs. In this study, we investigated the roles of MAPKs in the induction of phase II detoxifying enzymes by chemicals. Treatment of human hepatoma (HepG2) and murine hepatoma (Hepa1c1c7) cells with tert-butylhydroquinone (tBHQ) or sulforaphane (SUL), two potent phase II enzyme inducers, stimulated the activity of extracellular signal-regulated protein kinase 2 (ERK2) but not c-Jun N-terminal kinase 1. tBHQ and SUL also activated MAPK kinase. Inhibition of MAPK kinase with its inhibitor, PD98059, abolished ERK2 activation and impaired the induction of quinone reductase, a phase II detoxifying enzyme, and antioxidant response element (ARE)-linked reporter gene by tBHQ and SUL. Overexpression of a dominant-negative mutant of ERK2 also attenuated tBHQ and SUL induction of ARE reporter gene activity. Interestingly, although expression of Ras and its mutant forms showed distinct effects on basal ARE reporter gene activity, they did not affect the activation of reporter gene by the inducers. Furthermore, a dominant-negative mutant of Ras had little effect on ERK2 activation by tBHQ and SUL, implicating a Ras-independent mechanism. Indeed, both tBHQ and SUL were able to stimulate Raf-1 kinase activity in vivo as well as in vitro. Thus, our results indicate that the induction of ARE-dependent phase II detoxifying enzymes is mediated by a MAPK pathway, which may involve direct activation of Raf-1 by the inducers.
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.
GTP binding by class II transactivator: role in nuclear import.
Class II transactivator (CIITA) is a global transcriptional coactivator of human leukocyte antigen-D (HLA-D) genes. CIITA contains motifs similar to guanosine triphosphate (GTP)-binding proteins. This report shows that CIITA binds GTP, and mutations in these motifs decrease its GTP-binding and transactivation activity. Substitution of these motifs with analogous sequences from Ras restores CIITA function. CIITA exhibits little GTPase activity, yet mutations in CIITA that confer GTPase activity reduce transcriptional activity. GTP binding by CIITA correlates with nuclear import. Thus, unlike other GTP-binding proteins, CIITA is involved in transcriptional activation that uses GTP binding to facilitate its own nuclear import.
M-Ras/R-Ras3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPase-activating protein, interacts with the putative Ras effector AF6.
M-Ras is a Ras-related protein that shares approximately 55% identity with K-Ras and TC21. The M-Ras message was widely expressed but was most predominant in ovary and brain. Similarly to Ha-Ras, expression of mutationally activated M-Ras in NIH 3T3 mouse fibroblasts or C2 myoblasts resulted in cellular transformation or inhibition of differentiation, respectively. M-Ras only weakly activated extracellular signal-regulated kinase 2 (ERK2), but it cooperated with Raf, Rac, and Rho to induce transforming foci in NIH 3T3 cells, suggesting that M-Ras signaled via alternate pathways to these effectors. Although the mitogen-activated protein kinase/ERK kinase inhibitor, PD98059, blocked M-Ras-induced transformation, M-Ras was more effective than an activated mitogen-activated protein kinase/ERK kinase mutant at inducing focus formation. These data indicate that multiple pathways must contribute to M-Ras-induced transformation. M-Ras interacted poorly in a yeast two-hybrid assay with multiple Ras effectors, including c-Raf-1, A-Raf, B-Raf, phosphoinositol-3 kinase delta, RalGDS, and Rin1. Although M-Ras coimmunoprecipitated with AF6, a putative regulator of cell junction formation, overexpression of AF6 did not contribute to fibroblast transformation, suggesting the possibility of novel effector proteins. The M-Ras GTP/GDP cycle was sensitive to the Ras GEFs, Sos1, and GRF1 and to p120 Ras GAP. Together, these findings suggest that while M-Ras is regulated by similar upstream stimuli to Ha-Ras, novel targets may be responsible for its effects on cellular transformation and differentiation.
Cellular functions of TC10, a Rho family GTPase: regulation of morphology, signal transduction and cell growth.
The small Ras-related GTPase, TC10, has been classified on the basis of sequence homology to be a member of the Rho family. This family, which includes the Rho, Rac and CDC42 subfamilies, has been shown to regulate a variety of apparently diverse cellular processes such as actin cytoskeletal organization, mitogen-activated protein kinase (MAPK) cascades, cell cycle progression and transformation. In order to begin a study of TC10 biological function, we expressed wild type and various mutant forms of this protein in mammalian cells and investigated both the intracellular localization of the expressed proteins and their abilities to stimulate known Rho family-associated processes. Wild type TC10 was located predominantly in the cell membrane (apparently in the same regions as actin filaments), GTPase defective (75L) and GTP-binding defective (31N) mutants were located predominantly in cytoplasmic perinuclear regions, and a deletion mutant lacking the carboxyl terminal residues required for post-translational prenylation was located predominantly in the nucleus. The GTPase defective (constitutively active) TC10 mutant: (1) stimulated the formation of long filopodia; (2) activated c-Jun amino terminal kinase (JNK); (3) activated serum response factor (SRF)-dependent transcription; (4) activated NF-kappaB-dependent transcription; and (5) synergized with an activated Raf-kinase (Raf-CAAX) to transform NIH3T3 cells. In addition, wild type TC10 function is required for full H-Ras transforming potential. We demonstrate that an intact effector domain and carboxyl terminal prenylation signal are required for proper TC10 function and that TC10 signals to at least two separable downstream target pathways. In addition, TC10 interacted with the actin-binding and filament-forming protein, profilin, in both a two-hybrid cDNA library screen, and an in vitro binding assay. Taken together, these data support a classification of TC10 as a member of the Rho family, and in particular, suggest that TC10 functions to regulate cellular signaling to the actin cytoskeleton and processes associated with cell growth.
Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.
We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrin-coated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is co-localized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.
TC21 and Ras share indistinguishable transforming and differentiating activities.
Constitutively activated mutants of the Ras-related protein TC21/R-Ras2 cause tumorigenic transformation of NIH3T3 cells. However, unlike Ras, TC21 fails to bind to and activate the Raf-1 serine-threonine kinase. Thus, whereas Ras transformation is critically dependent on Raf-1 TC21 activity is promoted by activation of Raf-independent signaling pathways. In the present study, we have further compared the functions of Ras and TC21. First we determined the basis for the inability of TC21 to activate Raf-1. Whereas Ras can interact with the two distinct Ras-binding sequences in NH2-terminus of Raf-1, designated RBS1 and Raf-Cys, TC21 could only bind Raf-Cys. Thus, the inability of TC21 to bind to RBS1 may prevent it from promoting the translocation of Raf-1 to the plasma membrane. Second, we found that TC21 is an activator of the JNK and p38, but not ERK, mitogen-activated protein kinase cascades and that TC21 transforming activity was dependent on Rac function. Thus, like Ras, TC21 may activate a Rac/JNK pathway. Third, we determined if TC21 could cause the same biological consequences as Ras in three distinct cell types. Like Ras, activated TC21 caused transformation of RIE-1 rat intestinal epithelial cells and terminal differentiation of PC12 pheochromocytoma cells. Finally, activated TC21 blocked serum starvation-induced differentiation of C2 myoblasts, whereas dominant negative TC21 greatly accelerated this differentiation process. Therefore, TC21 and Ras share indistinguishable biological activities in all cell types that we have evaluated. These results support the importance of Raf-independent pathways in mediating the actions of Ras and TC21.