Ian P. Whitehead

Dependence of Dbl and Dbs Transformation on MEK and NF-κB Activation

ABSTRACT Dbs was identified initially as a transforming protein and is a member of the Dbl family of proteins (>20 mammalian members). Here we show that Dbs, like its rat homolog Ost and the closely related Dbl, exhibited guanine nucleotide exchange activity for the Rho family members RhoA and Cdc42, but not Rac1, in vitro. Dbs transforming activity was blocked by specific inhibitors of RhoA and Cdc42 function, demonstrating the importance of these small GTPases in Dbs-mediated growth deregulation. Although Dbs transformation was dependent upon the structural integrity of its pleckstrin homology (PH) domain, replacement of the PH domain with a membrane localization signal restored transforming activity. Thus, the PH domain of Dbs (but not Dbl) may be important in modulating association with the plasma membrane, where its GTPase substrates reside. Both Dbs and Dbl activate multiple signaling pathways that include activation of the Elk-1, Jun, and NF-κB transcription factors and stimulation of transcription from the cyclin D1 promoter. We found that Elk-1 and NF-κB, but not Jun, activation was necessary for Dbl and Dbs transformation. Finally, we have observed that Dbl and Dbs regulated transcription from the cyclin D1 promoter in a NF-κB-dependent manner. Previous studies have dissociated actin cytoskeletal activity from the transforming potential of RhoA and Cdc42. These observations, when taken together with those of the present study, suggest that altered gene expression, and not actin reorganization, is the critical mediator of Dbl and Rho family protein transformation.

The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways.

We utilized a cDNA expression library derived from the B6SutA1 mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-1. In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-1 transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-1 cooperated with activated Raf-1 and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-κB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA. Finally, PAR-1 transforming activity was blocked by pertussis toxin and by co-expression of the RGS domain of Lsc, implicating Gαi and Gα12/Gα13 subunits, respectively, as mediators of PAR-1 transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.

Expression Cloning of lsc, a Novel Oncogene with Structural Similarities to the Dbl Family of Guanine Nucleotide Exchange Factors

In a screen for genes with oncogenic potential expressed by the murine B6SUtA1 myeloid progenitor cell line, we isolated a 2.5-kilobase pair cDNA whose expression causes strong morphological transformation and deregulated proliferation of NIH 3T3 cells. The transforming cDNA encodes a truncated protein (designated Lsc) with a region of sequence similarity to the product of the lbc oncogene. This region includes the tandem Dbl homology and pleckstrin homology domains that are hallmarks of the Dbl-like proteins, a family of presumptive or demonstrated guanine nucleotide exchange factors that act on Rho family GTPases. Lsc requires intact Dbl homology and pleckstrin homology domains for its oncogenic activity. The transforming activity of Lsc in NIH 3T3 cells is reduced by cotransfection with p190 (a GTPase activating protein for Rho family GTPases) and the Rho family dominant-negative mutants RhoA(19N), CDC42(17N), and Rac1(17N). These results indicate a role for the Rho family of GTPases in mediating the transforming activity of Lsc and are consistent with the exchange specificities that have been attributed to Dbl family members. The lsc gene is expressed in a variety of tissues and is particularly abundant in hemopoietic tissues (thymus, spleen, and bone marrow). Lsc is a member of a growing family of proteins that may function as activators of Rho family GTPases in a developmental or tissue-specific manner.

Multifunctional Roles for the PH Domain of Dbs in Regulating Rho GTPase Activation

Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivodespite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.

Rho GTPase-dependent transformation by G protein-coupled receptors

G protein coupled receptors (GPCRs) constitute the largest family of cell surface receptors, with more than 1000 members, and are responsible for converting a diverse array of extracellular stimuli into intracellular signaling events. Most members of the family have defined roles in intermediary metabolism and generally perform these functions in well-differentiated cells. However, there is an increasing awareness that some GPCRs can also regulate proliferative signaling pathways and that chronic stimulation or mutational activation of receptors can lead to oncogenic transformation. Activating mutations in GPCRs are associated with several types of human tumors and some receptors exhibit potent oncogenic activity due to agonist overexpression. Additionally, expression screening analyses for novel oncogenes identified GPCRs whose expression causes the oncogenic transformation of NIH3T3 mouse fibroblasts. These include Mas, G2A, and the PAR-1 thrombin receptor. In this review we summarize the signaling and transforming properties of these GPCR oncoproteins. What has emerged from these studies is the delineation of a GTPase cascade where transforming GPCRs cause aberrant growth regulation via activation of Rho family small GTPases.

p38 MAPK-mediated activation of NF-κB by the RhoGEF domain of Bcr

The oncogenic fusion protein p210 Bcr-Abl is causally associated with virtually all cases of chronic myelogenous leukemia. The wild-type Bcr product has several recognizable structural and functional motifs including a domain that contains guanine nucleotide exchange activity for Rho family GTPases (DH/PH domain). Although this domain is retained within p210 Bcr-Abl, it has no known signaling activities in vivo. Here we report that a fragment of Bcr that encodes the isolated DH/PH domain is a potent activator of the NF-κB transcription factor. Within the context of full length Bcr, this activity is regulated by proximal flanking sequences that suppress the DH/PH domain encoded guanine nucleotide exchange activity. NF-κB activation by Bcr is not mediated by nuclear translocation, but rather by p38 mitogen-activated protein kinase (MAPK)-dependent modification of the RelA/p65 transactivation domain. Although we were able to demonstrate that Bcr can function as an exchange factor for Cdc42 in vivo, NF-κB activation appears to occur via a Cdc42-independent mechanism. These studies constitute direct evidence that the Bcr RhoGEF domain can function in vivo, and identify a new signaling activity that may contribute to the transforming potential of p210 Bcr-Abl.

Bone Marrow Stroma Influences Transforming Growth Factor-β Production in Breast Cancer Cells to Regulate c-myc Activation of the Preprotachykinin-I Gene in Breast Cancer Cells

Breast cancer cells (BCCs) have preference for the bone marrow (BM). This study used an in vitro coculture of BCCs and BM stroma to represent a model of early breast cancer metastasis to the BM. The overarching hypothesis states that once BCCs are in the BM, microenvironmental factors induce changes in the expression of genes for cytokines and preprotachykinin-I (PPT-I) in both BCCs and stromal cells. Consequently, the expression of both PPT-I and cytokines are altered to facilitate BCC integration within BM stroma. Cytokine and transcription factor arrays strongly suggested that transforming growth factor-β (TGF-β) and c-myc regulate the expression of PPT-I so as to facilitate BCC integration among stroma. Northern analyses and TGF-β bioassays showed that stromal cells and BCCs influence the level of PPT-I and TGF-β in each other. In cocultures, PPT-I and TGF-β expressions were significantly (P < 0.05) increased and decreased, respectively. TGF-β and PPT-I were undetectable in separate stromal cultures but were expressed as cocultures. Two consensus sequences for c-myc in the 5′ flanking region of the PPT-I gene were shown to be functional using gel shift and reporter gene assays. Mutagenesis of c-myc sites, neutralization studies with anti-TGF-β, and transient tranfections all showed that c-myc is required for TGF-β-mediated induction of PPT-I in BCCs. TGF-β was less efficient as a mediator of BCC integration within stroma for c-myc-BCCs. Because the model used in this study represents BCC integration within BM stroma, these studies suggest that TGF-β is important to the regulation of PPT-I in the early events of bone invasion by BCCs.

Involvement of NH2-terminal Sequences in the Negative Regulation of Vav Signaling and Transforming Activity

Deletion of the NH2-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 NH2-terminal sequences and whether additional NH2-terminal sequences are also involved in negative regulation have not been determined. Therefore, we generated additional NH2-terminal deletion mutants of proto-Vav that lack the NH2-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 NH2-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-κB 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 NH2-terminal Vav deletion mutant lacking this tyrosine residue (ΔN-186 Vav) retained the ability to be phosphorylated by Lckin vivo and Lck still caused enhancement of ΔN-186 Vav signaling and transforming activity. Thus, Lck can stimulate Vav via a mechanism that does not involve Tyr174 or removal of NH2-terminal regulatory activity. Finally, we found that NH2-terminal deletion enhanced the degree of Vav association with the membrane-containing particulate fraction and that an isolated NH2-terminal fragment (residues 1–186) could impair ΔN-186 Vav signaling. Taken together, these observations suggest that the NH2 terminus may serve as a negative regulator of Vav by intramolecular interaction with COOH-terminal sequences to modulate efficient membrane association.

Pleckstrin homology domain-mediated activation of the Rho-specific guanine nucleotide exchange factor Dbs by Rac1

Dbs is a Rho-specific guanine nucleotide exchange factor that was identified in a screen for proteins whose expression causes deregulated growth in NIH 3T3 mouse fibroblasts. Although Rac1 has not been shown to be a substrate for Dbs in either in vitro or in vivo assays, the Rat ortholog of Dbs (Ost) has been shown to bind specifically to GTP·Rac1 in vitro. The dependence of the Rac1/Dbs interaction on GTP suggests that Dbs may in fact be an effector for Rac1. Here we show that the interaction between activated Rac1 and Dbs can be recapitulated in mammalian cells and that the Rac1 docking site resides within the pleckstrin homology domain of Dbs. This interaction is specific for Rac1 and is not observed between Rac1 and several other members of the Rho-specific guanine nucleotide exchange factor family. Co-expression of Dbs with activated Rac1 causes enhanced focus forming activity and elevated levels of GTP·RhoA in NIH 3T3 cells, indicating that Dbs is activated by the interaction. Consistent with this, activated Rac1 co-localizes with Dbs in NIH 3T3 cells, and natively expressed Rac1 relocalizes in response to Dbs expression. To summarize, we have characterized a surprisingly direct pleckstrin homology domain-mediated mechanism through which Rho GTPases can become functionally linked.

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco- or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco- and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130Cas on Tyr-410 and an increase in the abundance of the Crk·p130Cas complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130Cas and disrupts the Crk·p130Cas complex. We further determined that suppression of expression of Cdc42, Ack1, p130Cas, or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk·p130Cas complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.