Rho GTPases cycle between inactive and active states based upon conformational alterations imposed by the state of bound guanine nucleotide. Rho GTPases bound to GDP are inactive in downstream signaling, while GTP-bound versions modulate a plethora of downstream effectors typically associated with morphological alterations of the cytoskeleton and activation of stress response genes (1-5). Consistent with their central role in regulating cellular differentiation and proliferation, constitutively active Rho GTPases are sufficient to promote cellular transformation. Similarly, Ras-induced transformation is dependent on Rac1, a Rho GTPase (6 -9). Since the proper control of a multitude of signaling cascades by G proteins depends critically upon the state of bound nucleotide, G proteins have evolved several, tightly controlled processes for regulating the binding and hydrolysis of guanine nucleotides. For Rho GTPases, the exchange of bound GDP for GTP is catalyzed by a large class of guanine nucleotide exchange factors (GEFs) 1 related to the gene product for Dbl (diffuse B-cell lymphoma) (10). Similarly to constitutively active forms of Rho GTPases, the unregulated activation of Dbl family members is generally associated with cellular transformation, and many Dbl family members are proto-oncogenic (7, 11).Dbl family proteins invariantly contain an ϳ300-amino acid span composed of a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain (12, 13). DH domains are sufficient to catalyze nucleotide exchange; however, exchange activity is often enhanced by inclusion of the adjacent PH domain (14). While DH domains serve as the major docking site for Rho GTPases, roles for the adjacent, conserved PH domains remain unclear. The invariant DH/PH domain architecture in all Dbl family members strongly suggests that associated PH domains have a unique and highly conserved role in regulating nucleotide exchange. Simple structural stabilization of the DH domain by the adjacent PH domain as suggested by the Tiam1-DH/PH⅐Rac1 crystal structure (15) is an unsatisfactory explanation for the universal pairing of DH and PH domains. A multitude of other domains could easily be imagined to serve this purpose.In many other proteins, PH domains bind phosphoinositides to function as regulated tethers to cellular membranes (16 -20). Although PH domains typically share very low sequence identity, all possess a common ␤-sandwich fold capped at one end with a C-terminal helix. Numerous studies indicate that PH domains generally bind phosphoinositides with a wide degree of affinity and specificity via clusters of basic residues located within the highly variable loops between strands ␤ 1 /␤ 2 and ␤ 3 /␤ 4 (21-23). Several reports present conflicting data describ-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ Supported by an NIH Molecular and Cellul...

Dbl family guanine nucleotide exchange factors (GEFs) for Rho family small GTPases invariably contain a pleckstrin homology (PH) domain that immediately follows their Dbl homology (DH) domain. Although the DH domain is responsible for GEF activity, the role of the PH domain is less clear. We previously reported that PH domains from several Dbl family members bind phosphoinositides with very low affinity (K d values in the 10 M range). This suggests that, unlike several other PH domains, those from Dbl proteins will not function as independent membrane-targeting modules. To determine the functional relevance of low affinity phosphoinositide binding, we mutated the corresponding PH domain from Tiam-1 to abolish its weak, specific binding to phosphatidylinositol 3-phosphate. We first confirmed in vitro that phosphoinositide binding by the isolated DH/PH domain was impaired by the mutations but that intrinsic GEF activity was unaffected. We then introduced the PH domain mutations into full-length Tiam-1 and found that its ability to activate Rac1 or serum response factor in vivo was abolished. Immunofluorescence studies showed that membrane targeting of Tiam-1 was essentially unaffected by mutations in the C-terminal PH domain. Our studies therefore indicate that low affinity phosphatidylinositol 3-phosphate binding by the C-terminal PH domain may be critical for in vivo regulation and activity of Tiam-1 but that the PH domain exerts its regulatory effects without altering membrane targeting. We suggest instead that ligand binding to the PH domain induces conformational and/or orientational changes at the membrane surface that are required for maximum exchange activity of its adjacent DH domain.Tiam-1 (T-lymphoma invasion and metastasis 1) was first identified in a screen for genes that, when amplified, can induce invasiveness of normally noninvasive lymphoma cells (1). Its 1591-amino acid protein product, Tiam-1, is myristoylated at the N terminus (2) and contains two N-terminal PEST sequences (3), a PDZ (postsynaptic density-96/Discs large/Zona occludens-1) domain (4), a Ras-binding domain (5), two pleckstrin homology (PH) 1 domains (6), plus a Dbl (diffuse B-cell lymphoma) homology (DH) domain (7) (Fig.

Glycosphingolipid-enriched domains (GEDs) are believed to act as platforms for transduction of B cell Ag receptor (BCR)-induced signals from the cell surface. We sought to study whether differential sequestration of BCR into GEDs may contribute to the described intrinsic signaling differences between mature and immature B cells. In this study we found that mature B cells copolarize the BCR with GEDs following BCR aggregation, whereas transitional immature B cells do not. Although anti-BCR treatment leads to receptor aggregation by immature stage B cells, the aggregated complexes do not colocalize with GEDs. We found this difference to be independent of the isotype of the receptor, thereby associating this difference in BCR-GED colocalization to the developmental stage of the B cell. These findings suggest a structural basis for the developmentally regulated differences observed in Ag receptor-mediated signal transduction.

Dbl family GEFs (guanine nucleotide-exchange factors) for the Rho GTPases almost invariably contain a PH (pleckstrin homology) domain adjacent to their DH (Dbl homology) domain. The DH domain is responsible for GEF activity, and the PH domain plays a regulatory role that remains poorly understood. We demonstrated previously that Dbl family PH domains bind phosphoinositides with low affinity and cannot function as independent membrane targeting modules. In the present study, we show that dimerization of a Dbs (Dbl's big sister) DH/PH domain fragment is sufficient to drive it to the plasma membrane through a mechanism involving PH domain-phosphoinositide interactions. Thus, the Dbs PH domain could play a significant role in membrane targeting if it co-operates with other domains in the protein. We also show that mutations that prevent phosphoinositide binding by the Dbs PH domain significantly impair cellular GEF activity even in chimaeric proteins that are robustly membrane targeted by farnesylation or by the PH domain of phospholipase C-delta1. This finding argues that the Dbs PH domain plays a regulatory role that is independent of its ability to aid membrane targeting. Thus, we suggest that the PH domain plays dual roles, contributing independently to membrane localization of Dbs (as part of a multi-domain interaction) and allosteric regulation of the DH domain.

Zika virus (ZIKV) is a single-stranded RNA arbovirus belonging to the Flavivirus genus. 1,2 Similar to other zoonotic arboviruses, ZIKV is transmitted by the Aedes mosquito. It causes Zika fever, a usually non-fatal condition with symptoms including headache, fever, malaise, maculopapular rash, joint pains, and conjunctivitis. 2 Recently, it was revealed that ZIKV may have additional pathogenic effects on pregnant women and their fetuses. Reports from South America suggest that ZIKV infection during pregnancy causes fetal microcephaly, 2 which has negative impacts up to and including death. 2,3 In addition to mosquito-borne transmission, it has been reported that ZIKV may be transmitted through heterosexual intercourse, 2 with concomitant implications for global women's health. This is the first example of a mosquito-borne arbovirus that may also utilize a sexual route of transmission. This knowledge has initiated a massive effort by the translational science community to develop more effective detection methods and treatment strategies.Currently there are no approved vaccines or drugs for preventing or treating ZIKV infection or ZIKV-induced pathologies. Recent diagnostics development efforts have focused on ELISA and highly sensitive quantitative reverse transcription PCR