Biomedical subjects

Jason T Snyder

Publications and source records attributed to Jason T Snyder.

11 recordsLinked to original sources

PubMed2006

Crystal structure of Rac1 bound to its effector phospholipase C-beta2.

Although diverse signaling cascades require the coordinated regulation of heterotrimeric G proteins and small GTPases, these connections remain poorly understood. We present the crystal structure of the GTPase Rac1 bound to phospholipase C-beta2 (PLC-beta2), a classic effector of heterotrimeric G proteins. Rac1 engages the pleckstrin-homology (PH) domain of PLC-beta2 to optimize its orientation for substrate membranes. Gbetagamma also engages the PH domain to activate PLC-beta2, and these two activation events are compatible, leading to additive stimulation of phospholipase activity. In contrast to PLC-delta, the PH domain of PLC-beta2 cannot bind phosphoinositides, eliminating this mode of regulation. The structure of the Rac1-PLC-beta2 complex reveals determinants that dictate selectivity of PLC-beta isozymes for Rac GTPases over other Rho-family GTPases, and substitutions within PLC-beta2 abrogate its stimulation by Rac1 but not by Gbetagamma, allowing for functional dissection of this integral signaling node.

Crystallography, X-Ray↗

PubMed2006

Direct activation of purified phospholipase C epsilon by RhoA studied in reconstituted phospholipid vesicles.

Phospholipase C-epsilon (PLC-epsilon) was shown recently to be a downstream effector of Rho GTPases, and we have used an in vitro phospholipid vesicle reconstitution system with purified proteins to show this regulation to be direct. This chapter describes high-level expression of a hexahistidine-tagged fragment of PLC-epsilon encompassing the catalytic core of the enzyme through the tandem RA domains by use of a recombinant baculovirus and High Five insect cells. The recombinant protein is purified to homogeneity using metal chelate affinity and size exclusion chromatography. The small GTPase RhoA also is expressed to high levels in a lipidated form after baculovirus expression in High Five cells and is purified to near homogeneity after detergent extraction and metal chelate affinity chromatography. The capacity of GTPgammaS-bound RhoA to stimulate the phospholipase activity of PLC-epsilon is assessed by reconstitution of the RhoA in mixed-detergent phospholipid micelles containing PtdIns(4,5)P2 substrate.

Animals↗

PubMed2006

Regulation of PLCbeta isoforms by Rac.

Small GTPases function as molecular switches, which transduce cellular signals from upstream regulators to downstream effectors in a guanine nucleotide-dependent manner. Direct binding partners of small GTPases fall into four classes of both regulators and effectors that can be differentiated on the basis of the state of nucleotide required for binding. Here we describe a procedure for the rapid screening and quantitative assessment of direct interactions of the Rho family of small GTPases with effector molecules of the phospholipase Cbeta class of enzymes using surface plasmon resonance technology. The experimental format described is also readily adaptable toward characterizing guanine nucleotide-dependent binding events of both small and heterotrimeric G proteins with various classes of GTPase regulatory proteins.

Animals↗

PubMed2005

A Cdc42 mutant specifically activated by intersectin.

The Rho family GTPase Cdc42 functions as a molecular switch and controls many fundamental cellular processes such as cytoskeletal regulation, cell polarity, and vesicular trafficking. Guanine nucleotide exchange factors of the Dbl family activate Cdc42 and other Rho GTPases by catalyzing the removal of bound GDP, allowing for GTP loading, and subsequent effector recognition ultimately leading to downstream signaling events. Analysis of existing structural data reveals that the Dbl exchange factor intersectin engages a strictly conserved GTPase residue of Cdc42 (tyrosine 32) in a unique mode with respect to all other visualized exchange factor-Rho GTPase interfaces. To investigate this differential binding architecture, we analyzed the role of tyrosine 32 of Cdc42 in binding, and stimulation by Dbl family exchange factors. Deletion of the hydroxyl side chain of tyrosine 32 substantially increases the affinity of Cdc42 for intersectin, yet severely cripples interaction with Dbs, a normally potent exchange factor of Cdc42. Moreover, Cdc42(Y32F) is exclusively activated by intersectin, while virtually unresponsive to other Cdc42-activating exchange factors in vitro and in vivo. Further, the structural determinants unique to intersectin, which permit selective recognition and concomitant stimulation of Cdc42(Y32F), have been defined. Cdc42 and other individual Rho GTPases receive input stimulatory signals from a multitude of Dbl exchange factors, and therefore, Cdc42(Y32F) could act as a valuable reagent for understanding the specific influence of ITSN on Cdc42-mediated signaling phenomena.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

RhoA activates purified phospholipase C-epsilon by a guanine nucleotide-dependent mechanism.

Phospholipase C-epsilon (PLC-epsilon) is a recently identified PLC isoform activated by subunits of heterotrimeric G proteins (Galpha(12), Galpha(13), and Gbetagamma) as well as by the low molecular weight GTPases, Rho and Ras. To define the enzymatic activity and substrate specificity of PLC-epsilon as well as its potential direct activation by Rho family GTPases, a major fragment of PLC-epsilon encompassing the catalytic core (EF-hand repeats through the tandem Ras-associating domains; approximately 118 kDa) was purified to near homogeneity and assayed after reconstitution under various conditions. Similar to the enzymatic profiles of previously purified PLC-beta isozymes, the purified fragment of PLC-epsilon maximally hydrolyzed phosphatidylinositol 4-phosphate at a rate of approximately 10 mumol/mg of protein/min, exhibited phospholipase activity dependent on the concentration of free calcium, and favored phosphatidylinositol 4,5-bisphosphate as substrate relative to other phosphoinositides. Furthermore, in mixed detergent phospholipid micelles, RhoA stimulated the phospholipase activity of the PLC-epsilon fragment in both a concentration-dependent and guanosine 5'-O-(3-thiotriphosphate)-dependent manner. This activation was abolished by the deletion of a unique approximately 65 amino acid-insert within the catalytic core of PLC-epsilon. Although Rac1 activated purified PLC-beta2ina guanine nucleotide-dependent manner, Rac1 failed to promote guanine nucleotide-dependent activation of purified PLC-epsilon. These results indicate that PLC-epsilon is a direct downstream effector for RhoA and that RhoA-dependent activation of PLC-epsilon depends on a unique insert within the catalytic core of the phospholipase.

Animals↗

PubMed2003

Direct activation of phospholipase C-epsilon by Rho.

Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-epsilon, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.

Amino Acid Sequence↗

PubMed2003

The pleckstrin homology domain of phospholipase C-beta2 as an effector site for Rac.

Increasing evidence links the activation of Rho family GTPases to the stimulation of lipid hydrolysis catalyzed by phospholipase C (PLC)-beta isozymes. To better define this relationship, members of a library of recombinant Rho GTPases were screened for their capacity to directly engage various purified PLC-beta isozymes. Of the 17 tested members of the Rho family, only the active isoforms of Rac (Rac1, Rac2, and Rac3) both stimulate PLC-beta activity in vivo and bind PLC-beta2 and PLC-beta3, but not PLC-beta1, in vitro. Furthermore, the recognition site for Rac GTPases was localized to the pleckstrin homology (PH) domain of PLC-beta2, and this PH domain is fully sufficient to selectively interact with the active versions of the Rac GTPases, but not with other similar Rho GTPases. Together, these findings present a quantitative evaluation of the direct interactions between Rac GTPases and PLC-beta isozymes and define a novel role for the PH domain of PLC-beta2 as a putative effector site for Rac GTPases.

Animals↗

PubMed2003

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 vivo despite 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.

3T3 Cells↗

PubMed2002

Functional analysis of cdc42 residues required for Guanine nucleotide exchange.

Guanine nucleotide exchange factors (GEFs) directly engage small GTPases to facilitate the exchange of bound GDP for GTP, leading to GTPase activation. Several recent crystal structures of GEFs in complex with Rho family GTPases highlight the conserved interactions and conformational alterations necessary for catalyzing exchange. In the present study, functional roles were defined for specific residues within Cdc42 implicated by the crystal structures as important for physiological exchange of guanine nucleotides within Rho GTPases. In particular, this study highlights the paramount importance of the phosphate-binding loop and interactions with the magnesium co-factor as critical for proper regulation of RhoGEF-catalyzed exchange. Other conformational alterations of the GTPases affecting interactions with the sugar and base of guanine nucleotides are also important but are secondary. Of particular note, substitution of alanine for cysteine at position 18 of Cdc42 leads to a fast cycling phenotype for Cdc42 with heightened affinity for RhoGEFs and produces a dominant negative form of Cdc42 capable of inhibiting RhoGEFs both in vitro and in vivo.

Cloning, Molecular↗

PubMed2002

A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange.

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1.Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs.

Amino Acid Sequence↗

PubMed2002

Structural basis for the selective activation of Rho GTPases by Dbl exchange factors.

Activation of Rho-family GTPases involves the removal of bound GDP and the subsequent loading of GTP, all catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl-family. Despite high sequence conservation among Rho GTPases, Dbl proteins possess a wide spectrum of discriminatory potentials for Rho-family members. To rationalize this specificity, we have determined crystal structures of the conserved, catalytic fragments (Dbl and pleckstrin homology domains) of the exchange factors intersectin and Dbs in complex with their cognate GTPases, Cdc42 and RhoA, respectively. Structure-based mutagenesis of intersectin and Dbs reveals the key determinants responsible for promoting exchange activity in Cdc42, Rac1 and RhoA. These findings provide critical insight into the structural features necessary for the proper pairing of Dbl-exchange factors with Rho GTPases and now allow for the detailed manipulation of signaling pathways mediated by these oncoproteins in vivo.

Amino Acid Sequence↗