Novel regulatory roles of small G protein GDP dissociation stimulator (smgGDS) in insulin secretion from pancreatic ß-cells.

Emerging evidence implicates novel roles for small G protein GDP dissociation stimulator (smgGDS) in G protein activation and subsequent targeting to relevant subcellular compartments for effector regulation. Given the well-established roles of small G proteins in insulin secretion, we undertook this investigation to determine the putative roles of smgGDS in insulin secretion. Immunoblotting studies revealed that both splice variants of smgGDS are expressed in human islets, rat islets and INS-1 832/13 cells. A significant inhibition (-52%) of glucose-stimulated insulin secretion (GSIS) was observed in INS-1 832/13 cells following siRNA-mediated depletion of smgGDS. In addition, insulin secretion elicited by a membrane depolarizing concentration of KCl (via increased calcium influx), forskolin (via increased cAMP generation) or IBMX (via inhibition of phosphodiesterase) was inhibited by -49%, -27%, and -28%, respectively. Subcellular distribution studies revealed no significant alterations in the abundance of smgGDS in the cytosolic and membrane fractions during the 45-min exposure of INS-1 832/13 cells to an insulinotropic concentration of glucose. Together, we present the first evidence of expression of smgGDS in human islets, rodent islets, and clonal ß-cells. We also demonstrate novel regulatory roles of these proteins in insulin secretion derived from glucose metabolic events, including calcium- and cAMP-dependent signaling steps.

Synthesis, Enzymatic Peptide Incorporation, and Applications of Diazirine-Containing Isoprenoid Diphosphate Analogues.

Prenylated proteins contain C15 or C20 isoprenoids linked to cysteine residues positioned near their C-termini. Here we describe the preparation of isoprenoid diphosphate analogues incorporating diazirine groups that can be used to probe interactions between prenylated proteins and other proteins that interact with them. Studies using synthetic peptides and whole proteins demonstrate that these diazirine analogues are efficient substrates for prenyltransferases. Photo-cross-linking experiments using peptides incorporating the diazirine-functionalized isoprenoids selectively cross-link to several different proteins. These new isoprenoid analogues should be broadly useful in the studies of protein prenylation.

GTPase splice variants RAC1 and RAC1B display isoform-specific differences in localization, prenylation, and interaction with the chaperone protein SmgGDS.

Identifying events that regulate the prenylation and localization of small GTPases will help define new strategies for therapeutic targeting of these proteins in disorders such as cancer, cardiovascular disease, and neurological deficits. Splice variants of the chaperone protein SmgGDS (encoded by RAP1GDS1) are known to regulate prenylation and trafficking of small GTPases. The SmgGDS-607 splice variant regulates prenylation by binding preprenylated small GTPases but the effects of SmgGDS binding to the small GTPase RAC1 versus the splice variant RAC1B are not well defined. Here we report unexpected differences in the prenylation and localization of RAC1 and RAC1B and their binding to SmgGDS. Compared to RAC1, RAC1B more stably associates with SmgGDS-607, is less prenylated, and accumulates more in the nucleus. We show that the small GTPase DIRAS1 inhibits binding of RAC1 and RAC1B to SmgGDS and reduces their prenylation. These results suggest that prenylation of RAC1 and RAC1B is facilitated by binding to SmgGDS-607 but the greater retention of RAC1B by SmgGDS-607 slows RAC1B prenylation. We show that inhibiting RAC1 prenylation by mutating the CAAX motif promotes RAC1 nuclear accumulation, suggesting that differences in prenylation contribute to the different nuclear localization of RAC1 versus RAC1B. Finally, we demonstrate RAC1 and RAC1B that cannot be prenylated bind GTP in cells, indicating that prenylation is not a prerequisite for activation. We report differential expression of RAC1 and RAC1B transcripts in tissues, consistent with these two splice variants having unique functions that might arise in part from their differences in prenylation and localization.

The complex, dynamic SpliceOme of the small GTPase transcripts altered by technique, sex, genetics, tissue specificity, and RNA base editing.

The small GTPase family is well-studied in cancer and cellular physiology. With 162 annotated human genes, the family has a broad expression throughout cells of the body. Members of the family have multiple exons that require splicing. Yet, the role of splicing within the family has been underexplored. We have studied the splicing dynamics of small GTPases throughout 41,671 samples by integrating Nanopore and Illumina sequencing techniques. Within this work, we have made several discoveries. 1). Using the GTEx long read data of 92 samples, each small GTPase gene averages two transcripts, with 83 genes (51%) expressing two or more isoforms. 2). Cross-tissue analysis of GTEx from 17,382 samples shows 41 genes (25%) expressing two or more protein-coding isoforms. These include protein-changing transcripts in genes such as RHOA, RAB37, RAB40C, RAB4B, RAB5C, RHOC, RAB1A, RAN, RHEB, RAC1, and KRAS. 3). The isolation and library technique of the RNAseq influences the abundance of non-sense-mediated decay and retained intron transcripts of small GTPases, which are observed more often in genes than appreciated. 4). Analysis of 16,243 samples of "Blood PAXgene" identified seven genes (3.7%; RHOA, RAB40C, RAB4B, RAB37, RAB5B, RAB5C, RHOC) with two or more transcripts expressed as the major isoform (75% of the total gene), suggesting a role of genetics in altering splicing. 5). Rare (ARL6, RAB23, ARL13B, HRAS, NRAS) and common variants (GEM, RHOC, MRAS, RAB5B, RERG, ARL16) can influence splicing and have an impact on phenotypes and diseases. 6). Multiple genes (RAB9A, RAP2C, ARL4A, RAB3A, RAB26, RAB3C, RASL10A, RAB40B, and HRAS) have sex differences in transcript expression. 7). Several exons are included or excluded for small GTPase genes (RASEF, KRAS, RAC1, RHEB, ARL4A, RHOA, RAB30, RHOBTB1, ARL16, RAP1A) in one or more forms of cancer. 8). Ten transcripts are altered in hypoxia (SAR1B, IFT27, ARL14, RAB11A, RAB10, RAB38, RAN, RIT1, RAB9A) with RHOA identified to have a transient 3'UTR RNA base editing at a conserved site found in all of its transcripts. Overall, we show a remarkable and dynamic role of splicing within the small GTPase family that requires future explorations.

Structural and biophysical properties of farnesylated KRas interacting with the chaperone SmgGDS-558.

KRas is a small GTPase and membrane-bound signaling protein. Newly synthesized KRas is post-translationally modified with a membrane-anchoring prenyl group. KRas chaperones are therapeutic targets in cancer due to their participation in trafficking oncogenic KRas to membranes. SmgGDS splice variants are chaperones for small GTPases with basic residues in their hypervariable domain (HVR), including KRas. SmgGDS-607 escorts pre-prenylated small GTPases, while SmgGDS-558 escorts prenylated small GTPases. We provide a structural description of farnesylated and fully processed KRas (KRas-FMe) in complex with SmgGDS-558 and define biophysical properties of this interaction. Surface plasmon resonance measurements on biomimetic model membranes quantified the thermodynamics of the interaction of SmgGDS with KRas, and small-angle x-ray scattering was used to characterize complexes of SmgGDS-558 and KRas-FMe structurally. Structural models were refined using Monte Carlo and molecular dynamics simulations. Our results indicate that SmgGDS-558 interacts with the HVR and the farnesylated C-terminus of KRas-FMe, but not its G-domain. Therefore, SmgGDS-558 interacts differently with prenylated KRas than prenylated RhoA, whose G-domain was found in close contact with SmgGDS-558 in a recent crystal structure. Using immunoprecipitation assays, we show that SmgGDS-558 binds the GTP-bound, GDP-bound, and nucleotide-free forms of farnesylated and fully processed KRas in cells, consistent with SmgGDS-558 not engaging the G-domain of KRas. We found that the dissociation constant, Kd, for KRas-FMe binding to SmgGDS-558 is comparable with that for the KRas complex with PDEδ, a well-characterized KRas chaperone that also does not interact with the KRas G-domain. These results suggest that KRas interacts in similar ways with the two chaperones SmgGDS-558 and PDEδ. Therapeutic targeting of the SmgGDS-558/KRas complex might prove as useful as targeting the PDEδ/KRas complex in KRas-driven cancers.

Rap1A, Rap1B, and ß-Adrenergic Signaling in Autologous HCT: A Randomized Controlled Trial of Propranolol.

Successful hematopoietic cell transplantation (HCT) depends on rapid engraftment of the progenitor and stem cells that will reestablish hematopoiesis. Rap1A and Rap1B are two closely related small GTPases that may affect platelet and neutrophil engraftment during HCT through their roles in cell adhesion and migration. ß-adrenergic signaling may regulate the participation of Rap1A and Rap1B in engraftment through their inhibition or activation. We conducted a correlative study of a randomized controlled trial evaluating the effects of the nonselective ß-antagonist propranolol on expression and prenylation of Rap1A and Rap1B during neutrophil and platelet engraftment in 25 individuals receiving an autologous HCT for multiple myeloma. Propranolol was administered for 1 week prior to and 4 weeks following HCT. Blood was collected 7 days (baseline) and 2 days (Day -2) before HCT, and 28 days after HCT (Day +28). Circulating polymorphonuclear cells (PMNC) were isolated and analyzed via immunoblotting to determine levels of prenylated and total Rap1A versus Rap1B. Twelve participants were randomized to the intervention and 13 to the control. Rap1A expression significantly correlated with Rap1B expression. Rap1B expression significantly correlated with slower platelet engraftment; however, this association was not observed in the propranolol-treated group. There were no significant associations between neutrophil engraftment and Rap1A or Rap1B expression. Post hoc exploratory analyses did not reveal an association between social health variables and Rap1A or Rap1B expression. This study identifies a greater regulatory role for Rap1B than Rap1A in platelet engraftment and suggests a possible role for ß-adrenergic signaling in modulating Rap1B function during HCT.

SmgGDS: An Emerging Master Regulator of Prenylation and Trafficking by Small GTPases in the Ras and Rho Families.

Newly synthesized small GTPases in the Ras and Rho families are prenylated by cytosolic prenyltransferases and then escorted by chaperones to membranes, the nucleus, and other sites where the GTPases participate in a variety of signaling cascades. Understanding how prenylation and trafficking are regulated will help define new therapeutic strategies for cancer and other disorders involving abnormal signaling by these small GTPases. A growing body of evidence indicates that splice variants of SmgGDS (gene name RAP1GDS1) are major regulators of the prenylation, post-prenylation processing, and trafficking of Ras and Rho family members. SmgGDS-607 binds pre-prenylated small GTPases, while SmgGDS-558 binds prenylated small GTPases. This review discusses the history of SmgGDS research and explains our current understanding of how SmgGDS splice variants regulate the prenylation and trafficking of small GTPases. We discuss recent evidence that mutant forms of RabL3 and Rab22a control the release of small GTPases from SmgGDS, and review the inhibitory actions of DiRas1, which competitively blocks the binding of other small GTPases to SmgGDS. We conclude with a discussion of current strategies for therapeutic targeting of SmgGDS in cancer involving splice-switching oligonucleotides and peptide inhibitors.

Splice switching an oncogenic ratio of SmgGDS isoforms as a strategy to diminish malignancy.

The chaperone protein SmgGDS promotes cell-cycle progression and tumorigenesis in human breast and nonsmall cell lung cancer. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, facilitate the activation of oncogenic members of the Ras and Rho families of small GTPases through membrane trafficking via regulation of the prenylation pathway. SmgGDS-607 interacts with newly synthesized preprenylated small GTPases, while SmgGDS-558 interacts with prenylated small GTPases. We determined that cancer cells have a high ratio of SmgGDS-607:SmgGDS-558 (607:558 ratio), and this elevated ratio is associated with reduced survival of breast cancer patients. These discoveries suggest that targeting SmgGDS splicing to lower the 607:558 ratio may be an effective strategy to inhibit the malignant phenotype generated by small GTPases. Here we report the development of a splice-switching oligonucleotide, named SSO Ex5, that lowers the 607:558 ratio by altering exon 5 inclusion in SmgGDS pre-mRNA (messenger RNA). Our results indicate that SSO Ex5 suppresses the prenylation of multiple small GTPases in the Ras, Rho, and Rab families and inhibits ERK activity, resulting in endoplasmic reticulum (ER) stress, the unfolded protein response, and ultimately apoptotic cell death in breast and lung cancer cell lines. Furthermore, intraperitoneal (i.p.) delivery of SSO Ex5 in MMTV-PyMT mice redirects SmgGDS splicing in the mammary gland and slows tumorigenesis in this aggressive model of breast cancer. Taken together, our results suggest that the high 607:558 ratio is required for optimal small GTPase prenylation, and validate this innovative approach of targeting SmgGDS splicing to diminish malignancy in breast and lung cancer.

Differences in the Phosphorylation-Dependent Regulation of Prenylation of Rap1A and Rap1B.

Two isoforms of the small GTPase Rap1, Rap1A and Rap1B, participate in cell adhesion; Rap1A promotes steady state adhesion, while Rap1B regulates dynamic changes in cell adhesion. These events depend on the prenylation of Rap1, which promotes its membrane localization. Here, we identify previously unsuspected differences in the regulation of prenylation of Rap1A versus Rap1B, due in part to their different phosphorylation-dependent interactions with the chaperone protein SmgGDS-607. Previous studies indicate that the activation of Gαs protein-coupled receptors (GPCRs) phosphorylates S-179 and S-180 in the polybasic region (PBR) of Rap1B, which inhibits Rap1B binding to SmgGDS-607 and diminishes Rap1B prenylation and membrane localization. In this study, we investigate how phosphorylation in the PBR of multiple small GTPases, including K-Ras4B, RhoA, Rap1A, and Rap1B, affects their binding to SmgGDS, with emphasis on differences between Rap1A and Rap1B. We identify the amino acids in SmgGDS-607 necessary for binding of Rap1A and Rap1B, and present homology models examining the binding between Rap1A or Rap1B and SmgGDS-607. Unlike Rap1B, phosphorylation in the PBR of Rap1A does not detectably inhibit its prenylation or its binding to SmgGDS-607. Activation of GPCRs suppresses Rap1A prenylation, but unlike this effect on Rap1B, the GPCR-mediated suppression of Rap1A prenylation can occur independently of Rap1A phosphorylation and does not detectably diminish Rap1A membrane localization. These data demonstrate unexpected evolutionarily conserved differences in the ability of GPCRs to regulate the prenylation of Rap1B compared to Rap1A.

Alternative scenarios of bioenergy crop production in an agricultural landscape and implications for bird communities.

Increased demand and government mandates for bioenergy crops in the United States could require a large allocation of agricultural land to bioenergy feedstock production and substantially alter current landscape patterns. Incorporating bioenergy landscape design into land-use decision making could help maximize benefits and minimize trade-offs among alternative land uses. We developed spatially explicit landscape scenarios of increased bioenergy crop production in an 80-km radius agricultural landscape centered on a potential biomass-processing energy facility and evaluated the consequences of each scenario for bird communities. Our scenarios included conversion of existing annual row crops to perennial bioenergy grasslands and conversion of existing grasslands to annual bioenergy row crops. The scenarios explored combinations of four biomass crop types (three potential grassland crops along a gradient of plant diversity and one annual row crop [corn]), three land conversion percentages to bioenergy crops (10%, 20%, or 30% of row crops or grasslands), and three spatial configurations of biomass crop fields (random, clustered near similar field types, or centered on the processing plant), yielding 36 scenarios. For each scenario, we predicted the impact on four bird community metrics: species richness, total bird density, species of greatest conservation need (SGCN) density, and SGCN hotspots (SGCN birds/ha ≥ 2). Bird community metrics consistently increased with conversion of row crops to bioenergy grasslands and consistently decreased with conversion of grasslands to bioenergy row crops. Spatial arrangement of bioenergy fields had strong effects on the bird community and in some cases was more influential than the amount converted to bioenergy crops. Clustering grasslands had a stronger positive influence on the bird community than locating grasslands near the central plant or at random. Expansion of bioenergy grasslands onto marginal agricultural lands will likely benefit grassland bird populations, and bioenergy landscapes could be designed to maximize biodiversity benefits while meeting targets for biomass production.

The Tumor-suppressive Small GTPase DiRas1 Binds the Noncanonical Guanine Nucleotide Exchange Factor SmgGDS and Antagonizes SmgGDS Interactions with Oncogenic Small GTPases.

The small GTPase DiRas1 has tumor-suppressive activities, unlike the oncogenic properties more common to small GTPases such as K-Ras and RhoA. Although DiRas1 has been found to be a tumor suppressor in gliomas and esophageal squamous cell carcinomas, the mechanisms by which it inhibits malignant phenotypes have not been fully determined. In this study, we demonstrate that DiRas1 binds to SmgGDS, a protein that promotes the activation of several oncogenic GTPases. In silico docking studies predict that DiRas1 binds to SmgGDS in a manner similar to other small GTPases. SmgGDS is a guanine nucleotide exchange factor for RhoA, but we report here that SmgGDS does not mediate GDP/GTP exchange on DiRas1. Intriguingly, DiRas1 acts similarly to a dominant-negative small GTPase, binding to SmgGDS and inhibiting SmgGDS binding to other small GTPases, including K-Ras4B, RhoA, and Rap1A. DiRas1 is expressed in normal breast tissue, but its expression is decreased in most breast cancers, similar to its family member DiRas3 (ARHI). DiRas1 inhibits RhoA- and SmgGDS-mediated NF-κB transcriptional activity in HEK293T cells. We also report that DiRas1 suppresses basal NF-κB activation in breast cancer and glioblastoma cell lines. Taken together, our data support a model in which DiRas1 expression inhibits malignant features of cancers in part by nonproductively binding to SmgGDS and inhibiting the binding of other small GTPases to SmgGDS.

ß-Adrenergic receptors suppress Rap1B prenylation and promote the metastatic phenotype in breast cancer cells.

A greater understanding of the molecular basis of breast cancer metastasis will lead to identification of novel therapeutic targets and better treatments. Rap1B is a small GTPase that suppresses the metastasis of breast cancer cells by increasing cell-cell adhesion. In breast cancer, a decrease in Rap1B prenylation and subsequent loss of Rap1B at the plasma membrane decreases cell-cell adhesion and increases cell scattering, which promotes the metastatic phenotype. Protein kinase A (PKA) was recently found to phosphorylate Rap1B and inhibit its prenylation. PKA is activated by G protein-coupled receptors (GPCR) that stimulate Gαs. In this study, we investigated whether the general Gαs activator, cholera toxin, and agonists of the ß-adrenergic receptor (ßAR), which is a Gαs-coupled GPCR, promote Rap1B phosphorylation and inhibit its prenylation. We show here that cholera toxin and ßAR activation phosphorylate Rap1B and inhibit its prenylation and membrane localization, reducing cell-cell adhesion and promoting cell scattering. Furthermore, we report that breast cancer cell migration is decreased by the FDA-approved ß-blocker, propranolol. Pharmacological targeting of GPCRs, especially those such as the ßAR that are regulated by FDA-approved drugs, to increase cell adhesion and decrease cell scattering could provide a promising therapeutic approach to reduce breast cancer metastasis.

Cyclic AMP regulates the migration and invasion potential of human pancreatic cancer cells.

Aggressive dissemination and metastasis of pancreatic ductal adenocarcinoma (PDAC) results in poor prognosis and marked lethality. Rho monomeric G protein levels are increased in pancreatic cancer tissue. As the mechanisms underlying PDAC malignancy are little understood, we investigated the role for cAMP in regulating monomeric G protein regulated invasion and migration of pancreatic cancer cells. Treatment of PDAC cells with cAMP elevating agents that activate adenylyl cyclases, forskolin, protein kinase A (PKA), 6-Bnz-cAMP, or the cyclic nucleotide phosphodiesterase inhibitor cilostamide significantly decreased migration and Matrigel invasion of PDAC cell lines. Inhibition was dose-dependent and not significantly different between forskolin or cilostamide treatment. cAMP elevating drugs not only blocked basal migration, but similarly abrogated transforming-growth factor-ß-directed PDAC cell migration and invasion. The inhibitory effects of cAMP were prevented by the pharmacological blockade of PKA. Drugs that increase cellular cAMP levels decreased levels of active RhoA or RhoC, with a concomitant increase in phosphorylated RhoA. Diminished Rho signaling was correlated with the appearance of thickened cortical actin bands along the perimeter of non-motile forskolin or cilostamide-treated cells. Decreased migration did not reflect alterations in cell growth or programmed cell death. Collectively these data support the notion that increased levels of cAMP specifically hinder PDAC cell motility through F-actin remodeling.

Bird communities and biomass yields in potential bioenergy grasslands.

Demand for bioenergy is increasing, but the ecological consequences of bioenergy crop production on working lands remain unresolved. Corn is currently a dominant bioenergy crop, but perennial grasslands could produce renewable bioenergy resources and enhance biodiversity. Grassland bird populations have declined in recent decades and may particularly benefit from perennial grasslands grown for bioenergy. We asked how breeding bird community assemblages, vegetation characteristics, and biomass yields varied among three types of potential bioenergy grassland fields (grass monocultures, grass-dominated fields, and forb-dominated fields), and assessed tradeoffs between grassland biomass production and bird habitat. We also compared the bird communities in grassland fields to nearby cornfields. Cornfields had few birds compared to perennial grassland fields. Ten bird Species of Greatest Conservation Need (SGCN) were observed in perennial grassland fields. Bird species richness and total bird density increased with forb cover and were greater in forb-dominated fields than grass monocultures. SGCN density declined with increasing vertical vegetation density, indicating that tall, dense grassland fields managed for maximum biomass yield would be of lesser value to imperiled grassland bird species. The proportion of grassland habitat within 1 km of study sites was positively associated with bird species richness and the density of total birds and SGCNs, suggesting that grassland bioenergy fields may be more beneficial for grassland birds if they are established near other grassland parcels. Predicted total bird density peaked below maximum biomass yields and predicted SGCN density was negatively related to biomass yields. Our results indicate that perennial grassland fields could produce bioenergy feedstocks while providing bird habitat. Bioenergy grasslands promote agricultural multifunctionality and conservation of biodiversity in working landscapes.

Membrane phospholipid redistribution in cancer micro-particles and implications in the recruitment of cationic protein factors.

Cancer cell-derived micro-particles (MPs) play important regulatory roles on cellular and system levels. These activities are attributed in part to protein factors carried by MPs. However, recruitment strategies for sequestering certain protein factors in MPs are poorly understood. In the current study, using exogenous and endogenously expressed phospholipid-binding probes, we investigated the distribution of membrane phospholipids in MPs as a potential mechanism for electrostatically enriching cationic protein factors in MPs. We detected a significant level of externalised phosphatidylethanolamine (PE) at the outer surface of MPs. This was accompanied, in the inner leaflet of the MP membrane, by a greater density of negatively charged phospholipids, particularly phosphatidylserine (PS). The local enrichment of PS in the inner surface of MPs was correlated with an elevated presence of small GTPases in a polybasic region (PBR)-dependent fashion. By employing a series of RhoA derivatives, including constitutively active and RhoA derivatives lacking a PBR, we could demonstrate that the congregation of RhoA in MPs was dependent on the presence of the PBR. A chimer with the fusion of PBR sequence alone to GFP significantly enhanced GFP localisation in MPs, indicative of a positive contribution of electrostatic interactions in RhoA recruitment to MPs. Using in silico thermodynamic simulations, we characterised the electrostatic interactions between PBR and anionic lipid membrane surface. In summary, the redistribution of membrane phospholipids in MPs has an impact on the local ionic density, and is likely a contributing factor in the electrostatic recruitment of membrane-associated proteins to MPs in a PBR-dependent fashion.

SmgGDS-558 regulates the cell cycle in pancreatic, non-small cell lung, and breast cancers.

Oncogenic mutation or misregulation of small GTPases in the Ras and Rho families can promote unregulated cell cycle progression in cancer. Post-translational modification by prenylation of these GTPases allows them to signal at the cell membrane. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, promote the prenylation and membrane trafficking of multiple Ras and Rho family members, which makes SmgGDS a potentially important regulator of the cell cycle. Surprisingly little is known about how SmgGDS-607 and SmgGDS-558 affect cell cycle-regulatory proteins in cancer, even though SmgGDS is overexpressed in multiple types of cancer. To examine the roles of SmgGDS splice variants in the cell cycle, we compared the effects of the RNAi-mediated depletion of SmgGDS-558 vs. SmgGDS-607 on cell cycle progression and the expression of cyclin D1, p27, and p21 in pancreatic, lung, and breast cancer cell lines. We show for the first time that SmgGDS promotes proliferation of pancreatic cancer cells, and we demonstrate that SmgGDS-558 plays a greater role than SmgGDS-607 in cell cycle progression as well as promoting cyclin D1 and suppressing p27 expression in multiple types of cancer. Silencing both splice variants of SmgGDS in the cancer cell lines produces an alternative signaling profile compared with silencing SmgGDS-558 alone. We also show that loss of both SmgGDS-607 and SmgGDS-558 simultaneously decreases tumorigenesis of NCI-H1703 non-small cell lung carcinoma (NSCLC) xenografts in mice. These findings indicate that SmgGDS promotes cell cycle progression in multiple types of cancer, making SmgGDS a valuable target for cancer therapeutics.

The chaperone protein SmgGDS interacts with small GTPases entering the prenylation pathway by recognizing the last amino acid in the CAAX motif.

Ras family small GTPases localize at the plasma membrane, where they can activate oncogenic signaling pathways. Understanding the mechanisms that promote membrane localization of GTPases will aid development of new therapies to inhibit oncogenic signaling. We previously reported that SmgGDS splice variants promote prenylation and trafficking of GTPases containing a C-terminal polybasic region and demonstrated that SmgGDS-607 interacts with nonprenylated GTPases, whereas SmgGDS-558 interacts with prenylated GTPases in cells. The mechanism that SmgGDS-607 and SmgGDS-558 use to differentiate between prenylated and nonprenylated GTPases has not been characterized. Here, we provide evidence that SmgGDS-607 associates with GTPases through recognition of the last amino acid in the CAAX motif. We show that SmgGDS-607 forms more stable complexes in cells with nonprenylated GTPases that will become geranylgeranylated than with nonprenylated GTPases that will become farnesylated. These binding relationships similarly occur with nonprenylated SAAX mutants. Intriguingly, farnesyltransferase inhibitors increase the binding of WT K-Ras to SmgGDS-607, indicating that the pharmacological shunting of K-Ras into the geranylgeranylation pathway promotes K-Ras association with SmgGDS-607. Using recombinant proteins and prenylated peptides corresponding to the C-terminal sequences of K-Ras and Rap1B, we found that both SmgGDS-607 and SmgGDS-558 directly bind the GTPase C-terminal region, but the specificity of the SmgGDS splice variants for prenylated versus nonprenylated GTPases is diminished in vitro. Finally, we present structural homology models and data from functional prediction software to define both similar and unique features of SmgGDS-607 when compared with SmgGDS-558.

The SmgGDS splice variant SmgGDS-558 is a key promoter of tumor growth and RhoA signaling in breast cancer.

UNLABELLED: Breast cancer malignancy is promoted by the small GTPases RhoA and RhoC. SmgGDS is a guanine nucleotide exchange factor that activates RhoA and RhoC in vitro. We previously reported that two splice variants of SmgGDS, SmgGDS-607, and SmgGDS-558, have different characteristics in binding and transport of small GTPases. To define the role of SmgGDS in breast cancer, we tested the expression of SmgGDS in breast tumors, and the role of each splice variant in proliferation, tumor growth, Rho activation, and NF-κB transcriptional activity in breast cancer cells. We show upregulated SmgGDS protein expression in breast cancer samples compared with normal breast tissue. In addition, Kaplan-Meier survival curves indicated that patients with high SmgGDS expression in their tumors had worse clinical outcomes. Knockdown of SmgGDS-558, but not SmgGDS-607, in breast cancer cells decreased proliferation, in vivo tumor growth, and RhoA activity. Furthermore, we found that SmgGDS promoted a Rho-dependent activation of the transcription factor NF-κB, which provides a potential mechanism to define how SmgGDS-mediated activation of RhoA promotes breast cancer. This study demonstrates that elevated SmgGDS expression in breast tumors correlates with poor survival, and that SmgGDS-558 plays a functional role in breast cancer malignancy. Taken together, these findings define SmgGDS-558 as a unique promoter of RhoA and NF-κB activity and a novel therapeutic target in breast cancer. IMPLICATIONS: This study defines a new mechanism to regulate the activities of RhoA and NF-κB in breast cancer cells, and identifies SmgGDS-558 as a novel promoter of breast cancer malignancy.