Protein prenylation: unique fats make their mark on biology.

The modification of eukaryotic proteins by isoprenoid lipids, which is known as prenylation, controls the localization and activity of a range of proteins that have crucial functions in biological regulation. The roles of prenylated proteins in cells are well conserved across species, underscoring the biological and evolutionary importance of this lipid modification pathway. Genetic suppression and pharmacological inhibition of the protein prenylation machinery have provided insights into several cellular processes and into the aetiology of diseases in which prenylation is involved. The functional dependence of prenylation substrates, such as RAS proteins, on this modification and the therapeutic potential of targeting the prenylation process in pathological conditions accentuate the need to fully understand this form of post-translational modification.

GNA13 suppresses proliferation of ER+ breast cancer cells via ERα dependent upregulation of the MYC oncogene.

GNA13 (Gα13) is one of two alpha subunit members of the G12/13 family of heterotrimeric G-proteins which mediate signaling downstream of GPCRs. It is known to be essential for embryonic development and vasculogenesis and has been increasingly shown to be involved in mediating several steps of cancer progression. Recent studies found that Gα13 can function as an oncogene and contributes to progression and metastasis of multiple tumor types, including ovarian, head and neck and prostate cancers. In most cases, Gα12 and Gα13, as closely related α-subunits in the subfamily, have similar cellular roles. However, in recent years their differences in signaling and function have started to emerge. We previously identified that Gα13 drives invasion of Triple Negative Breast Cancer (TNBC) cells in vitro. As a highly heterogenous disease with various well-defined molecular subtypes (ER+ /Her2-, ER+ /Her2+, Her2+, TNBC) and subtype associated outcomes, the function(s) of Gα13 beyond TNBC should be explored. Here, we report the finding that low expression of GNA13 is predictive of poorer survival in breast cancer, which challenges the conventional idea of Gα12/13 being universal oncogenes in solid tumors. Consistently, we found that Gα13 suppresses the proliferation in multiple ER+ breast cancer cell lines (MCF-7, ZR-75-1 and T47D). Loss of GNA13 expression drives cell proliferation, soft-agar colony formation and in vivo tumor formation in an orthotopic xenograft model. To evaluate the mechanism of Gα13 action, we performed RNA-sequencing analysis on these cell lines and found that loss of GNA13 results in the upregulation of MYC signaling pathways in ER+ breast cancer cells. Simultaneous silencing of MYC reversed the proliferative effect from the loss of GNA13, validating the role of MYC in Gα13 regulation of proliferation. Further, we found Gα13 regulates the expression of MYC, at both the transcript and protein level in an ERα dependent manner. Taken together, our study provides the first evidence for a tumor suppressive role for Gα13 in breast cancer cells and demonstrates for the first time the direct involvement of Gα13 in ER-dependent regulation of MYC signaling. With a few exceptions, elevated Gα13 levels are generally considered to be oncogenic, similar to Gα12. This study demonstrates an unexpected tumor suppressive role for Gα13 in ER+ breast cancer via regulation of MYC, suggesting that Gα13 can have subtype-dependent tumor suppressive roles in breast cancer.

GPCR-Gα13 Involvement in Mitochondrial Function, Oxidative Stress, and Prostate Cancer.

Gα13 and Gα12, encoded by the GNA13 and GNA12 genes, respectively, are members of the G12 family of Gα proteins that, along with their associated Gßγ subunits, mediate signaling from specific G protein-coupled receptors (GPCRs). Advanced prostate cancers have increased expression of GPCRs such as CXC Motif Chemokine Receptor 4 (CXCR4), lysophosphatidic acid receptor (LPAR), and protease activated receptor 1 (PAR-1). These GPCRs signal through either the G12 family, or through Gα13 exclusively, often in addition to other G proteins. The effect of Gα13 can be distinct from that of Gα12, and the role of Gα13 in prostate cancer initiation and progression is largely unexplored. The oncogenic effect of Gα13 on cell migration and invasion in prostate cancer has been characterized, but little is known about other biological processes such as mitochondrial function and oxidative stress. Current knowledge on the link between Gα13 and oxidative stress is based on animal studies in which GPCR-Gα13 signaling decreased superoxide levels, and the overexpression of constitutively active Gα13 promoted antioxidant gene activation. In human samples, mitochondrial superoxide dismutase 2 (SOD2) correlates with prostate cancer risk and prognostic Gleason grade. However, overexpression of SOD2 in prostate cancer cells yielded conflicting results on cell growth and survival under basal versus oxidative stress conditions. Hence, it is necessary to explore the effect of Gα13 on prostate cancer tumorigenesis, as well as the effect of Gα13 on SOD2 in prostate cancer cell growth under oxidative stress conditions.

RAB4A GTPase regulates epithelial-to-mesenchymal transition by modulating RAC1 activation.

Epithelial-to-mesenchymal transition (EMT) is a critical underpinning process for cancer progression, recurrence and resistance to drug treatment. Identification of new regulators of EMT could lead to the development of effective therapies to improve the outcome of advanced cancers. In the current study we discovered, using a variety of in vitro and in vivo approaches, that RAB4A function is essential for EMT and related manifestation of stemness and invasive properties. Consistently, RAB4A suppression abolished the cancer cells' self-renewal and tumor forming ability. In terms of downstream signaling, we found that RAB4A regulation of EMT is achieved through its control of activation of the RAC1 GTPase. Introducing activated RAC1 efficiently rescued EMT gene expression, invasion and tumor formation suppressed by RAB4A knockdown in both the in vitro and in vivo cancer models. In summary, this study identifies a RAB4A-RAC1 signaling axis as a key regulatory mechanism for the process of EMT and cancer progression and suggests a potential therapeutic approach to controlling these processes.

Gα-13 induces CXC motif chemokine ligand 5 expression in prostate cancer cells by transactivating NF-κB.

GNA13, the α subunit of a heterotrimeric G protein, mediates signaling through G-protein-coupled receptors (GPCRs). GNA13 is up-regulated in many solid tumors, including prostate cancer, where it contributes to tumor initiation, drug resistance, and metastasis. To better understand how GNA13 contributes to tumorigenesis and tumor progression, we compared the entire transcriptome of PC3 prostate cancer cells with those cells in which GNA13 expression had been silenced. This analysis revealed that GNA13 levels affected multiple CXC-family chemokines. Further investigation in three different prostate cancer cell lines singled out pro-tumorigenic CXC motif chemokine ligand 5 (CXCL5) as a target of GNA13 signaling. Elevation of GNA13 levels consistently induced CXCL5 RNA and protein expression in all three cell lines. Analysis of the CXCL5 promoter revealed that the -505/+62 region was both highly active and influenced by GNA13, and a single NF-κB site within this region of the promoter was critical for GNA13-dependent promoter activity. ChIP experiments revealed that, upon induction of GNA13 expression, occupancy at the CXCL5 promoter was significantly enriched for the p65 component of NF-κB. GNA13 knockdown suppressed both p65 phosphorylation and the activity of a specific NF-κB reporter, and p65 silencing impaired the GNA13-enhanced expression of CXCL5. Finally, blockade of Rho GTPase activity eliminated the impact of GNA13 on NF-κB transcriptional activity and CXCL5 expression. Together, these findings suggest that GNA13 drives CXCL5 expression by transactivating NF-κB in a Rho-dependent manner in prostate cancer cells.

GNA13 loss in germinal center B cells leads to impaired apoptosis and promotes lymphoma in vivo.

GNA13 is the most frequently mutated gene in germinal center (GC)-derived B-cell lymphomas, including nearly a quarter of Burkitt lymphoma and GC-derived diffuse large B-cell lymphoma. These mutations occur in a pattern consistent with loss of function. We have modeled the GNA13-deficient state exclusively in GC B cells by crossing the Gna13 conditional knockout mouse strain with the GC-specific AID-Cre transgenic strain. AID-Cre(+) GNA13-deficient mice demonstrate disordered GC architecture and dark zone/light zone distribution in vivo, and demonstrate altered migration behavior, decreased levels of filamentous actin, and attenuated RhoA activity in vitro. We also found that GNA13-deficient mice have increased numbers of GC B cells that display impaired caspase-mediated cell death and increased frequency of somatic hypermutation in the immunoglobulin VH locus. Lastly, GNA13 deficiency, combined with conditional MYC transgene expression in mouse GC B cells, promotes lymphomagenesis. Thus, GNA13 loss is associated with GC B-cell persistence, in which impaired apoptosis and ongoing somatic hypermutation may lead to an increased risk of lymphoma development.

c-Jun Contributes to Transcriptional Control of GNA12 Expression in Prostate Cancer Cells.

Abstract: GNA12 is the α subunit of a heterotrimeric G protein that possesses oncogenic potential. Activated GNA12 also promotes prostate and breast cancer cell invasion in vitro and in vivo, and its expression is up-regulated in many tumors, particularly metastatic tissues. In this study, we explored the control of expression of GNA12 in prostate cancer cells. Initial studies on LnCAP (low metastatic potential, containing low levels of GNA12) and PC3 (high metastatic potential, containing high GNA12 levels) cells revealed that GNA12 mRNA levels correlated with protein levels, suggesting control at the transcriptional level. To identify potential factors controlling GNA12 transcription, we cloned the upstream 5' regulatory region of the human GNA12 gene and examined its activity using reporter assays. Deletion analysis revealed the highest level of promoter activity in a 784 bp region, and subsequent in silico analysis indicated the presence of transcription factor binding sites for C/EBP (CCAAT/enhancer binding protein), CREB1 (cAMP-response-element-binding protein 1), and c-Jun in this minimal element for transcriptional control. A small interfering RNA (siRNA) knockdown approach revealed that silencing of c-Jun expression significantly reduced GNA12 5' regulatory region reporter activity. In addition, chromatin immunoprecipitation assays confirmed that c-Jun binds to the GNA12 5' regulatory region in PC3 cells. Silencing of c-Jun expression reduced mRNA and protein levels of GNA12, but not the closely-related GNA13, in prostate cancer cells. Understanding the mechanisms by which GNA12 expression is controlled may aid in the development of therapies that target key elements responsible for GNA12-mediated tumor progression.

MicroRNA-31 controls G protein alpha-13 (GNA13) expression and cell invasion in breast cancer cells.

BACKGROUND: Gα13 (GNA13) is the α subunit of a heterotrimeric G protein that mediates signaling through specific G protein-coupled receptors (GPCRs). Our recent study showed that control of GNA13 expression by specific microRNAs (miRNAs or miRs) is important for prostate cancer cell invasion. However, little is known about the control of GNA13 expression in breast cancers. This project was carried out to determine (i) whether enhanced GNA13 expression is important for breast cancer cell invasion, and (ii) if so, the mechanism of deregulation of GNA13 expression in breast cancers. METHODS: To determine the probable miRNAs regulating GNA13, online miRNA target prediction tool Targetscan and Luciferase assays with GNA13-3'-UTR were used. Effect of miRNAs on GNA13 mRNA, protein and invasion was studied using RT-PCR, western blotting and in vitro Boyden chamber assay respectively. Cell proliferation was done using MTT assays. RESULTS: Overexpression of GNA13 in MCF-10a cells induced invasion, whereas knockdown of GNA13 expression in MDA-MB-231 cells inhibited invasion. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-31 exhibited an inverse correlation to GNA13 protein expression in breast cancer cells. Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3'-UTR-reporter activity. Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3'-UTR reporter activity. Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells. Examination of 48 human breast cancer tissues revealed that GNA13 mRNA levels were inversely correlated to miR-31 levels. CONCLUSIONS: These data provide strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms involving miR-31. Additionally our data shows that miR-31 regulates breast cancer cell invasion partially via targeting GNA13 expression in breast cancer cells. Loss of miR-31 expression and increased GNA13 expression could be used as biomarkers of breast cancer progression.

Gαz regulates BDNF-induction of axon growth in cortical neurons.

The disruption of neurotransmitter and neurotrophic factor signaling in the central nervous system (CNS) is implicated as the root cause of neuropsychiatric disorders, including schizophrenia, epilepsy, chronic pain, and depression. Therefore, identifying the underlying molecular mechanisms by which neurotransmitter and neurotrophic factor signaling regulates neuronal survival or growth may facilitate identification of more effective therapies for these disorders. Previously, our lab found that the heterotrimeric G protein, Gz, mediates crosstalk between G protein-coupled receptors and neurotrophin signaling in the neural cell line PC12. These data, combined with Gαz expression profiles--predominantly in neuronal cells with higher expression levels corresponding to developmental times of target tissue innervation--suggested that Gαz may play an important role in neurotrophin signaling and neuronal development. Here, we provide evidence in cortical neurons, both manipulated ex vivo and those cultured from Gz knockout mice, that Gαz is localized to axonal growth cones and plays a significant role in the development of axons of cortical neurons in the CNS. Our findings indicate that Gαz inhibits BDNF-stimulated axon growth in cortical neurons, establishing an endogenous role for Gαz in regulating neurotrophin signaling in the CNS.

Control of RhoA methylation by carboxylesterase I.

A number of proteins that play key roles in cell signaling are post-translationally modified by the prenylation pathway. The final step in this pathway is methylation of the carboxyl terminus of the prenylated protein by isoprenylcysteine carboxylmethyltransferase. Due to the impact of methylation on Rho function, we sought to determine if the process was reversible and hence could control Rho function in a dynamic fashion. Elevating isoprenylcysteine carboxylmethyltransferase activity in cells has profound effects on MDA-MB-231 cell morphology, implying the presence of a pool of unmethylated prenyl proteins in these cells under normal conditions. Using a knockdown approach, we identified a specific esterase, carboxylesterase 1, whose function had a clear impact not only on the methylation status of RhoA but also RhoA activation and cell morphology. These data provide compelling evidence that C-terminal modification of prenyl proteins, rather than being purely a constitutive process, can serve as a point of regulation of function for this important class of protein.

MicroRNA-182 and microRNA-200a control G-protein subunit α-13 (GNA13) expression and cell invasion synergistically in prostate cancer cells.

G protein-coupled receptors (GPCRs) and their ligands have been implicated in progression and metastasis of several cancers. GPCRs signal through heterotrimeric G proteins, and among the different types of G proteins, GNA12/13 have been most closely linked to tumor progression. In this study, we explored the role of GNA13 in prostate cancer cell invasion and the mechanism of up-regulation of GNA13 in these cells. An initial screen for GNA13 protein expression showed that GNA13 is highly expressed in the most aggressive cancer cell lines. Knockdown of GNA13 in highly invasive PC3 cells revealed that these cells depend on GNA13 expression for their invasion, migration, and Rho activation. As mRNA levels in these cells did not correlate with protein levels, we assessed the potential involvement of micro-RNAs (miRNAs) in post-transcriptional control of GNA13 expression. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-182 and miR-141/200a showed an inverse correlation to the protein expression in LnCAP and PC3 cells. Ectopic expression of miR-182 and miR-141/200a in PC3 cells significantly reduced protein levels, GNA13-3'-UTR reporter activity and in vitro invasion of these cells. This effect was blocked by restoration of GNA13 expression in these cells. Importantly, inhibition of miR-182 and miR-141/200a in LnCAP cells using specific miRNA inhibitors elevated the expression of GNA13 and enhanced invasion of these cells. These data provide strong evidence that GNA13 is an important mediator of prostate cancer cell invasion, and that miR-182 and miR-200 family members regulate its expression post-transcriptionally.

Post-prenylation-processing enzymes as new targets in oncogenesis.

RAS and many other oncogenic proteins undergo a complex series of post-translational modifications that are initiated by the addition of an isoprenoid lipid through a process known as prenylation. Following prenylation, these proteins usually undergo endoproteolytic processing by the RCE1 protease and then carboxyl methylation by a unique methyltransferase known as isoprenylcysteine carboxyl methyltransferase (ICMT). Although inhibitors that have been designed to target the prenylation step are now in advanced-stage clinical trials, their utility and efficacy seem to be limited. Recent findings, however, indicate that the inhibition of these post-prenylation-processing steps--particularly that of ICMT-catalysed methylation--might provide a better approach to the control of cancer-cell proliferation.

Deletion of GαZ protein protects against diet-induced glucose intolerance via expansion of ß-cell mass.

Insufficient plasma insulin levels caused by deficits in both pancreatic ß-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed ß-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating ß-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric G(z) protein, Gα(z), impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gα(z)-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gα(z) is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gα(z) are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in ß-cell proliferation is correlated with significantly increased ß-cell mass. Further, an endogenous Gα(z) signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gα(z) to both major aspects of ß-cell decompensation: insufficient ß-cell function and mass.

Protein farnesyltransferase in embryogenesis, adult homeostasis, and tumor development.

Protein farnesyltransferase (FTase) is an enzyme responsible for posttranslational modification of proteins carrying a carboxy-terminal CaaX motif. Farnesylation allows substrates to interact with membranes and protein targets. Using gene-targeted mice, we report that FTase is essential for embryonic development, but dispensable for adult homeostasis. Six-month-old FTase-deficient mice display delayed wound healing and maturation defects in erythroid cells. Embryonic fibroblasts lacking FTase have a flat morphology and reduced motility and proliferation rates. Ablation of FTase in two ras oncogene-dependent tumor models has no significant consequences for tumor initiation. However, elimination of FTase during tumor progression had a limited but significant inhibitory effect. These results should help to better understand the role of protein farnesylation in normal tissues and in tumor development.

Positionality and Its Problems: Questioning the Value of Reflexivity Statements in Research.

There has been a remarkable push for the use of positionality statements-also known as reflexivity statements-in scientific-journal articles and other research literatures. Grounded in reputable philosophical traditions, positionality statements are meant to address genuine concerns about the limits of knowledge production. However, there are at least three reasons why they should be avoided in scholarship. First, it is impossible to construct credible positionality statements because they are constrained by the very positionality they seek to address. Second, positionality statements are unnecessary because reducing bias-positional or otherwise-in scientific literatures does not hinge on the biographical details of individual scholars but on the integrity of the collective process of truth-seeking. Third, by asking scholars to disclose information about themselves, positionality statements undermine the very norms and practices that safeguard the impartiality of research. Instead of asking individual scholars to issue subjective declarations about their positionalities, scholarly communities should focus on improving the rules of intersubjective competition at the heart of scientific progress. In our view, the most productive path to increasing representation and reducing positional bias in research is to protect the freedom of scholarly inputs while insisting on methodological transparency and rigor.

Prenylated c17orf37 induces filopodia formation to promote cell migration and metastasis.

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.

A role for Rac3 GTPase in the regulation of autophagy.

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.

The effect of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on in vitro maturation of equine oocytes.

The objective of this study was to test the hypothesis that equine growth hormone (eGH), in combination with insulin growth factor-I (IGF-I), influences positively in vitro nuclear and cytoplasmic maturation of equine oocytes. Cumulus-oocyte complexes were recovered from follicles that were < 25 mm in diameter, characterized by morphology and were allocated randomly as follow: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 400 ng/ml anti-IGF-I antibody. Oocytes were matured for 30 h at 38.5°C in air with 5% CO2 and then stained with 10 µg/ml propidium iodide (PI) to evaluate nuclear status and 10 µg/ml Lens culinaris agglutinin-fluorescein complex (FITC-LCA) to assess cortical granule migration by confocal microscopy. The proportion of immature oocytes that developed to the metaphase II (MII) stage in the eGH + IGF-I group (15 of 45) was greater than in the groups that were treated only with IGF-I (7 of 36, p = 0.03). Oocytes that reached MII in the control group (20 of 56; 35.7%) showed a tendency to be different when compared with eGH + IGF-I group (15 of 45; 33.3%, p = 0.08). The treated group that contained anti-IGF-I (15 of 33; 45.4%) decreased the number of oocytes reaching any stage of development when compared with eGH (47 of 72; 65.3%) and eGH + IGF-I (33 of 45; 73.3%) groups (p = 0.05) when data from MI and MII were combined. We concluded that the addition of eGH to in vitro maturation (IVM) medium influenced the in vitro nuclear and cytoplasmic maturation of equine oocytes. The use of GH and IGF-I in vitro may represent a potential alternative for IVM of equine oocytes.

The emerging roles of Gα12/13 proteins on the hallmarks of cancer in solid tumors.

G12 proteins comprise a subfamily of G-alpha subunits of heterotrimeric GTP-binding proteins (G proteins) that link specific cell surface G protein-coupled receptors (GPCRs) to downstream signaling molecules and play important roles in human physiology. The G12 subfamily contains two family members: Gα12 and Gα13 (encoded by the GNA12 and GNA13 genes, respectively) and, as with all G proteins, their activity is regulated by their ability to bind to guanine nucleotides. Increased expression of both Gα12 and Gα13, and their enhanced signaling, has been associated with tumorigenesis and tumor progression of multiple cancer types over the past decade. Despite these strong associations, Gα12/13 proteins are underappreciated in the field of cancer. As our understanding of G protein involvement in oncogenic signaling has evolved, it has become clear that Gα12/13 signaling is pleotropic and activates specific downstream effectors in different tumor types. Further, the expression of Gα12/13 proteins is regulated through a series of transcriptional and post-transcriptional mechanisms, several of which are frequently deregulated in cancer. With the ever-increasing understanding of tumorigenic processes driven by Gα12/13 proteins, it is becoming clear that targeting Gα12/13 signaling in a context-specific manner could provide a new strategy to improve therapeutic outcomes in a number of solid tumors. In this review, we detail how Gα12/13 proteins, which were first discovered as proto-oncogenes, are now known to drive several "classical" hallmarks, and also play important roles in the "emerging" hallmarks, of cancer.

Site-specific analysis of protein S-acylation by resin-assisted capture.

Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells.