Small GTPases最新文献

The small GTPase Arf4-based ciliary membrane-targeting complex recognizes specific targeting signals within sensory receptors and regulates their directed movement to primary cilia. Activated Arf4 directly binds the VxPx ciliary-targeting signal (CTS) of the light-sensing receptor rhodopsin. Recent findings revealed that at the trans-Golgi, marked by the small GTPase Rab6, activated Arf4 forms a functional complex with rhodopsin and the Arf guanine nucleotide exchange factor (GEF) GBF1, providing positive feedback that drives further Arf4 activation in ciliary trafficking. Arf4 function is conserved across diverse cell types; however, it appears that not all its aspects are conserved across species, as mouse Arf4 is a natural mutant in the conserved α3 helix, which is essential for its interaction with rhodopsin. Generally, activated Arf4 regulates the assembly of the targeting nexus containing the Arf GAP ASAP1 and the Rab11a-FIP3-Rabin8 dual effector complex, which controls the assembly of the highly conserved Rab11a-Rabin8-Rab8 ciliary-targeting module. It was recently found that this module interacts with the R-SNARE VAMP7, likely in its activated, c-Src-phosphorylated form. Rab11 and Rab8 bind VAMP7 regulatory longin domain (LD), whereas Rabin8 interacts with the SNARE domain, capturing VAMP7 for delivery to the ciliary base and subsequent pairing with the cognate SNAREs syntaxin 3 and SNAP-25. This review will focus on the implications of these novel findings that further illuminate the role of well-ordered Arf and Rab interaction networks in targeting of sensory receptors to primary cilia. Abbreviations: CTS: Ciliary-Targeting Signal; GAP: GTPase Activating Protein; GEF: Guanine Nucleotide Exchange Factor; RTC(s), Rhodopsin Transport Carrier(s); SNARE: Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor; TGN: Trans-Golgi Network.

During development of the brain, neuronal circuits are formed through the projection of axons and dendrites in response to guidance signals. Rho GTPases (Rac1/RhoA/Cdc42) are major regulators of axo-dendritic outgrowth and guidance due to their role in controlling actin cytoskeletal dynamics, cell adhesion and motility. Functional redundancy of Rho GTPase-regulated pathways in neuronal development can mask the roles of specific GTPases. To examine potential Rho GTPase redundancy, we utilized a recently isolated hypomorphic mutation in a Caenorhabditis elegans Rac1 protein - CED-10(G30E) - which reduces the GTP binding and inhibits axon outgrowth of the PVQ interneurons. Here, we show that the CDC-42-specific guanine nucleotide exchange factor UIG-1 acts in parallel to CED-10/Rac1 to control PVQ axon outgrowth. UIG-1 performs this function in a cell-autonomous manner. Further, we found that transgenic expression of CDC-42 can compensate for aberrant CED-10(G30E)-regulated signalling during PVQ axon outgrowth. Together, our study reveals a previously unappreciated function for CDC-42 in PVQ axon outgrowth in C. elegans.

Jnks are mitogen activated protein kinases that are best known for regulating transcription and apoptotic signaling. However, they also play important roles in controlling cell motility and invasion by phosphorylating many actin and microtubule regulatory proteins. These mechanisms have important implications for normal cell motility as well as cancer metastasis. Jnks are activated by growth factors and cytokines that stimulate cell motility, and this often requires upstream activation of Rho GTPases. Our recent work indicates that Jnks may also regulate Rho GTPase activation. Specifically, we found that Jnk-dependent phosphorylation of the RhoA guanine nucleotide exchange factor (RhoGEF) Net1A promotes its cytosolic accumulation to drive RhoA activation and actin cytoskeletal reorganization. Net1A is unusual among RhoGEFs in that it is sequestered in the nucleus to prevent aberrant RhoA activation. Importantly, Jnk-stimulated cytosolic localization of Net1A is sufficient to stimulate cell motility and extracellular matrix invasion in non-invasive breast cancer cells. Since Net1A expression is critical for cancer cell motility and invasion in vitro, and breast cancer metastasis in vivo, these data uncover a previously unappreciated regulatory mechanism that may contribute to metastasis in multiple types of cancer.

RAS signaling pathways govern diverse cellular processes, are dynamic, and exhibit marked plasticity. Yet, these features also present a considerable obstacle to their study. Here, we report the use of a recently described RAS rheostat, Chemically Inducible Activator of RAS (CIAR), to study two poorly understood phenomena in RAS biology. First, we show that short-term activation of wild type endogenous RAS can desensitize cells to EGF stimulation. Second, we examine the phenomena of paradoxical activation of RAS/ERK signaling by RAF inhibitors. Specifically, we characterize the effects on RAS/ERK signaling kinetics of four RAF inhibitors, which stabilize distinct ATP-binding site conformations. These results demonstrate the utility of CIAR in conducting quantitative studies of complex features of RAS biology.

The CDC42 small GTPase is a major influence on actin-myosin cytoskeleton organization and dynamics, signalling via effector proteins including the Myotonic dystrophy related CDC42-binding protein kinases (MRCK) α and β. We previously identified Serine 1003 of MRCKα as a site of autophosphorylation, and showed that a phosphorylation-sensitive antibody raised against this site could be used as a surrogate indicator of kinase activity. In this study, a kinase-dead version of MRCKβ was established by mutation of the conserved Lysine 105 to Methionine (K105M), which was then used for mass spectrometry analysis to identify phosphorylation events that occurred in catalytically-competent MRCKβ but not in the kinase-dead form. A total of ten phosphorylations were identified on wild-type MRCKβ, of which the previously undescribed Threonine 1108 (Thr1108) was not found on kinase-dead MRCKβ K105M, consistent with this being due to autophosphorylation. Mutation of Thr1108 to non-phosphorylatable Alanine (T1108A) or phosphomimetic Glutamate (T1108E) did not affect the ability of MRCKβ to phosphorylate recombinant myosin light chain in vitro, or observably alter the subcellular localization of green fluorescent protein (GFP)-tagged MRCKβ expressed in MDA MB 231 human breast cancer cells. Although phosphorylation of Thr1108 did not appear to contribute to MRCKβ function or regulation, the identification of this phosphorylation does make it possible to characterize whether this site could be used as a surrogate biomarker of kinase activity and inhibitor efficacy as we previously demonstrated for Ser 1003 in MRCKα.

Light-induced dimerizing systems, e.g. iLID, are an increasingly utilized optogenetics tool to perturb cellular signaling. The major benefit of this technique is that it allows external spatiotemporal control over protein localization with sub-cellular specificity. However, when it comes to local recruitment of signaling components to the plasmamembrane, this precision in localization is easily lost due to rapid diffusion of the membrane anchor. In this study, we explore different approaches of countering the diffusion of peripheral membrane anchors, to the point where we detect immobilized fractions with iFRAP on a timescale of several minutes. One method involves simultaneous binding of the membrane anchor to a secondary structure, the microtubules. The other strategy utilizes clustering of the anchor into large immobile structures, which can also be interlinked by employing tandem recruitable domains. For both approaches, the anchors are peripheral membrane constructs, which also makes them suitable for in vitro use. Upon combining these slower diffusing anchors with recruitable guanine exchange factors (GEFs), we show that we can elicit much more localized morphological responses from Rac1 and Cdc42 as compared to a regular CAAX-box based membrane anchor in living cells. Thanks to these new slow diffusing anchors, more precisely defined membrane recruitment experiments are now possible.

When B lymphocytes encounter antigen-bearing surfaces, B-cell receptor (BCR) signaling initiates remodeling of the F-actin network and reorientation of the microtubule-organizing center (MTOC) towards the antigen contact site. We have previously shown that the Rap1 GTPase, an evolutionarily conserved regulator of cell polarity, is essential for these processes and that Rap1-regulated actin remodeling is required for MTOC polarization. The role of Rap2 proteins in establishing cell polarity is not well understood. We now show that depleting Rap2c, the only Rap2 isoform expressed in the A20 B-cell line, impairs BCR-induced MTOC reorientation as well as the actin remodeling that supports MTOC polarization. Thus Rap1 and Rap2 proteins may have similar but non-redundant functions in coupling the BCR to MTOC polarization.

We examined sequence conservation and signatures of selection in Rab7 proteins across 11 Paramecium aurelia species, and determined the localization patterns of two P. tetraurelia Rab7 paralogs when expressed as GFP fusions in live cells. We found that, while there is a variable number of Rab7 paralogs per genome, Rab7 genes are highly conserved in sequence and appear to be under strong purifying selection across aurelias. Additionally, and surprisingly based on earlier studies, we found that two P. tetraurelia Rab7 proteins have virtually identical localization patterns. Consistent with this, when we examined the gene family of a highly conserved Rab binding partner across aurelias (Rab-Interacting Lysosomal Protein, or RILP), we found that residues in key binding sites in RILPs were absolutely conserved in 13 of 21 proteins, representing genes from 9 of the 11 species examined. Of note, RILP gene number appears to be even more constrained than Rab7 gene number per genome. Abbreviation: WGD: Whole genome duplication.

The Rho GTPase Cdc42 is highly conserved in structure and function. Mechanical or chemical cues in the microenvironment stimulate the localized activation of Cdc42 to rearrange the actin cytoskeleton and establish cell polarity. A role for Cdc42 in cell polarization was first discovered in the budding yeast Saccharomyces cerevisiae, and subsequently shown to also regulate directional motility in animal cells. Accordingly, in cancer Cdc42 promotes migration, invasion, and spread of tumor cells. Therefore, we targeted Cdc42 as a therapeutic strategy to treat metastatic breast cancer and designed the small molecule MBQ-167 as a potent inhibitor against Cdc42 and the homolog Rac. MBQ-167 inhibited cancer cell proliferation and migration in-vitro, and tumor growth and spread in-vivo in a mouse xenograft model of metastatic breast cancer. Since haploid budding yeast express a single Cdc42 gene, and do not express Rac, we used this well characterized model of polarization to define the contribution of Cdc42 inhibition to the effects of MBQ-167 in eukaryotic cells. Growth, budding pattern, and Cdc42 activity was determined in wildtype yeast or cells expressing a conditional knockdown of Cdc42 in response to vehicle or MBQ-167 treatment. As expected, growth and budding polarity were reduced by knocking-down Cdc42, with a parallel effect observed with MBQ-167. Cdc42 activity assays confirmed that MBQ-167 inhibits Cdc42 activation in yeast, and thus, bud polarity. Hence, we have validated MBQ-167 as a Cdc42 inhibitor in another biological context and present a method to screen Cdc42 inhibitors with potential as anti-metastatic cancer drugs.

Adult central nervous system (CNS) axons do not regenerate after injury because of extrinsic inhibitory factors, and a low intrinsic capacity for axon growth. Developing CNS neurons have a better regenerative ability, but lose this with maturity. This mini-review summarises recent findings which suggest one reason for regenerative failure is the selective distribution of growth machinery away from axons as CNS neurons mature. These studies demonstrate roles for the small GTPases ARF6 and Rab11 as intrinsic regulators of polarised transport and axon regeneration. ARF6 activation prevents the axonal transport of integrins in Rab11 endosomes in mature CNS axons. Decreasing ARF6 activation permits axonal transport, and increases regenerative ability. The findings suggest new targets for promoting axon regeneration after CNS injury.