ABSTRACT
Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.
Subject(s)
Prenylation/drug effects , rac1 GTP-Binding Protein/chemistry , rho GTP-Binding Proteins/chemistry , rho-Specific Guanine Nucleotide Dissociation Inhibitors/chemistry , Amino Acid Sequence/genetics , Guanine Nucleotide Dissociation Inhibitors/chemistry , Guanine Nucleotide Dissociation Inhibitors/pharmacology , Humans , Hydrophobic and Hydrophilic Interactions/drug effects , Kinetics , Static Electricity , rac1 GTP-Binding Protein/genetics , rho GTP-Binding Proteins/genetics , rho-Specific Guanine Nucleotide Dissociation Inhibitors/geneticsABSTRACT
RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation of RAS signalling predominantly through the RAF/MEK/ERK (MAPK) cascade. We report on two germline mutations (p.Gly39dup and p.Val55Met) in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare (2 subjects among 504 individuals analysed) and variable phenotype with features partially overlapping Noonan syndrome, the most common RASopathy. We also identified somatic RRAS mutations (p.Gly39dup and p.Gln87Leu) in 2 of 110 cases of non-syndromic juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in Caenorhabditis elegans enhanced RAS signalling and engendered protruding vulva, a phenotype previously linked to the RASopathy-causing SHOC2(S2G) mutant. Overall, these findings provide evidence of a functional link between RRAS and MAPK signalling and reveal an unpredicted role of enhanced RRAS function in human disease.
Subject(s)
Carcinogenesis/genetics , Mutation/physiology , Phenotype , ras Proteins/genetics , Animals , Caenorhabditis elegans , Cohort Studies , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myelomonocytic, Juvenile/genetics , MAP Kinase Kinase Kinases/metabolism , Noonan Syndrome/genetics , Oncogene Protein v-akt/metabolism , Signal Transduction/genetics , ras Proteins/chemistry , ras Proteins/metabolismABSTRACT
The three deleted in liver cancer genes (DLC1-3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins.
Subject(s)
Catalytic Domain , GTPase-Activating Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/antagonists & inhibitors , p120 GTPase Activating Protein/chemistry , Alanine/chemistry , DNA Mutational Analysis , GTPase-Activating Proteins/chemistry , GTPase-Activating Proteins/genetics , Humans , Metabolic Networks and Pathways , Protein Binding , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , p120 GTPase Activating Protein/geneticsABSTRACT
Activating somatic and germline mutations of closely related RAS genes (H, K, N) have been found in various types of cancer and in patients with developmental disorders, respectively. The involvement of the RAS signalling pathways in developmental disorders has recently emerged as one of the most important drivers in RAS research. In the present study, we investigated the biochemical and cell biological properties of two novel missense KRAS mutations (Y71H and K147E). Both mutations affect residues that are highly conserved within the RAS family. KRAS(Y71H) showed no clear differences to KRAS(wt), except for an increased binding affinity for its major effector, the RAF1 kinase. Consistent with this finding, even though we detected similar levels of active KRAS(Y71H) when compared with wild-type protein, we observed an increased activation of MEK1/2, irrespective of the stimulation conditions. In contrast, KRAS(K147E) exhibited a tremendous increase in nucleotide dissociation generating a self-activating RAS protein that can act independently of upstream signals. As a consequence, levels of active KRAS(K147E) were strongly increased regardless of serum stimulation and similar to the oncogenic KRAS(G12V). In spite of this, KRAS(K147E) downstream signalling did not reach the level triggered by oncogenic KRAS(G12V), especially because KRAS(K147E) was downregulated by RASGAP and moreover exhibited a 2-fold lower affinity for RAF kinase. Here, our findings clearly emphasize that individual RAS mutations, despite being associated with comparable phenotypes of developmental disorders in patients, can cause remarkably diverse biochemical effects with a common outcome, namely a rather moderate gain-of-function.
Subject(s)
Ectodermal Dysplasia/genetics , Ectodermal Dysplasia/metabolism , Failure to Thrive/genetics , Failure to Thrive/metabolism , Heart Defects, Congenital/genetics , Heart Defects, Congenital/metabolism , Mutation , Noonan Syndrome/genetics , Noonan Syndrome/metabolism , ras Proteins/genetics , ras Proteins/metabolism , Cell Line , Facies , Humans , Protein Binding , Protein Conformation , Protein Stability , Signal Transduction , ras Proteins/chemistryABSTRACT
Plexin-B1 regulates various cellular processes interacting directly with several Rho proteins. Molecular details of these interactions are, however, not well understood. In this study, we examined in vitro and in silico the interaction of the Rho binding domain (B1RBD) of human Plexin-B1 with 11 different Rho proteins. We show that B1RBD binds in a GTP-dependent manner to Rac1, Rac2, Rac3, Rnd1, Rnd2, Rnd3, and RhoD, but not to RhoA, Cdc42, RhoG, or Rif. Interestingly, Rnd1 competitively displaces the Rac1 from B1RBD but not vice versa. Structure-function analysis revealed a negatively charged loop region, called B1L(31), which may facilitate a selective B1RBD interaction with Rho proteins.
Subject(s)
Nerve Tissue Proteins/chemistry , Receptors, Cell Surface/chemistry , rho GTP-Binding Proteins/chemistry , Amino Acid Sequence , Binding Sites/genetics , Binding, Competitive , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Sequence Homology, Amino Acid , rac1 GTP-Binding Protein/chemistry , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
In a variety of normal and pathological cell types, Rho-kinases I and II (ROCKI/II) play a pivotal role in the organization of the nonmuscle and smooth muscle cytoskeleton and adhesion plaques as well as in the regulation of transcription factors. Thus, ROCKI/II activity regulates cellular contraction, motility, morphology, polarity, cell division, and gene expression. Emerging evidence suggests that dysregulation of the Rho-ROCK pathways at different stages is linked to cardiovascular, metabolic, and neurodegenerative diseases as well as cancer. This review focuses on the current status of understanding the multiple functions of Rho-ROCK signaling pathways and various modes of regulation of Rho-ROCK activity, thereby orchestrating a concerted functional response.
Subject(s)
rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Cell Proliferation , Down-Regulation/genetics , Down-Regulation/physiology , Gene Expression Regulation, Enzymologic/genetics , Gene Expression Regulation, Enzymologic/physiology , Humans , Nitric Oxide Synthase Type III/antagonists & inhibitors , Nitric Oxide Synthase Type III/metabolism , Phosphorylation/genetics , Phosphorylation/physiology , Protein Stability , Signal Transduction/genetics , Signal Transduction/physiology , rho GTP-Binding Proteins/physiology , rho-Associated Kinases/physiology , rhoA GTP-Binding Protein/physiology , rhoB GTP-Binding Protein/physiology , rhoC GTP-Binding ProteinABSTRACT
Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.
Subject(s)
Guanine Nucleotide Dissociation Inhibitors/chemistry , Guanine Nucleotide Exchange Factors/chemistry , Liposomes/chemistry , Protein Processing, Post-Translational , p21-Activated Kinases/chemistry , rac1 GTP-Binding Protein/chemistry , Animals , Baculoviridae/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Guanine Nucleotide Dissociation Inhibitors/genetics , Guanine Nucleotide Dissociation Inhibitors/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Humans , Liposomes/metabolism , Models, Biological , Protein Prenylation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spodoptera , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
A new psychrophilic marine protease was found from a marine bacterium Flavobacterium YS-80 in the Chinese Yellow Sea. The protease is about 49 kD with an isoelectric point about 4.5. It consists of 480 amino acids and is homologous to a psychrophilic alkaline protease (PAP) from an Antarctic Pseudomonas species. The protein was purified from the natural bacterium fermented and crystallized. Its crystal structure (PDB ID 3U1R) was solved at 2.0 Å by Molecular Replacement using a model based on PAP, and was refined to a crystallographic R(work) of 0.16 and an R(free) of 0.21. The marine protease consists of a two domain structure with an N-terminal domain including residues 37-264 and a C-terminal domain including residues 265-480. Similar to PAP, the N-terminal domain is responsible for proteolysis and the C-terminal is for stability. His186, His190, His196 and Tyr226 are ligands for the Zn(2+) ion in the catalytic center. The enzyme's Tyr226 is closer to the Zn(2+) ion than in PAP and it shows a stronger Zn(2+)-Tyr-OH bond. There are eight calcium ions in the marine protease molecule and they have significantly shorter bond distances to their ligands compared to their counterparts in all three crystal forms of PAP. On the other hand, the loops in the marine protease are more compact than in PAP. This makes the total structure stable and less flexible, resulting in higher thermo stability. These properties are consistent with the respective environments of the proteases. The structural analysis of this new marine protease provides new information for the study of psychrophilic proteases and is helpful for elucidating the structure-environment adaptation of these enzymes.