GTP hydrolases (GTPases) are hydrolase enzymes capable of binding GTP and hydrolyzing GTP to GDP. The GTPase family contains sub families that are responsible for many fundamental cellular processes such as differentiation, proliferation, signal transduction, and migration. The subfamilies include Rho, Rac, and Ras. Antibodies specific to each of these subfamily targets allow for study of cellular processes mentioned previously. Provided below are overviews of some of the key members of these subfamilies.


Arf1-6

Arf1 is one of the ADP ribosylation factor proteins 1-6, which are members of the Ras superfamily of small GTPases. Although structurally similar, the cellular roles of Arf1-6 are different from the other Arf family members; their endogenous roles are not ADP ribosylation, but rather regulation of heterotrimeric G proteins. The Arf proteins can be divided into three classes: Class I – Arf 1-3; Class II – Arf 4,5; Class III – Arf6. Class I and II Arfs are associated with trans-Golgi network (TGN) and are involved in recruiting effector proteins to the Golgi membrane and forming vesicles. Unlike other GTPases, Arf GTPases are modified by myristoylation at the amino-terminus to allow insertion into the membrane. The Class III protein Arf6 is associated with the plasma membrane and is involved in vesicle formation at the plasma membrane, vesicle recycling and remodeling of the actin cytoskeleton.

Immunocytochemistry analysis of ARF6 using MA5-32558
Immunocytochemical analysis of ARF6 in Hela cells using a ARF6 Monoclonal antibody (Product # MA5-32558) as seen in red. The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, permeabilised with 0.25% Triton X100/PBS.

Cdc42

The Cdc42 GTPase belongs to the Rho subfamily and Ras superfamily of GTPases. Cdc42 activation results in the polymerization of actin filaments and filopodia formation. Like other small GTPases, Cdc42 acts as a molecular switch activated by GEFs, which catalyze the exchange of bound GDP for GTP, and inhibited by GAPs, which catalyze the hydrolysis of GTP to GDP. Another level of regulation comes through the binding of Rho GDP dissociation inhibitor (RhoGDI), which retains Rho family GTPases in their inactive GDP-bound state.

Both Cdc42 and Rac1 (described below) are activated through tyrosine receptor kinase signaling leading to SAPK and p38 stress kinase pathway activation. Cdc42 is also activated by the chemoattractant fMLP in neutrophils. Fibronectin activates Cdc42 and Rac1 to induce cell spreading, and stimulation with TNF-alpha and IL-1 results in changes in the actin cytoskeleton. There is significant cross-talk between Cdc42 and Rac1, as they act in overlapping pathways, and in some cases, Cdc42 may act upstream of Rac1 during signal transduction. Some of the main effector proteins of Cdc42 are Pak1, N-WASP and IQGAP. Pak1 is a kinase involved in the activation of JNK in the SAPK stress pathway. N-WASP is an effector that induces filopodia formation, and IQGAP interacts with F-actin filaments. In differentiating neurons, Cdc42 plays an active role in neurite outgrowth. However, the Rho family of GTPases can work agonistically during cell signaling and antagonistically during differentiation.

Western blot analysis of Cdc42 using PA1-092
Western blot analysis of cdc42 was performed by loading 50 µg of various whole cell lysate onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% Milk/TBST for at least 1 hour. Membranes were probed with a rabbit polyclonal antibody recognizing cdc42 (Product # PA1-092) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Membranes were then washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody (Product # 31460) at a dilution of 1:10000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Super Signal West Pico (Product # 34580).

Rac1

The Rac1 GTPase is part of the Rho subfamily and Ras superfamily of GTPases. Activation of Rac1 results in actin polymerization and appears as membrane ruffling at the cellular periphery. Rac1 activation also results in lamelipodia formation. The Rac1 GTPase transduces signals through tyrosine kinases, adhesion molecules or cytokine/chemokine receptors after stimulation with growth factors (EGF, insulin, PDGF, NGF), integrins (fibronectin) or chemoattractants (fMLP). For example, stimulation with EGF results in PI3 kinase activation, resulting in cell growth and reorganization at the cell periphery (membrane ruffling).

Alternatively, tyrosine receptor kinase signaling through Rac1 leads to activation of the MAPK stress response pathways SAPK (JNK) and p38. Stimulation of cells with fibronectin results in integrin-mediated cell spreading. Two of the main effector proteins of Rac1 are Pak1 and phosphoinositol 4-phosphate 5-kinase. Pak1 (p65 Pak) is a kinase that activates the JNK pathway, while phosphoinositol 4-phosphate 5-kinase promotes actin filament assembly. Rac is critical for T-cell development and for promoting differentiating cells. However, the Rho family of GTPases can work agonistically during cell signaling and and antagonistically during differentiation.

Immunohistochemistry analysis of RAC1 using PA1-091
Immunohistochemistry was performed on human breast tissue. To expose target protein, antigen was retreived using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Rac-1 Rabbit polyclonal antibody (Product # PA1-091) at a dilution of 1:200 overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

Rap1

The Rap GTPases are part of the Ras superfamily of GTPases and are encoded by Rap1a, Rap1b and Rap2. Rap GTPases are structurally similar to Ras GTPases and have similar effector and activator proteins, although Rap GTPases have different functional activities than Ras. While Ras is involved in cell proliferation and survival, Rap1 regulates cytoskeletal rearrangements, cell adhesion and cell junction formation.

The specificity of Rap1 and Ras is mediated by their respective upstream regulators and downstream effectors. The GEFs for Rap contain a CDC25 homology domain that mediates the GDP/GTP exchange reaction and a REM (Ras exchange motif) domain. Some Rap GEFs include C3G, Epac1 and 2, RasGRP2, PDZ-GEF1 and 2 and PLC epsilon. The binding domain used in the active Rap1 Pull-Down and Detection kit is RalGDS, a GEF that contains an RA domain to which Rap1 has a higher binding affinity than Ras. The RapGAPs, including Rap1GAP and the Spa-1 family, insert an asparagine side chain into the nucleotide-binding pocket to catalyze the GTP hydrolysis reaction. Rap effector proteins, including RAPL, Riam, AF-6, Krit1, RacGEFs, Tiam1, Vav2, Rho GAPs and ARAP3, are involved in cell-cell junctions and adhesion and are often localized to the membrane or at cell-cell junctions. Besides Ras, there is also cross-talk between Rap and Rho GTPases.

Flow cytometry analysis of Rap1 using MA5-17164
Flow cytometric analysis of A431 cells using RAP1A monoclonal antibody (Product # MA5-17164) (green) and negative control (red).

Ras protein family

The Ras subfamily of GTPases is part of the Ras superfamily of GTPases, named after the rat sarcoma viral oncogene. The Ras subfamily contains many Ras isoforms, including K-Ras, H-Ras, and N-Ras. While the three isoforms are expressed at different levels in different types of cells, in general, activating mutations of at least one of these isoforms are present in approximately 15 percent of all cancers. The Ras proteins serve as initiators of intracellular signal transduction from extracellular molecules and associate with the plasma membrane via lipid modification and prenylation of its carboxy terminus. These modifications at the carboxy terminus determine the localization of Ras to distinct membrane microdomains, which dictates subsequent downstream signaling. Ras signaling pathways affect many cellular processes including proliferation, survival, vesicular trafficking and gene expression.

Signaling through Ras is central to many cellular responses, and activation of Ras is regulated by several GEF and GAP proteins. The GEF proteins mediate GTPase activation by dissociating GDP from the inactive Ras to allow binding of Ras to GTP. Conversely, GAP proteins inactivate GTPases by hydrolyzing GTP to GDP. Ras mediates downstream signaling by interacting with effector proteins including Raf and PI3 kinase. Raf1 is a serine/threonine protein kinase that is part of the MAP kinase kinase signaling pathway that leads to the activation of ERK and p38, which influences proliferation and survival. PI3 kinase signaling results in activation of AKT and mTOR, which are central for cell growth and survival. Ras is also integral for cellular differentiation and development, including immune cell development and function. Mutations in the Ras genes (H-Ras, N-Ras, and K-Ras) are found in a variety of cancers, with the highest incidences in lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, colorectal carcinoma, thyroid tumors, and myeloid leukemia.

Immunofluorescent analysis of Ras using MA1-012
Immunofluorescent analysis of Ras in HeLa Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Ras monoclonal antibody (Product # MA1-012) at a dilution of 1:100 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Ras staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.