Signal Transduction

Normal cell function, and its contribution to overall physiology, depends on the proper response of cells to stimuli or extracellular signals. The first critical component of cell signaling is communication of signal from its origin outside the cell across the cell membrane to evoke a response inside the cell, a function known as signal transduction. Hence, the stimulation of signal transduction pathways requires the receptor to be activated through binding to the specific ligand or hormone. Once the receptor is activated, the signal will be transduced inside the cells and result in the stimulation of different signal transduction pathways.

Small GTPases

One important subset of proteins involved in signal transduction is the small GTPases. Small GTPases of the Ras superfamily are monomeric guanine nucleotide-binding proteins with molecular masses of 20-25 kDa and serve as molecular switches to regulate growth, morphogenesis, cell mobility, axonal guidance, cytokinesis and trafficking. The first small GTPase to be discovered was Ras, and there are now approximately 60 different small GTPases that have been identified in mammalian cells. Among them, the Ras and Rho families are of special interest because they transduce the signals from extracellular stimuli to intracellular signal transduction pathways. The small GTPases cycle between inactive (GDP-bound) and active (GTP-bound) states. Under basal conditions, GTPases are GDP-bound and inactive. Upon stimulation, GTPases release GDP and bind to GTP, a reaction accomplished by guanine nucleotide exchange factors (GEFs). In their active GTP-bound state, Ras and Rho GTPases interact with a variety of effector proteins to promote cellular responses. The active state of GTPases is transient because of their intrinsic GTPase activity, which is stimulated further by GTPase activating proteins (GAPs)^1-4.

Small GTPase cycle. Under basal conditions, the small GTPase exists in an inactive, GDP-bound state. In response to stimuli, the small GTPase releases GDP and binds to GTP, a reaction catalyzed by GEF. The activity of the small GTPase is transient because of intrinsic GTPase activity, which is stimulated by GAP.

Bi-phasic activation of Ras in response to serum. NIH3T3 cells were serum starved for 24 h, followed by treatment with 10% FBS at the indicated time. The cells were lysed and a Ras pull-down assay was carried out using Active Ras Pull-Down and Detection Kit (Product # 89855 ). Western blotting of pulled-down active Ras with anti-Pan-Ras antibody showed that activation of Ras was detected at 1 min and then declined at 2 min. However, activation of Ras reccurred at 30–60 min. This data shows that the immediate activation of Ras in response to serum is transient.

Ras GTPase Family

Ras is a key regulator of cell growth in all eukaryotic cells in response to peptide growth factors, cytokines and hormones. Three members (H-, K- and N-ras) encoding proteins of 21 kDa have been identified in the mammalian Ras family. Mutations in Ras at amino acids 12, 13 or 61 make Ras insensitive to GAP, resulting in a constitutively activated Ras which, in turn, leads to malignant transformation. It has been estimated that 30% of human tumors contain an active mutation in Ras. The first signaling pathway discovered involving Ras was the Raf/MEK/ERK cascade of protein kinases that leads to stimulation of certain transcription factors and results in stimulation of cellular proliferation and differentiation. Ras activates this kinase cascade by directly binding to Raf. The binding of Ras to Raf requires active, GTP-bound Ras and an intact effector domain, located in the NH₂-terminal region of Raf.^2,3

Rap1 is another member of the Ras family of small GTPases that is activated in response to a variety of extracellular signals. It has been identified as an antagonist of Ras-induced cell transformation and has been implicated to be in control of cell morphogenesis, cell-cell adhesion and regulation of the cell cycle.^5-7

Rap1 is activated by a diverse array of extracellular stimuli and secondary messengers. The resulting signal cascade can lead to both the activation and inhibition of ERK (extracellular signal-regulated kinase). Like other small GTPases, Rap1 is active when bound to GTP and inactive when bound to GDP. Upon binding to GTP, Rap1 interacts with the Rap1-binding domain (RBD) of RalGDS (guanine dissociation stimulator) and leads to the activation of RalGDS.

Signaling pathways mediated by Ras, Rac1/Cdc42 or Rho GTPase.

Rho GTPase Family

The mammalian Rho GTPase family currently consists of three subfamilies, Rho (RhoA, RhoB and RhoC), Rac (Rac1, Rac2 and Rac3) and Cdc42 (Cdc42Hs and G25K). Active Rho GTPases interact with cellular target proteins to regulate a variety of cellular responses, including reorganization of the actin cytoskeleton and gene transcription. Cdc42, Rac1 and RhoA are the most extensively characterized members of the Rho family. Activation of Rac induces actin polymerization to form lamellipodia (broad web-like extensions), whereas activation of Cdc42 stimulates the polymerization of actin to filopodia or microspikes (long and thin extensions). In contrast, Rho regulates bundling of actin filaments into stress fibers and the formation of focal adhesion complexes. Rho, Rac and Cdc42 have also been reported to stimulate serum response factor (SRF)-dependent transcription and to activate the transcription factor NF-kB. One of the target proteins for Rac and Cdc42 is p21-activated kinase (Pak), which plays a key role in the linkage of Rac signaling pathways to Ras signaling pathways through the phosphorylation of Raf and MEK (Figure 5). Several targets have been identified for Rho, including p160 Rho kinase (ROCK) and Rhotekin. The activity of ROCK is enhanced upon binding to GTP-Rho and leads to phosphorylation of myosin light chain and myosin light chain phosphatase. Phosphorylation of these two substrates leads to an increase in phosphorylation of myosin light chain, myosin filament assembly, and F-actin bundling, thereby leading to stress fiber formation^2,4.

Analysis of GTPase Activation

The traditional assay to monitor the activation of GTPases involves measuring the GTP/GDP ratio. This is achieved by labeling cells with ³²P, immunoprecipitating the GTPase, and measuring the ratio of bound ³²P-GTP and ³²P-GDP by thin layer chromatography. The disadvantages of this approach include its use of radioactivity, the rapid turnover of the GTP-bound state in the absence of effector proteins and the lack of efficient immunoprecipitating antibodies. To overcome these problems, an alternative method for measuring GTPase-GTP levels was developed. This nonradioactive approach is based on the principle that only the active form of the GTPase interacts with downstream effectors. Binding of the active GTPase to the GST-GTPase binding domain has been shown to either inhibit or decrease GTP hydrolysis, which is an advantage for preserving the active state of the small GTPase.^8-10 For example, the N-terminal region of Raf1 protein kinase contains the Ras–binding domain (RBD),⁸ the N-terminal regulatory region in p21-activated protein kinase 1 and 2 (Pak1 and 2) contains the p21-binding domain (PBD) for Rac and Cdc42,⁹ and the N-terminal region of Rhotekin contains the Rho-binding domain (RBD).¹⁰ The GTPase-binding domains from these downstream effectors are expressed as recombinant glutathione S-transferase (GST) fusion proteins immobilized on glutathione resin and can be used to affinity precipitate (pull-down) the active GTPase from cell lysates. Pulled-down active GTPases are eluted from the resin and detected by immunoblotting with a specific antibody.

Active GTPase Pull-Down and Detection Kits
The Pierce Active GTPase (Rho, Ras, Rac1 and Cdc42) Pull-Down and Detection Kits (Product # 89854, 89855, 89856, 89857 and 89872) were developed based on the aforementioned principle that active GTPases can be pulled-down from a lysate via their specific downstream effectors and then detected by immunoblotting with a specific antibody (Figure 6). The specificity and function of GST-Raf1- RBD, GST-Pak1-PBD or GST-Rhotekin-RBD or GST-RalGDS-RBD were analyzed by incubation of GST-fusion proteins with SwellGel Immobilized Glutathione Discs. NIH3T3 cell lysate that was pre-treated with GTPgS to activate endogenous small GTPases or with GDP to inactivate small GTPases. A strong signal for Rho, Ras, Rac1 or Cdc42 was detected in a chemiluminescent Western blot of pulled-down GTPases using GTPgS-treated lysate; while very low or no signal was detected when GDP-treated lysate was used. These results show that Active GTPase (Rho, Ras, Rac1 and Cdc42) Pull-Down and Detection Kits specifically detect active small GTPases.


Schematic illustration of the pull-down assay used to detect active small GTPases. The small GTPase binding domain from each down-stream effector is expressed as a GST-fusion protein (GST-PBD or GST-RBD). Once immobilized to glutathione resin (e.g. SwellGel Immobilized Glutathione), GST-PBD or GST-RBD is incubated with cell lysate containing active GTPases. The unbound proteins are removed and the resin is washed. The pulled-down active GTPases are eluted and analyzed by Western blotting using anti-bodies specific for the GTPase of interest.

Detection of active Rho, Ras, Rac1 and Cdc42 using Active Rho, Ras, Rac1 and Cdc42 Pull-Down and Detection Kits (Product # 89854, 89855, 89856 and 89857). NIH3T3 cell lysate (500 µg) treated with GTPgS or GDP was incubated with GST-Raf1-RBD, GST-Rhotekin-RBD or GST-Pak1-PBD and SwellGel Immobilized Glutathione. Half of the eluted pull-down samples (25 µl) and 20 µg of lysate were analyzed by Western blotting using anti-Pan-Ras, anti-Rho, anti-Rac1 or anti-Cdc42 antibody.

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