As is already known, multiple families of small GTPases (Ras, Arf and Rho families) make up the Ras GTPase superfamily. Due to their role as molecular switches in cell signalling (active GTP-bound state vs inactive GDP-bound states) and in many other cellular responses (cytoskeletal reorganization, regulation of transcription, apoptosis…), these small GTPases are the subject of intense investigation to try to understand the mechanisms that regulate activation and inactivation of this proteins.

In this post, I invite you to discover a revolutionary way to study these cell signalling events by quantifying the level of active small GTPases in your experimental model.

Small GTP-binding proteins such as Rho and Ras GTPase family members are involved in regulating cell signalling pathways and impact a wide range of cellular processes, functions, and morphology. In this post, you’ll discover two types of Small G-protein Activation Assays to easily measure the GTP-bound form of your protein of interest, as well as some tips for choosing between both solutions and finding the assays that fit best with your cell signalling experiments.

Fig. 1: Small G protein activation cycle

In this post, I invite you to discover some state-of-the art methods to measure the activation of the most prominent small G proteins. In future posts, we’ll then be looking at methods to measure the activity of Guanine nucleotide exchange factors (GEFs) which stimulate the release of GDP to allow the binding of GTP (which activates the small G protein) and of GTPase-activating proteins (GAPs) which stimulate the GTPase activity of small G proteins to inactive them (see Fig. 1).

Post-translational Modifications of RhoA

Rho family GTPases are key regulators in a wide range of physiological processes, including cell motility, cell division, and neuronal development. Rho activity is regulated temporally and spatially by a variety of direct post-translational modifications (PTMs) that include prenylation, ubiquitination, oxidation, nitrosylation, and phosphorylation.

Cytoskeleton Inc. recently released a newsletter highlighting the control of RhoA function through phosphorylation. RhoA is a target for a growing number of kinases and as such, phosphorylation is emerging as a central theme in the regulation of this family of proteins (2).

The newsletter focussed on the mechanism of RhoA phosphorylation at Serine 188, which is mainly conducted by kinases like PKA and PKG (protein kinase A and protein kinase G) which are cyclic AMP-dependent and cyclic GMP-dependent respectively.

Furthermore, it looks at the physiological consequences of RhoA phosphorylation and future directions especially concerning the RhoA PTM involvement in diseases and potential  therapeutic options.

You can download a copy of this newsletter, or if you have any questions or comments, don’t hesitate to contact me through the form below.

Related to RhoA and PTM research:

• G-LISA kits to measure the activation of RhoA
• Cell permeable RhoA inhibitor (C3Transferase)
• RhoA activators
• Anti Acetyl Lysine Mouse Monoclonal Antibody
• Anti-SUMO1 Mouse Monoclonal Antibody
• Anti-Ubiquitin Mouse Monoclonal Antibody

References:

1. Stankiewicz T. & Linseman D. 2014. Rho family GTPases: key players in neuronal development, neuronal survival and neurodegeneration. Front. Cell. Neurosci. doi: 10.3389/fncel.2014.00314.
2. Boulter E. et al. 2012. Off the beaten paths: alternative and crosstalk regulation of Rho GTPases. FASEB J. 26, 469-479.