RalA and RalB GTPases regulate cell motility, morphology, signaling, vesicular trafficking, and endo/exocytosis. The regulation of these functions is critical for the development and spread of cancer, implicating Ral in oncogenesis and metastasis. Both isoforms are integral for Ras-mediated tumorigenesis, metastasis, and invasion. Despite sharing 82% amino acid sequence identity, effectors, and structural/biochemical properties, RalA and RalB have their own unique functions in oncogenesis due to distinct subcellular localization and differential effector interactions. Ral localization, binding partners, and function are regulated by post-translational modifications (PTMs).

In their recent newsletter, Cytoskeleton Inc. summarize recent findings about the relevance of geranylgeranylation, carboxymethylation, palmitoylation, phosphorylation, and ubiquitination in regulating Ral activity, subcellular localization, effector binding, and ultimately, function.

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

Kits to measure RalA activation

• G-LISA RalA Activation Assay Biochem Kit (colorimetric format)
• RalA Activation Assay Biochem Kit (bead pull-down format)

If you’d like to get an overview about what’s available in the small G protein field, take a look at this Small GTPase product guide.

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.

Recently, R. Palanivel et al. investigated the role that RhoA-mediated re-organization of the actin cytoskeleton has in adiponectin-regulated glucose uptake in cardiomyocytes. Adiponectin is a protein secreted by adipose tissue that modulates glucose and fatty acid metabolism.  In concert with APPL1, an adiponectin receptor binding partner, adiponectin carries out these functions which are important in obesity and type 2 diabetes, two diseases that reduce cardiac energy metabolism. The authors found that adiponectin (both full-length and globular) elevates RhoA activity which correlates with increased actin polymerization and glucose uptake.  Changes in the G-/F-actin ratio likely involve APPL1 as adiponectin increases colocalization of actin and APPL1.  Inhibition of actin polymerization or RhoA signaling significantly reduces the adiponectin-mediated increase in glucose uptake.  Thus, RhoA-mediated actin cytoskeleton remodeling is required for adiponectin-regulated glucose uptake in cardiomyocytes.  Increased glucose uptake is cardioprotective in diabetes. A number of products by Cytoskeleton Inc. (“The Protein Experts”) were essential in this study, providing accurate and sensitive assays for quantifying levels of activated RhoA and changes in G- and F-actin levels or binding partners in cardiomyocytes under conditions of RhoA inhibition.

• RhoA G-LISA activation assayÂ
• G-/F-actin In Vivo Assay Kit
• Anti-actin antibody
• Cell-permeable Rho inhibitor

These reagents are available in Europe through tebu-bio, who have also compiled useful selection tools:

• Actin Product Guide

• Small G Protein Product Guide

Reference: Palanivel et al. 2014. Adiponectin stimulates Rho-mediated actin cytoskeleton remodeling and glucose uptake via APPL1 in primary cardiomyocytes. Metabolism. 63, 1363-1373.

Ras and Rho family members are small G proteins involved in the regulation of actin-dependent cell processes such as motility, growth, and intracellular trafficking. Furthermore, dysfunctions of Ras and Rho proteins are known to be correlated with a number of diseases (cancer, neurodegeneration).

Small G proteins cycle between the inactive, GDP-bound form and the active, GTP-bound form.

G-LISA technology: state of the art small G protein activation measurement

Cytoskeleton, Inc. offers activation kits for a number of small G proteins (RhoA, Rac1, Cdc42, Ras, RalA, Arf1, Arf6). All these assays are available as G-LISA formats, a 96 well based technology, in which a protein sequence specifically binding to the activated for of the respective small G protein is coupled to the bottom of the wells and “catches” activated, GTP-bound proteins from cell lysates derived from cultured cells. The activation status of the small G protein can thus be detected in an ELISA like, quantitative approach.

A number of recently published papers using Cytoskeleton’s G-LISA kits show that not only RhoA and Rac1 can be measured with the RhoA-G-LISA and Rac1 G-LISA, respectively. By changing the antibody which is finally used to detect the activated small G protein bound to the binding protein one can e.g. differentiate between the isoforms RhoA, RhoB, and RhoC, and even RhoJ (which shows a high homology with Cdc42).

In their November newsletter Cytoskeleton Inc. summarized these publications and give valuable information and tips how to broaden the target specificity of their G-LISA kits.

Download your free copy of the newsletter GTPase Activation Assays: Detecting Different Isoforms

Any questions about using G-LISA? Fire away below!