Post-translational modifications regulate Ral GTPases

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).

print_logoIn 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

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.

Phosphorylation of RhoA as a Signal Transduction Regulator

PTMs on Rho A

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:

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.

RhoA mediates cardiomyocyte actin cytoskeleton and glucose uptake

Recently, R. Palanivel et al. investigated the role that RhoA-mediated re-organization of the actin cytoskeleton has in adiponectin-regulated glucose uptake in cardiomyocytesAdiponectin 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 small-g-protein-inactivationreduce 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.

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

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

Detect different isoforms of Rho, Ras and Rac

small-g-protein-inactivationRas 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!

Activation of RhoA, Rac1 and Cdc42 – New G-LISA Trial kits

Small GTP-binding proteins such as RhoA, Rac1, and Cdc42 are involved in regulating cell signalling pathways and impact a wide range of cellular processes, functions, and morphology. They bind and hydrolyze GTP, thus being switched from the activated form to the inactivated form.small-g-protein-inactivation

The most prominent family of small G proteins is represented by the Ras superfamily of proteins. The Rho subfamily belonging to this superfamily consists of proteins like RhoA, Rac1, and Cdd42. These proteins have been shown to be involved in the regulation of actin dynamics, thus playing a crucial role in processes like cell movement, intracellular transport, and organelle development. While RhoA affects actin stress fibers, Rac1 exhibits effects on lamellipodia and Cdc42 on filopodia.

Measuring the activation of small G proteins

In the past, the activation of small G proteins (i.e. the transformation of the GDP-bound form to the GTP-bound form) could only be measured in pull-down assays. The assays make use of effector proteins which specifically bind to the activated forms of small G proteins. Coupled to beads these proteins can be used to pull-down the activated small G protein from a cell lysate.

Cytoskeleton Inc. introduced a new format of this approach a few years ago. With the G-LISA technology, the effector proteins which selectively bind to activated small G proteins are coupled to 96 well plates, making it possible to run an ELISA-like activation assay. To help you to choose the right activation assay format for your application, take a look at this short video.

New trial sizes for the most commonly investigated small G proteins

Up to now, researchers had to order full 96 well plates to start using the G-LISA technology.

Now Cytoskeleton Inc. have launched trial kits (which are attractively priced) to enable you to establish the method in your lab:

# RhoA G-LISA Kit (Colorim.) Trial Size

# Rac1 G-LISA Activation Assay (Colorim.) Trial Size

# Cdc42 G-LISA Kit (Colorim.) Trial Size

And you can even try all 3 small G proteins with one kit!

# RhoA/Rac1/Cdc42 G-LISA Activation Assay Bundle

What about you?

Are you interested in the G-LISA technology? Or if you’re already using it, how does it compare to other methods?
Share your comments below!

New hope for Ras inhibitors

Ras belongs to the family of small G-proteins and plays an important role in several signal transduction pathways involved in normal cell growth and differentiation. Over 30 years ago three isoforms of Ras – H-Ras, N-Ras, and K-Ras, have been identified for their oncogenic activation in human cancer cells. In fact aberrant Ras signaling could be shown in more than 30% of all human cancers including lung, colon, and pancreatic cancer. K-Ras has been identified as the most important Ras protein in cancer research.

As other small G proteins, Ras cycles between inactive (GDP-bound) and active state (GTP-bound) forms (see figure).

Ras and Inhibitors

Although extensive research activities have been dedicated to Ras, no effective Ras inhibitor has been identified so far. But, there may be new hope in developing a therapeutic inhibitor to Ras proteins by targeting the upstream guanine nucleotide exchange factor (GEF) protein SOS (Son of sevenless). [Read more…]

Is there a link between members of Rho family of small G proteins and reactive oxygen species?

Redox schematic representation and crosstalk between GTPase pathwaysBoth ROS (Reactive Oxygen Species) and RNS (Reactive Nitrogen Species) on the one hand, and the small G proteins belonging to the Rho family on the other hand, are key regulators in various signal transduction pathways.

More recently it has been suggested that crosstalk between reactive species and Rho GTPases plays a crucial role in some of their physiological functions. Furthermore, these crosstalk events have been linked to pathological processes, e.g. lung injury and cancer.

Our partner Cytoskeleton Inc.  has summarized recent findings in the newsletter Rho GTPases and Reactive Oxygen Species: Crosstalk and Feedback. Let’s take a look at what’s available for research in this area.

[Read more…]