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Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Published on: November 11, 2018

Splice variants of SmgGDS control small GTPase prenylation and membrane localization

Tracy J Berg¹, Adam J Gastonguay, Ellen L Lorimer

¹Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

The Journal of Biological Chemistry

|August 17, 2010

Abstract:

Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.

Insights

Cellular signals regulate small GTPase prenylation via SmgGDS splice variants. This challenges the belief that GTPases are prenylated upon synthesis, revealing a new regulatory mechanism for cancer-related proteins.

Area of Science:

Molecular Biology
Cell Signaling
Cancer Biology

Background:

Ras and Rho small GTPases are crucial signaling proteins involved in cell functions.
Their misregulation, particularly their prenylation and membrane trafficking, is linked to cancer development.
It was previously assumed that GTPases with a C-terminal polybasic region (PBR) are prenylated immediately after synthesis.

Purpose of the Study:

To investigate the regulation of the prenylation pathway for small GTPases.
To challenge the prevailing belief regarding the constitutive prenylation of PBR-possessing GTPases.
To identify regulatory factors controlling GTPase entry into the prenylation pathway.

Main Methods:

Utilized dominant-negative mutations to inhibit GDP/GTP exchange in small GTPases.
Analyzed the impact of these mutations on the prenylation of Rap1A, RhoA, Rac1, and K-Ras.
Investigated the differential roles of SmgGDS splice variants (SmgGDS-558 and SmgGDS-607) in GTPase prenylation and trafficking.

Main Results:

Inhibition of GDP/GTP exchange reduced Rap1A and RhoA prenylation but enhanced Rac1 prenylation, with no effect on K-Ras.
Identified two SmgGDS splice variants that differentially regulate GTPase prenylation.
SmgGDS-607 associates with nonprenylated GTPases and controls their entry into the prenylation pathway, while SmgGDS-558 binds prenylated GTPases and mediates Rap1A trafficking.

Conclusions:

Guanine nucleotide exchange and SmgGDS splice variants critically regulate the prenylation of PBR-possessing small GTPases.
This provides a novel mechanism for controlling GTPase signaling and offers potential therapeutic targets for cancer.
The differential functions of SmgGDS splice variants highlight a complex regulatory network governing GTPase activation and localization.

Read full analysis

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