Cooperative transformation of NIH3T3 cells by G alpha12 and Rac1

Area of Science:

• Molecular Biology
• Cell Signaling
• Oncology

Background:

• G alpha12 and G alpha13 are G12 class alpha-subunits mediating signals from seven transmembrane domain-spanning receptors.
• Evidence links G alpha12 and G alpha13 to RHO gene family signaling pathways.

Purpose of the Study:

• To investigate the role of Rac1, RhoA, and Cdc42Hs in the transforming properties of G alpha12.
• To elucidate the specific interactions between G alpha12 and Rho GTPases in cellular transformation and signaling.

Main Methods:

• Utilized dominant inhibitory mutants (Asn 17) of Rac1 and RhoA to assess their effect on G alpha12-induced transformation in NIH3T3 cells.
• Employed constitutively active mutants (Val 12 for Rac1, Leu 63 for RhoA) to examine cooperation with wild-type G alpha12 in NIH3T3 cell transformation.
• Assessed G alpha12-induced mitogenesis using C3 ribosyltransferase.
• Investigated JNK/SAPK activation via transient co-transfection in COS7 cells.
• Measured transcription from the c-fos serum response element (SRE).

Main Results:

• Rac1 (Asn 17) and, to a lesser extent, RhoA (Asn 17) inhibited focus formation by a GTPase-deficient G alpha12 mutant.
• Wild-type G alpha12 cooperated with Rac1 (Val 12) but not RhoA (Leu 63) in NIH3T3 cell transformation.
• G alpha12-induced mitogenesis was not significantly affected by C3 ribosyltransferase, suggesting a minor role for RhoA in these events.
• Co-transfection of G alpha12 and Rac1 (Val 12) enhanced JNK/SAPK activation.
• Cooperation between G alpha12 and Rac1 correlated with increased c-fos SRE transcription.

Conclusions:

• G alpha12 utilizes Rac1, rather than RhoA, for significant cellular transformation.
• The G alpha12-Rac1 interaction activates JNK/SAPK signaling and promotes transcription via the c-fos SRE.
• These findings clarify the specific Rho GTPase involved in G alpha12-mediated cellular transformation and signaling.