Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate

Abstract

Ras family GTPases (RFGs) regulate signaling pathways that control multiple biological processes. How signaling specificity among the closely related family members is achieved is poorly understood. We have taken a proteomics approach to signaling by RFGs, and we have analyzed interactions of a panel of RFGs with a comprehensive group of known and potential effectors. We have found remarkable differences in the ability of RFGs to regulate the various isoforms of known effector families. We have also identified several proteins as novel effectors of RFGs with differential binding specificities to the various RFGs. We propose that specificity among RFGs is achieved by the differential regulation of combinations of effector families as well as by the selective regulation of different isoforms within an effector family. An understanding of this new level of complexity in the signaling pathways regulated by RFGs is necessary to understand how they carry out their many cellular functions. It will also likely have critical implications in the treatment of human diseases such as cancer.

FIG. 1.

Activation of the Raf/Erk pathway by RFGs. (A) Activation of Raf kinases by RFGs. Constructs expressing HA-tagged Raf kinases were cotransfected into 293T cells with constitutively active myc-tagged RFGs or empty vector (control). Two days later, Raf kinase activity on HA immunoprecipitates was measured in a coupled assay for its ability to activate MEK. (B) Expression levels of transfected proteins were measured by Western blotting with anti-HA or anti-myc antibodies. (C) Stimulation of Erk phosphorylation by RFGs. Erk phosphorylation was measured in lysates from transfections as described for panel A by Western blotting with phospho-specific Erk antibodies. (D) Activation of Erk1 activity by RFGs. HA-tagged Erk1 was contransfected with RFGs, and Erk kinase activity was measured in HA immunoprecipitates by using myelin basic protein as a substrate. (E) Interaction of Raf kinases with RFGs. GST-tagged RFGs were cotransfected into 293T cells with myc-tagged Raf kinases, and interactions were measured by pulling down RFGs with glutathione beads and detecting bound Raf in Western blots with anti-myc antibodies.

FIG. 2.

Activation of RalGEF pathway by RFGs. (A) Flag tagged-RalA was cotransfected into 293T cells with empty vector (background), HA-tagged RalGEFs, and empty vector or constitutively active myc-tagged RFGs. Two days after transfection, the levels of GTP-bound flag-RalA were measured in total cell lysates in pulldown assays with GST-RalBP1-RBD. In each gel, 1/100 of the lysate used was run. Expression levels of transfected proteins were measured by Western blotting with anti-Flag or anti-HA antibodies. Results shown are representative of at least three independent experiments. (B) The levels of Ral-GTP shown in panel A were quantified with a STORM phosphorimager. (C) Interaction of RalGEFS with RFGs. GST-tagged RFGs were cotransfected into 293T cells with myc-tagged RalGEFs, and interactions were measured by pulling down RFGs with glutathione beads and detecting bound RalGEFs with anti-myc antibodies in Western blots.

FIG. 3.

Activation of class I PI3Ks by RFGs. Constitutively active RFGs were cotransfected into 293T cells with PI3K isoforms. Two days after transfection, cells were labeled with [³²P]orthophosphate, and total cellular PIP3 levels were measured by high-pressure liquid chromatography. The levels of PI(4,5)P2 were standardized to 200,000 cpm. Results shown are representative of at least three independent experiments.

FIG. 4.

Proteins with RA domains. Domain structures of RA domain-containing protein families according to the SMART protein domain database (http://smart.embl-heidelberg.de). Proteins are not drawn to scale. Note that some genes have splice variants that give rise to proteins lacking some of the depicted domains. Asterisks indicate proteins that have been analyzed in this study.

FIG. 5.

Interaction of RFGs with RA domain-containing proteins. Constitutively active GST-tagged RFGs were cotransfected into 293T cells with myc-tagged RA domain-containing proteins, and interactions were measured by pulling down RFGs with glutathione beads and detecting bound proteins with anti-myc antibodies in Western blots. All proteins were full length except those shown with asterisks (APBB1IP/RIAM [amino acids 1 to 261] and PLCɛ [amino acids 1383 to 2303]).

FIG. 6.

Interaction of H-Ras effector mutants with RA domain-containing proteins. GST-tagged H-Ras effector mutants in a V12 backbone were cotransfected into 293T cells with myc-tagged RA domain-containing proteins, and interactions were measured in GST pulldown assays and with anti-myc Western blots.