The 19-Amino Acid Insertion in the Tumor-associated Splice Isoform Rac1b Confers Specific Binding to p120 Catenin

Lidiya Orlichenko,Rory Geyer,Masahiro Yanagisawa,Davitte Khauv,Evette S Radisky,Panos Z Anastasiadis,Derek C Radisky

doi:10.1074/jbc.m109.099382

Abstract

The Rac1b splice isoform contains a 19-amino acid insertion not found in Rac1; this insertion leads to decreased GTPase activity and reduced affinity for GDP, resulting in the intracellular predominance of GTP-bound Rac1b. Here, using co-precipitation and proteomic methods, we find that Rac1b does not bind to many common regulators of Rho family GTPases but that it does display enhanced binding to SmgGDS, RACK1, and p120 catenin (p120(ctn)), proteins involved in cell-cell adhesion, motility, and transcriptional regulation. We use molecular modeling and structure analysis approaches to determine that the interaction between Rac1b and p120(ctn) is dependent upon protein regions that are predicted to be unstructured in the absence of molecular complex formation, suggesting that the interaction between these two proteins involves coupled folding and binding. We also find that directed cell movement initiated by Rac1b is dependent upon p120. These results define a distinct binding functionality of Rac1b and provide insight into how the distinct phenotypic program activated by this protein may be implemented through molecular recognition of effectors distinct from those of Rac1.

Highlights

We hypothesized that the 19-aa insertion and its influence on the conformation and dynamics of the switch regions might confer upon Rac1b the ability to interact with a unique set of binding partners
We propose that the 19-amino acid insertion and adjacent switch regions are natively unstructured or sample a multiplicity of conformations in Rac1b but undergo disorder-to-order transition in the molecular recognition of several unique binding partners, distinct from those of Rac1
Structure coordinates are from Protein Data Bank entries for Rac1-GDP bound to arfaptin (1I4L), Rac1-GPPNP (1MH1), Rac1b-GDP (1RYF), and Rac1b-GPPNP (1RYH)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Reagents—Murine mammary epithelial (SCp2) cells were cultured as described previously [8]. Mass spectrometry analysis of protein bands excised from the silver-stained gels was performed as described previously [17]. GST-Rac1, -Rac1V12, -Rac1N17, -Rac1b, and -19 aa of Rac1b fusion proteins were incubated for 1 h with glutathione-agarose beads, thoroughly washed three times in phosphate-buffered saline, and incubated with murine mammary epithelial SCp2 cell lysate for protein binding. For GST-p120ctn pull-downs, GST-p120ctn proteins attached to the glutathione-agarose beads were incubated with cell lysate from SCp2 cells transfected with YFP-Rac1, -Rac1V12, -Rac1N17, -Rac1b, and -19 aa of Rac1b fusion constructs. GDP- or GTP␥S-bound His-Rac1s were eluted from the nickel-agarose gel using 100 ␮l of elution buffer (50 mM TrisHCl, pH 6.8, 300 mM NaCl, 200 mM imidazole, 10% glycerol), diluted 25 times with protein binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM MgCl2, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100), and added to GST-p120ctn-agarose beads in protein binding buffer for direct protein binding assay. Structure coordinates are from Protein Data Bank entries for Rac1-GDP bound to arfaptin (1I4L), Rac1-GPPNP (1MH1), Rac1b-GDP (1RYF), and Rac1b-GPPNP (1RYH)

RESULTS

Sequence coverage

DISCUSSION