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

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.The Ras superfamily of small GTPases mediates numerous biological functions through specific binding to a plethora of effector proteins when associated with GTP. The function of the small GTPases in diverse cellular processes, such as signal transduction, cytoskeletal organization, cell migration, transcription, vesicle transport, and cytokinesis, is carefully regulated by regulator proteins: guanine nucleotide exchange factors (GEFs), 2 GDP dissociation stimulators (GDSs), and GTPase-activating proteins (GAPs). Rac1b is a splice isoform of Rac1 that results from the inclusion of exon 3b, which contains 57 nucleotides and which leads to a 19-amino acid in-frame insertion (1, 2). Rac1b was first identified in human skin and epithelial tissue of the intestinal tract and has been found to be up-regulated in malignant colorectal (1) and breast cancer (2). When assessed in cultured cells, Rac1b has been found to have many of the properties of GTPase-defective Rac1 mutants. For example, exogenously expressed Rac1b promotes density-and anchorage-independent cell growth of NIH 3T3 cells (3), cell survival and cell cycle progression in mouse fibroblasts (4), and Dishevelled-3-mediated detachment of colorectal cancer cells (5). However, Rac1b also shows distinct activities from Rac1. Unlike Rac1, Rac1b does not promote cadherin-dependent disassembly of adherens junctions in keratinocytes (6), it does not induce cytoskeletal rearrangements in colorectal cancer cells as effectively as GTPase-defective mutants of Rac1 (3), and it is not as effective as Rac1 for activation of RelB (7). Furthermore, Rac1b has distinct positive activities, because expression of Rac1b has been shown to mediate epithelial-mesenchymal transition and genomic instability in mouse mammary epithelial cells exposed to MMP3 (matrix metalloproteinase 3) (8, 9) and to sustain colorectal t...

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