A Cdc42 Mutant Specifically Activated by Intersectin

Abstract

The Rho family GTPase Cdc42 functions as a molecular switch and controls many fundamental cellular processes such as cytoskeletal regulation, cell polarity, and vesicular trafficking. Guanine nucleotide exchange factors of the Dbl family activate Cdc42 and other Rho GTPases by catalyzing the removal of bound GDP, allowing for GTP loading, and subsequent effector recognition ultimately leading to downstream signaling events. Analysis of existing structural data reveals that the Dbl exchange factor intersectin engages a strictly conserved GTPase residue of Cdc42 (tyrosine 32) in a unique mode with respect to all other visualized exchange factor-Rho GTPase interfaces. To investigate this differential binding architecture, we analyzed the role of tyrosine 32 of Cdc42 in binding, and stimulation by Dbl family exchange factors. Deletion of the hydroxyl side chain of tyrosine 32 substantially increases the affinity of Cdc42 for intersectin, yet severely cripples interaction with Dbs, a normally potent exchange factor of Cdc42. Moreover, Cdc42(Y32F) is exclusively activated by intersectin, while virtually unresponsive to other Cdc42-activating exchange factors in vitro and in vivo. Further, the structural determinants unique to intersectin, which permit selective recognition and concomitant stimulation of Cdc42(Y32F), have been defined. Cdc42 and other individual Rho GTPases receive input stimulatory signals from a multitude of Dbl exchange factors, and therefore, Cdc42(Y32F) could act as a valuable reagent for understanding the specific influence of ITSN on Cdc42-mediated signaling phenomena.

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor, Modulation by Allosteric Factors, and the Uncompetitive Inhibitor Brefeldin A

“…Moreover, for the present study, the GDP-bound conformational state of the purified protein has been monitored through 1 (Table 1). In accordance with the allosteric competition between nucleotides and GEFs for the binding to G proteins as well as previous reports (9,44), the complex observed in this first experience is assumed to correspond to the free nucleotide Arf1-Arno complex (named binary complex in scheme 1) (44,45). As the terms affinity and equilibrium constant (K D ) of a complex referred to association/dissociation of two equivalent partners and because in this SPR experiment association occurs with Arf1-GDP whereas dissociation is related to Arf1 in its free nucleotide form, we reported for each complex observed the k d /k a ratio to illustrate the complex stability.…”

Control of protein signaling using a computationally designed GTPase/GEF orthogonal pair

“…S4). The interaction between Cdc42 WT and ITSN WT had a K D of 29 AE 2 nM, similar to that determined in a previous study (33 nM) (26). The K D of orthoCdc42 and orthoITSN was 478 AE 22 nM, approximately 16-fold weaker.…”

“…Proteins for in vitro experiments were expressed and purified from Escherichia coli, and nucleotide dissociation and association assays were performed as detailed in SI Appendix, Methods. Cdc42-ITSN binding affinities were determined by surface plasmon resonance (SPR) experiments similar to those described in Smith, et al (26), and the N-WASP translocation assay was performed as described by Co, et al (27). (For more details on protein in vitro assays see SI Appendix, Methods).…”

Progress in the therapeutic inhibition of Cdc42 signalling

“…The complex which has been targeted most frequently is Cdc42-Intersectin (ITSN1). ITSN1 has been identified as a Cdc42 specific GEF in in vitro studies [ 24 , 25 ] and is one of several RhoGEFs showing a high frequency of alterations in some cancer types [ 26 , 27 ]. The small molecule ZCL278 was identified by a high throughput virtual screen where candidates were docked into the ITSN1 binding groove of Cdc42 ( Figure 2 ).…”