HER2/ErbB2-induced Breast Cancer Cell Migration and Invasion Require p120 Catenin Activation of Rac1 and Cdc42

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.     

Structural basis of Rnd1 binding to plexin Rho GTPase binding domains (RBDs)

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.     

A Conserved residue in the yeast Bem1p SH3 domain maintains the high level of binding specificity required for function

The yeast Bem1p SH3b and Nbp2p SH3 domains are unusual because they bind to peptides containing the same consensus sequence, yet they perform different functions and display low sequence similarity. In this work, by analyzing the interactions of these domains with six biologically relevant peptides containing the consensus sequence, they are shown to possess finely tuned and distinct binding specificities. We also identify a residue in the Bem1p SH3b domain that inhibits binding, yet is highly conserved for the purpose of preventing nonspecific interactions. Substitution of this residue results in a marked reduction of in vivo function that is caused by titration of the domain away from its proper targets through nonspecific interactions with other proteins. This work provides a clear illustration of the importance of intrinsic binding specificity for the function of protein-protein interaction modules, and the key role of "negative" interactions in determining the specificity of a domain.     

Evidence for two CRIB domains in phospholipase D2 (PLD2) that the enzyme uses to specifically bind to the small GTPase Rac2

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named CRIB-1 and CRIB-2 in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent K(d) of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a termination signal leading to PLD2 inactivation.     

Target of rapamycin complex 2 signals to downstream effector yeast protein kinase 2 (Ypk2) through adheres-voraciously-to-target-of-rapamycin-2 protein 1 (Avo1) in Saccharomyces cerevisiae

The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.     

Involvement of 14-3-3 proteins in the second epidermal growth factor-induced wave of Rac1 activation in the process of cell migration

Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ζ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ζ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ζ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1.     

Structure of the Mouse Sex Peptide Pheromone ESP1 Reveals a Molecular Basis for Specific Binding to the Class C G-protein-coupled Vomeronasal Receptor

Exocrine gland-secreting peptide 1 (ESP1) is a sex pheromone that is released in male mouse tear fluids and enhances female sexual receptive behavior. ESP1 is selectively recognized by a specific class C G-protein-coupled receptor (GPCR), V2Rp5, among the hundreds of receptors expressed in vomeronasal sensory neurons (VSNs). The specific sensing mechanism of the mammalian peptide pheromone by the class C GPCR remains to be elucidated. Here we identified the minimal functional region needed to retain VSN-stimulating activity in ESP1 and determined its three-dimensional structure, which adopts a helical fold stabilized by an intramolecular disulfide bridge with extensive charged patches. We then identified the amino acids involved in the activation of VSNs by a structure-based mutational analysis, revealing that the highly charged surface is crucial for the ESP1 activity. We also demonstrated that ESP1 specifically bound to an extracellular region of V2Rp5 by an in vitro pulldown assay. Based on homology modeling of V2Rp5 using the structure of the metabotropic glutamate receptor, we constructed a docking model of the ESP1-V2Rp5 complex in which the binding interface exhibited good electrostatic complementarity. These experimental results, supported by the molecular docking simulations, reveal that charge-charge interactions determine the specificity of ESP1 binding to V2Rp5 in the large extracellular region characteristic of class C GPCRs. The present study provides insights into the structural basis for the narrowly tuned sensing of mammalian peptide pheromones by class C GPCRs.     

LKB1 Suppresses p21-activated Kinase-1 (PAK1) by Phosphorylation of Thr109 in the p21-binding Domain

The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr¹⁰⁹ in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr¹⁰⁹ by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/−) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.     

The epidermal growth factor receptor mediates tumor necrosis factor-alpha-induced activation of the ERK/GEF-H1/RhoA pathway in tubular epithelium

Tumor necrosis factor (TNF)-α induces cytoskeleton and intercellular junction remodeling in tubular epithelial cells; the underlying mechanisms, however, are incompletely explored. We have previously shown that ERK-mediated stimulation of the RhoA GDP/GTP exchange factor GEF-H1/Lfc is critical for TNF-α-induced RhoA stimulation. Here we investigated the upstream mechanisms of ERK/GEF-H1 activation. Surprisingly, TNF-α-induced ERK and RhoA stimulation in tubular cells were prevented by epidermal growth factor receptor (EGFR) inhibition or silencing. TNF-α also enhanced phosphorylation of the EGFR. EGF treatment mimicked the effects of TNF-α, as it elicited potent, ERK-dependent GEF-H1 and RhoA activation. Moreover, EGF-induced RhoA activation was prevented by GEF-H1 silencing, indicating that GEF-H1 is a key downstream effector of the EGFR. The TNF-α-elicited EGFR, ERK, and RhoA stimulation were mediated by the TNF-α convertase enzyme (TACE) that can release EGFR ligands. Further, EGFR transactivation also required the tyrosine kinase Src, as Src inhibition prevented TNF-α-induced activation of the EGFR/ERK/GEF-H1/RhoA pathway. Importantly, a bromodeoxyuridine (BrdU) incorporation assay and electric cell substrate impedance-sensing (ECIS) measurements revealed that TNF-α stimulated cell growth in an EGFR-dependent manner. In contrast, TNF-α-induced NFκB activation was not prevented by EGFR or Src inhibition, suggesting that TNF-α exerts both EGFR-dependent and -independent effects. In summary, in the present study we show that the TNF-α-induced activation of the ERK/GEF-H1/RhoA pathway in tubular cells is mediated through Src- and TACE-dependent EGFR activation. Such a mechanism could couple inflammatory and proliferative stimuli and, thus, may play a key role in the regulation of wound healing and fibrogenesis.