Ana Ruiz-Saenz

INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells

The formin INF2 promotes the formation of stabilized, detyrosinated microtubules, which are important for centrosome reorientation to the immunological synapse of T cells.

MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration.

Rac1 requires compartmentalization into specialized, condensed membranes to mediate cell migration. We show that myeloid-associated differentiation marker (MYADM), a member of the MAL family of proteins with ubiquitous expression, regulates membrane condensation required for Rac1 targeting and, subsequently, cell spreading and migration.

Protein 4.1R regulates cell migration and IQGAP1 recruitment to the leading edge

In red blood cells, multifunctional protein 4.1R stabilizes the spectrin–actin network and anchors it to the plasma membrane. To contribute to the characterization of functional roles of 4.1R in nonerythroid cells, we have analyzed the participation of protein 4.1R in cell migration. The distribution of endogenous 4.1R is polarized towards the leading edge of migrating cells. Exogenous 4.1R isoforms containing a complete membrane-binding domain consistently localized to plasma membrane extensions enriched in F-actin. Silencing of 4.1R caused the loss of persistence of migration in subconfluent cells and of directional migration in cells moving into a wound. Coimmunoprecipitation and pull-down assays identified the scaffold protein IQGAP1 as a partner for protein 4.1R and showed that the 4.1R membrane-binding domain is involved in binding IQGAP1. Importantly, we show that protein 4.1R is necessary for the localization of IQGAP1 to the leading edge of cells migrating into a wound, whereas IQGAP1 is not required for protein 4.1R localization. Collectively, our results indicate a crucial role for protein 4.1R in cell migration and in the recruitment of the scaffold protein IQGAP1 to the cell front.

MYADM controls endothelial barrier function through ERM-dependent regulation of ICAM-1 expression

Myeloid-associated differentiation marker (MYADM) protein belongs to the MAL family and regulates raft domains in epithelial cells. Membrane rafts participate in inflammatory responses. This study shows that MYADM is expressed in endothelial cells and controls the endothelial barrier by regulating ICAM-1 expression through ezrin, radixin, and moesin proteins, connectors between plasma membrane domains and actin cytoskeleton.

Protein 4.1R binds to CLASP2 and regulates dynamics, organization and attachment of microtubules to the cell cortex

Summary The microtubule (MT) cytoskeleton is essential for many cellular processes, including cell polarity and migration. Cortical platforms, formed by a subset of MT plus-end-tracking proteins, such as CLASP2, and non-MT binding proteins such as LL5&bgr;, attach distal ends of MTs to the cell cortex. However, the mechanisms involved in organizing these platforms have not yet been described in detail. Here we show that 4.1R, a FERM-domain-containing protein, interacts and colocalizes with cortical CLASP2 and is required for the correct number and dynamics of CLASP2 cortical platforms. Protein 4.1R also controls binding of CLASP2 to MTs at the cell edge by locally altering GSK3 activity. Furthermore, in 4.1R-knockdown cells MT plus-ends were maintained for longer in the vicinity of cell edges, but instead of being tethered to the cell cortex, MTs continued to grow, bending at cell margins and losing their radial distribution. Our results suggest a previously unidentified role for the scaffolding protein 4.1R in locally controlling CLASP2 behavior, CLASP2 cortical platform turnover and GSK3 activity, enabling correct MT organization and dynamics essential for cell polarity.

Effective treatment of HER2-amplified breast cancer by targeting HER3 and β1 integrin

Abstract The central role of HER2 as the disease driver and HER3 as its essential partner has made them rational targets for the treatment of HER2-amplifed breast cancers, and there is considerable interest in developing highly effective treatment regimens for this disease that consist of targeted therapies alone. Much of these efforts are focused on dual targeting approaches, particularly dual targeting of the HER2-HER3 tumor driver complex itself, or vertical combinations that target downstream PI3K or Akt in addition to HER2. There is also potential in lateral combinations based on evidence implicating cross-talk with other membrane receptor systems, particularly integrins, and such lateral combinations can potentially involve either HER2 or HER3. We established a preclinical model of targeting HER3 using doxycycline-inducible shRNA and determined the efficacy of a β1 integrin inhibitor in combination with targeting HER3. We report that targeting HER3 and β1 integrin provides a particularly effective combination therapy approach for HER2-amplified cancers, surpassing the combination of HER2 and β1 integrin targeting, and evading some of the safety concerns associated with direct HER2-targeting. This further validates HER3 as a major hub mediating the tumorigenic functions of HER2 and identifies it as a high value target for lateral combination therapy strategies.

Alternative polyadenylation in a family of paralogous EPB41 genes generates protein 4.1 diversity

ABSTRACT Alternative polyadenylation (APA) is a step in mRNA 3′-end processing that contributes to the complexity of the transcriptome by generating isoforms that differ in either their coding sequence or their 3′-untranslated regions (UTRs). The EPB41 genes, EPB41, EPB41L2, EPB41L3 and EPB41L1, encode an impressively complex array of structural adaptor proteins (designated 4.1R, 4.1G, 4.1B and 4.1N, respectively) by using alternative transcriptional promoters and tissue-specific alternative pre-mRNA splicing. The great variety of 4.1 proteins mainly results from 5′-end and internal processing of the EPB41 pre-mRNAs. Thus, 4.1 proteins can vary in their N-terminal extensions but all contain a highly homologous C-terminal domain (CTD). Here we study a new group of EPB41-related mRNAs that originate by APA and lack the exons encoding the CTD characteristic of prototypical 4.1 proteins, thereby encoding a new type of 4.1 protein. For the EPB41 gene, this type of processing was observed in all 11 human tissues analyzed. Comparative genomic analysis of EPB41 indicates that APA is conserved in various mammals. In addition, we show that APA also functions for the EPB41L2, EPB41L3 and EPB41L1 genes, but in a more restricted manner in the case of the latter 2 than it does for the EPB41 and EPB41L2 genes. Our study shows alternative polyadenylation to be an additional mechanism for the generation of 4.1 protein diversity in the already complex EPB41-related genes. Understanding the diversity of EPB41 RNA processing is essential for a full appreciation of the many 4.1 proteins expressed in normal and pathological tissues.

Chemical probing of HER2-amplified cancer cells identifies TORC2 as a particularly effective secondary target for combination with lapatinib.

The clinical impact of HER2 inhibitors in the treatment of HER2-amplified breast cancers has been largely confined to chemotherapy combination regimens, since HER2 inhibitors appear to have very modest efficacies by themselves. This is due to the resilient nature of the functionally relevant HER2-HER3 tumor driver, bidirectionally linked with downstream PI3K/Akt pathway signaling, which can break through the inhibitory effects of most current HER2 or HER3 targeting therapies. A vertical combination approach targeting HER2 and a downstream pathway is a highly rational strategy for much more effective targeted therapy of this disease. However the importance of these downstream pathways in many human tissues and cells significant limits their usefulness as secondary targets by narrowing the therapeutic index of such combination therapies. The secondary target that can afford the highest potential for clinical translation is the one with the highest synergy against tumor cells in combination with HER2-inhibition, allowing the widest therapeutic index for clinical translation. We conducted a comparative analysis of such secondary targets in combination with the HER2 inhibitor lapatinib and find that the inhibition of mTor affords the highest degree of synergy. In further dissecting the individual roles of TORC1 and TORC2 complexes using pharmacologic and genetic tools, we find that it is specifically the inactivation of TORC2 that most synergistically enhances the efficacy of lapatinib. Although inhibitors that selectively target TORC2 are not currently available, these data make a compelling case for their development.

A Dimerization Function in the Intrinsically Disordered N-Terminal Region of Src

SUMMARY The mode of regulation of Src kinases has been elucidated by crystallographic studies identifying conserved structured protein modules involved in an orderly set of intramolecular associations and ligand interactions. Despite these detailed insights, much of the complex behavior and diversity in the Src family remains unexplained. A key missing piece is the function of the unstructured N-terminal region. We report here the function of the N-terminal region in binding within a hydrophobic pocket in the kinase domain of a dimerization partner. Dimerization substantially enhances autophosphorylation and phosphorylation of selected substrates, and interfering with dimerization is disruptive to these functions. Dimerization and Y419 phosphorylation are codependent events creating a bistable switch. Given the versatility inherent in this intrinsically disordered region, its multisite phosphorylations, and its divergence within the family, the unique domain likely functions as a central signaling hub overseeing much of the activities and unique functions of Src family kinases.

HER2 Amplification in Tumors Activates PI3K/Akt Signaling Independent of HER3

Current evidence suggests that HER2-driven tumorigenesis requires HER3. This is likely due to the unique ability of HER3 to activate PI3K/Akt pathway signaling, which is not directly accessible to HER2. By genetic elimination of HER3 or shRNA knockdown of HER3 in HER2-amplified cancer cells, we find residual HER2-driven activation of PI3K/Akt pathway signaling that is driven by HER2 through direct and indirect mechanisms. Indirect mechanisms involved second messenger pathways, including Ras or Grb2. Direct binding of HER2 to PI3K occurred through p-Tyr1139, which has a weak affinity for PI3K but becomes significant at very high expression and phosphorylation. Mutation of Y1139 impaired the tumorigenic competency of HER2. Total elimination of HER3 expression in HCC1569 HER2-amplified cancer cells significantly impaired tumorigenicity only transiently, overcome by subsequent increases in HER2 expression and phosphorylation with binding and activation of PI3K. In contrast to activation of oncogenes by mutation, activation by overexpression was quantitative in nature: weak intrinsic activities were strengthened by overexpression, with additional gains observed through further increases in expression. Collectively, these data show that progressive functional gains by HER2 can increase its repertoire of activities such as the activation of PI3K and overcome its dependency on HER3.Significance: The intrinsic ability of HER2 to activate PI3K correlates with increased HER2 expression and can supplant the dependency upon HER3 for growth in HER2-amplified cancers. Cancer Res; 78(13); 3645-58. ©2018 AACR.