Jared L. Johnson

Cancer cell-derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells.

Tumor progression involves the ability of cancer cells to communicate with each other and with neighboring normal cells in their microenvironment. Microvesicles (MV) derived from human cancer cells have received a good deal of attention because of their ability to participate in the horizontal transfer of signaling proteins between cancer cells and to contribute to their invasive activity. Here we show that MV may play another important role in oncogenesis. In particular, we demonstrate that MV shed by two different human cancer cells, MDAMB231 breast carcinoma cells and U87 glioma cells, are capable of conferring onto normal fibroblasts and epithelial cells the transformed characteristics of cancer cells (e.g., anchorage-independent growth and enhanced survival capability) and that this effect requires the transfer of the protein cross-linking enzyme tissue transglutaminase (tTG). We further demonstrate that tTG is not sufficient to transform fibroblasts but rather that it must collaborate with another protein to mediate the transforming actions of the cancer cell-derived MV. Proteomic analyses of the MV derived from MDAMB231 and U87 cells indicated that both these vesicle preparations contained the tTG-binding partner and cross-inking substrate fibronectin (FN). Moreover, we found that tTG cross-links FN in MV from cancer cells and that the ensuing MV-mediated transfers of cross-linked FN and tTG to recipient fibroblasts function cooperatively to activate mitogenic signaling activities and to induce their transformation. These findings highlight a role for MV in the induction of cellular transformation and identify tTG and FN as essential participants in this process.

New Insights into How the Rho Guanine Nucleotide Dissociation Inhibitor Regulates the Interaction of Cdc42 with Membranes

The subcellular localization of the Rho family GTPases is of fundamental importance to their proper functioning in cells. The Rho guanine nucleotide dissociation inhibitor (RhoGDI) plays a key regulatory role by influencing the cellular localization of Rho GTPases and is essential for the transforming activity of oncogenic forms of Cdc42. However, the mechanism by which RhoGDI helps Cdc42 to undergo the transition between a membrane-associated protein and a soluble (cytosolic) species has been poorly understood. Here, we examine how RhoGDI influences the binding of Cdc42 to lipid bilayers. Despite having similar affinities for the signaling-inactive (GDP-bound) and signaling-active (GTP-bound) forms of Cdc42 in solution, we show that when RhoGDI interacts with Cdc42 along the membrane surface, it has a much higher affinity for GDP-bound Cdc42 compared with its GTP-bound counterpart. Interestingly, the rate for the dissociation of Cdc42·RhoGDI complexes from membranes is unaffected by the nucleotide-bound state of Cdc42. Moreover, the membrane release of Cdc42·RhoGDI complexes occurs at a similar rate as the release of Cdc42 alone, with the major effect of RhoGDI being to impede the re-association of Cdc42 with membranes. These findings lead us to propose a new model for how RhoGDI influences the ability of Cdc42 to move between membranes and the cytosol, which highlights the role of the membrane in helping RhoGDI to distinguish between the GDP- and GTP-bound forms of Cdc42 and holds important implications for how it functions as a key regulator of the cellular localization and signaling activities of this GTPase.

Establishment of a robust single axis of cell polarity by coupling multiple positive feedback loops

Establishment of cell polarity—or symmetry breaking—relies on local accumulation of polarity regulators. Although simple positive feedback is sufficient to drive symmetry breaking, it is highly sensitive to stochastic fluctuations typical for living cells. Here, by integrating mathematical modelling with quantitative experimental validations, we show that in the yeast Saccharomyces cerevisiae a combination of actin- and guanine nucleotide dissociation inhibitor-dependent recycling of the central polarity regulator Cdc42 is needed to establish robust cell polarity at a single site during yeast budding. The guanine nucleotide dissociation inhibitor pathway consistently generates a single-polarization site, but requires Cdc42 to cycle rapidly between its active and inactive form, and is therefore sensitive to perturbations of the GTPase cycle. Conversely, actin-mediated recycling of Cdc42 induces robust symmetry breaking but cannot restrict polarization to a single site. Our results demonstrate how cells optimize symmetry breaking through coupling between multiple feedback loops.

A unique role for heat shock protein 70 and its binding partner tissue transglutaminase in cancer cell migration

Cell migration is essential for several important biological outcomes and is involved in various developmental disorders and disease states including cancer cell invasiveness and metastasis. A fundamental step in cell migration is the development of a leading edge. By using HeLa carcinoma cells as an initial model system, we uncovered a surprising role for the heat shock protein 70 (Hsp70) and its ability to bind the protein cross-linking enzyme, tissue transglutaminase (tTG), in cancer cell migration. Treatment of HeLa cells with EGF results in the activation of a plasma membrane-associated pool of tTG and its redistribution to the leading edges of these cells, which are essential events for EGF-stimulated HeLa cell migration. However, we then found that the ability of tTG to be localized to the leading edge is dependent on Hsp70. Similarly, the localization of tTG to the leading edges of MDAMB231 breast carcinoma cells, where it also plays an essential role in their migration, has a strict requirement for Hsp70. Treatment of these different cell lines with inhibitors against the ATP hydrolytic activity of Hsp70 prevented tTG from localizing to their leading edges and thereby blocked EGF-stimulated HeLa cell migration, as well as the constitutive migration normally exhibited by MDAMB231 cells. These findings highlight a new and unconventional role for the chaperonin activity of Hsp70 in the localization of a key regulatory protein (tTG) at the leading edges of cancer cells and the important consequences that this holds for their ability to migrate.

Prenylation and membrane localization of Cdc42 are essential for activation by DOCK7

The unconventional guanine nucleotide exchange factor (GEF) family comprising 11 DOCK180 related proteins is classified into four subfamilies, A through D, based on their relative GEF activity toward the closely related Rac and Cdc42 GTPases. DOCK proteins participate in the remodeling of the actin cytoskeleton and are key regulators of cell motility, phagocytosis, and adhesion. Here we show that the guanine nucleotide exchange domain of DOCK7, DHR2 (for DOCK homology region 2), is a potent GEF for prenylated Cdc42 and Rac1 in a model liposome system, demonstrating that the prenylation and membrane localization of Cdc42 or Rac1 are necessary for their activation by DOCK7. Additionally, we identify DOCK7 residues that confer GTPase GEF specificity. Finally, using our liposome reconstitution assay, we show that a more narrowly defined GEF domain of DHR2 (designated DHR2s) harbors an N-terminal site distinct from the GEF active site that binds preferentially to the active, GTP-bound forms of Cdc42 and Rac1 and thereby recruits free DHR2s from solution to the membrane surface. This recruitment results in a progressive increase in the effective concentration of DHR2s at the membrane surface that in turn provides for an accelerated rate of guanine nucleotide exchange on Cdc42. The positive cooperativity observed in our reconstituted system suggests that the action of DOCK7 in vivo may involve the coordinated integration of Cdc42/Rac signaling in the context of the membrane recruitment of a DOCK7 GEF complex.

The C-terminal di-arginine motif of Cdc42 is essential for binding to phosphatidylinositol 4,5 bisphosphate-containing membranes and inducing cellular transformation

Background: We have examined the role of the polybasic domain of Cdc42 in its membrane association and transforming capability. Results: We show that a di-arginine motif within Cdc42 is essential for binding to PIP2-containing membranes and cellular transformation. Conclusion: These findings demonstrate that Cdc42 binds to specific membrane sites to trigger oncogenic transformation. Significance: These findings shed new light on how Cdc42 initiates transforming signals. Rho GTPases regulate a diverse range of processes that are dependent on their proper cellular localization. The membrane localization of these GTPases is due in large part to their carboxyl-terminal geranylgeranyl moiety. In addition, most of the Rho family members contain a cluster of positively charged residues (i.e. a “polybasic domain”), directly preceding their geranylgeranyl moiety, and it has been suggested that this domain serves to fine-tune their localization among different cellular membrane sites. Here, we have taken a closer look at the role of the polybasic domain of Cdc42 in its ability to bind to membranes and induce the transformation of fibroblasts. A FRET assay for the binding of Cdc42 to liposomes of defined composition showed that Cdc42 associates more strongly with liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP2) when compared either with uncharged control membranes or with liposomes containing a charge-equivalent amount of phosphatidylserine. The carboxyl-terminal di-arginine motif (Arg-186 and Arg-187) was shown to play an essential role in the binding of Cdc42 to PIP2-containing membranes. We further showed that substitutions for the di-arginine motif, when introduced within a constitutively active (“fast cycling”) Cdc42(F28L) background, had little effect on the ability of the activated Cdc42 mutant to induce microspikes/filopodia in NIH 3T3 cells, whereas they eliminated its ability to transform fibroblasts. Taken together, these findings suggest that the di-arginine motif within the carboxyl terminus of Cdc42 is necessary for this GTPase to bind at membrane sites containing PIP2, where it can initiate signaling activities that are essential for the oncogenic transformation of cells.

EGF-receptor specificity for phosphotyrosine-primed substrates provides signal integration with Src

Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK pathway and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Using peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, thus promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly than singly phosphorylated peptide to the Ras activator Grb2; this binding is a key step in activating the Ras-MAPK pathway. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras.

Human primary immunodeficiency caused by expression of a kinase-dead p110δ mutant

This case demonstrates the essential contribution of the p110δ catalytic domain in adaptive immunity function in a patient with expression of a kinase-dead p110δ mutant.

The chromatin remodeler RSF1 controls centromeric histone modifications to coordinate chromosome segregation

Chromatin remodelers regulate the nucleosome barrier during transcription, DNA replication, and DNA repair. The chromatin remodeler RSF1 is enriched at mitotic centromeres, but the functional consequences of this enrichment are not completely understood. Shugoshin (Sgo1) protects centromeric cohesion during mitosis and requires BuB1-dependent histone H2A phosphorylation (H2A-pT120) for localization. Loss of Sgo1 at centromeres causes chromosome missegregation. Here, we show that RSF1 regulates Sgo1 localization to centromeres through coordinating a crosstalk between histone acetylation and phosphorylation. RSF1 interacts with and recruits HDAC1 to centromeres, where it counteracts TIP60-mediated acetylation of H2A at K118. This deacetylation is required for the accumulation of H2A-pT120 and Sgo1 deposition, as H2A-K118 acetylation suppresses H2A-T120 phosphorylation by Bub1. Centromeric tethering of HDAC1 prevents premature chromatid separation in RSF1 knockout cells. Our results indicate that RSF1 regulates the dynamics of H2A histone modifications at mitotic centromeres and contributes to the maintenance of chromosome stability.The chromatin remodeler RSF1 is enriched at mitotic centromeres but its function there is poorly understood. Here, the authors show that RSF1 regulates H2A phosphorylation and acetylation at mitotic centromeres and contributes to chromosome stability.

Abstract 4714: Targeting p53 mutant cancers through inhibition of the phosphatidylinositol-5-phosphate 4-kinases

The bulk of cellular phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) is generated by the canonical pathway in which a 5-kinase converts phosphatidylinositol-4-phosphate (PI-4-P) to PI-4,5-P2. However, several years ago we discovered that PI-4, 5-P2 can also be generated at intracellular sites by a family of kinases that phosphorylate the 4 position of phosphatidylinositol-5-phosphate (PI-5-P), a lipid that was not previously known to exist in nature. These enzymes are called the type 2 phosphatidylinositol-5-phosphate 4-kinases (PI5P4Ks). To date, we have shown that a subset of breast cancers express high levels of PI5P4Kα and/or β, and have provided evidence that these kinases are essential for growth in the absence of p53 (Emerling et al., 2013). Furthermore, we showed that PI5P4Kα and β play critical roles in mediating changes in metabolism in response to cellular stress, in particular, stress that occurs in the absence of p53. Here, we disclose the discovery of potent and selective inhibitors of PI5P4K (Kis in the range of 10 to 200 nM). These novel PI5P4K inhibitors have provided us with powerful tools to further investigate the role of the PI5P4K enzymes in cancer metabolism. Through pharmacological inhibition of PI5P4K, we now have support that PI5P4K provides an alternative pathway to p53 in regard to mediating responses to metabolic and oxidative stress, thereby suggesting that the PI5P4K enzymes are essential for survival mechanisms when p53 function is lost. Above all, these inhibitors may be effective therapies not only for breast cancers with genetic aberrations in TP53, but in all cancer types with loss of p53 function. Supported by DOD Breast Research Program Breakthrough Award to B.M.E. References Emerling BM, Hurov JB, Poulogiannis G, Tsukazawa KS, Wulf G, Bell EL, Shim H, Choo-Wing R, Bellinger G, Lamia KA, Rameh LE, Sasaki A, Asara JM, Yuan X, Bullock A, Brown V, Signoretti S, and Cantley LC. (2013) Depletion of a Putatively Druggable Class of Phosphatidylinositol Kinases Inhibits Growth of p53 Null Tumors. Cell. Nov 7; 155 (4): 844-57. Citation Format: Brooke M. Emerling, Zhiwei Yang, Ryan Loughran, T.Jonathan Yang, Jared Johnson, Rajan Pragani, Mindy Davis, Min Shen, Matthew Boxer, Anton Simeonov, Lewis C. Cantley. Targeting p53 mutant cancers through inhibition of the phosphatidylinositol-5-phosphate 4-kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4714. doi:10.1158/1538-7445.AM2015-4714