Breast Cancer Antiestrogen Resistance 3 (BCAR3) Promotes Cell Motility by Regulating Actin Cytoskeletal and Adhesion Remodeling in Invasive Breast Cancer Cells

Metastatic breast cancer is incurable. In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility. Here, we focus on the role of the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) in cellular processes that contribute to cell motility, including protrusion, adhesion remodeling, and contractility. Previous work from our group showed that elevated BCAR3 protein levels enhance cell migration, while depletion of BCAR3 reduces the migratory and invasive capacities of breast cancer cells. In the current study, we show that BCAR3 is necessary for membrane protrusiveness, Rac1 activity, and adhesion disassembly in invasive breast cancer cells. We further demonstrate that, in the absence of BCAR3, RhoA-dependent signaling pathways appear to predominate, as evidenced by an increase in RhoA activity, ROCK-mediated phosphorylation of myosin light chain II, and large ROCK/mDia1-dependent focal adhesions. Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling. Considering that BCAR3 protein levels are elevated in advanced breast cancer cell lines and enhance breast cancer cell motility, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process.     

The Epsin Family of Endocytic Adaptors Promotes Fibrosarcoma Migration and Invasion

Abnormalities in the process of endocytosis are classically linked to malignant transformation through the deficient down-regulation of signaling receptors. The present study describes a non-classical mechanism that does not require internalization by which endocytic proteins affect cell migration and basement membrane invasion. Specifically, we found that the endocytic adaptor epsin binds and regulates the biological properties of the signaling molecule RalBP1 (Ral-binding protein 1). Epsin interacted with the N terminus of RalBP1 via its characteristic epsin N-terminal homology (ENTH) domain. A combination of siRNA-mediated knock-down and transfection of siRNA-resistant constructs in fibrosarcoma cells demonstrated that impairment of the epsin-RalBP1 interaction led to cell migration and basement membrane invasion defects. We found the ENTH domain was necessary and sufficient to sustain normal cell migration and invasion. Because all the epsin endocytic motifs reside in the C-terminal part of the molecule, these results suggest that this novel regulatory circuit does not require endocytosis. In addition, cells depleted of epsin-RalBP1 complex displayed deficient activation of Rac1 and Arf6 suggesting a signaling function for this novel interaction. Further, overexpression of either epsin or RalBP1 enhanced migration and invasion of fibrosarcoma cells. Collectively, our results indicate that epsin regulates RalBP1 function in Rac1- and Arf6-dependent pathways to ultimately affect cell migration and invasion. We propose that the observed up-regulation of both epsin and RalBP1 in certain cancers contributes to their invasive characteristics.     

Phosphorylation of the Cool-1/β-Pix Protein Serves as a Regulatory Signal for the Migration and Invasive Activity of Src-transformed Cells

Previously we showed that Cool-1 (Cloned out of library-1)/β-Pix (Pak-interactive exchange factor) is phosphorylated at a specific tyrosine residue (Tyr-442) in a Src-dependent manner and serves as a dual function guanine nucleotide exchange factor (GEF)/signaling-effector for Cdc42 that is essential for transformation by Src. Here, we show that knocking-down Cool-1 or overexpressing a Cool-1 mutant that contains substitutions within its Dbl homology domain and is defective for GEF activity, inhibits Src-promoted cell migration. Similarly, the expression of a Cool-1 mutant containing a tyrosine to phenylalanine substitution at position 442, making it incapable of being phosphorylated in response to serum, epidermal growth factor (EGF), or Src, also causes a significant inhibition of the migration and invasive activity of cells expressing oncogenic Src. We further demonstrate that the phosphorylation of Cool-1 at Tyr-442 weakens its ability to bind to one of its primary interaction-partners, Cat-1 (Cool-associated tyrosine phosphosubstrate-1)/Git-1 (G protein-coupled receptor kinase-interactor-1), thus making Cat more accessible for binding to paxillin. This enables cells to alternate between states where they contain large numbers of focal complexes (i.e. conditions favoring Cool-1-Cat interactions) versus reduced numbers of focal complexes (conditions favoring Cat-paxillin interactions). Overall, these findings show that the phosphorylation-dephosphorylation cycle of Cool-1 at Tyr-442 can serve as a key regulatory signal for focal complex assembly-disassembly, and consequently, for the migration and invasive activity of Src-transformed cells.     

The Linker for Activation of B Cells (LAB)/Non-T Cell Activation Linker (NTAL) Regulates Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Signaling and Macrophage Inflammatory Responses Independently of the Linker for Activation of T Cells

Triggering receptor expressed on myeloid cells-2 (TREM-2) is rapidly emerging as a key regulator of the innate immune response via its regulation of macrophage inflammatory responses. Here we demonstrate that proximal TREM-2 signaling parallels other DAP12-based receptor systems in its use of Syk and Src-family kinases. However, we find that the linker for activation of T cells (LAT) is severely reduced as monocytes differentiate into macrophages and that TREM-2 exclusively uses the linker for activation of B cells (LAB encoded by the gene Lat2^−/−) to mediate downstream signaling. LAB is required for TREM-2-mediated activation of Erk1/2 and dampens proximal TREM-2 signals through a novel LAT-independent mechanism resulting in macrophages with proinflammatory properties. Thus, Lat2^−/− macrophages have increased TREM-2-induced proximal phosphorylation, and lipopolysaccharide stimulation of these cells leads to increased interleukin-10 (IL-10) and decreased IL-12p40 production relative to wild type cells. Together these data identify LAB as a critical, LAT-independent regulator of TREM-2 signaling and macrophage development capable of controlling subsequent inflammatory responses.     

Attenuation of T Cell Receptor Signaling by Serine Phosphorylation-mediated Lysine 30 Ubiquitination of SLP-76 Protein

SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) is an adaptor protein that is essential for T cell development and T cell receptor (TCR) signaling activation. Previous studies have identified an important negative feedback regulation of SLP-76 by HPK1 (hematopoietic progenitor kinase 1; MAP4K1)-induced Ser-376 phosphorylation. Ser-376 phosphorylation of SLP-76 mediates 14-3-3 binding, resulting in the attenuation of SLP-76 activation and downstream signaling; however, the underlying mechanism of this action remains unknown. Here, we report that phosphorylated SLP-76 is ubiquitinated and targeted for proteasomal degradation during TCR signaling. SLP-76 ubiquitination is mediated by Ser-376 phosphorylation. Furthermore, Lys-30 is identified as a ubiquitination site of SLP-76. Loss of Lys-30 ubiquitination of SLP-76 results in enhanced anti-CD3 antibody-induced ERK and JNK activation. These results reveal a novel regulation mechanism of SLP-76 by ubiquitination and proteasomal degradation of activated SLP-76, which is mediated by Ser-376 phosphorylation, leading to down-regulation of TCR signaling.     

Down-Regulation of Filamin Ainteracting protein 1-like Is Associated with Promoter Methylation and an Invasive Phenotype in Breast,Colon, Lung and Pancreatic Cancers

Identifying key mediators of cancer cell invasion and metastasis is critical to the development of more effective cancer therapies. We previously identified Filamin A interacting protein 1-like (FILIP1L) as an important inhibitor of cell migration and invasion in ovarian cancer. FILIP1L expression was inversely correlated with the invasive potential of ovarian cancer cell lines and ovarian cancer specimens. We also demonstrated that DNA methylation in the FILIP1L promoter was a mechanism by which FILIP1L was down-regulated in ovarian cancer. In our present study, we tested this observation in other cancer histologies: breast, colon, lung and pancreatic cancers. Both mRNA and protein expression of FILIP1L were down-regulated in these cancer cells compared with their normal epithelial cells. As in ovarian cancer, DNA methylation is a mechanism by which FILIP1L is down-regulated in these cancer histologies. Methylation status of the FILIP1L promoter was inversely correlated with FILIP1L expression. Reduced methylation in the FILIP1L promoter following treatment with a DNA demethylating agent was associated with restoration of FILIP1L expression in these cancer cells. Further, FILIP1L expression was inversely correlated with the invasive potential of these cancer cells. Re-expression of FILIP1L in FILIP1L-low expressing, highly-invasive cancer cell lines resulted in inhibition of cell invasion. Correspondingly, knockdown of FILIP1L in FILIP1L-high expressing, low-invasive cancer cell lines resulted in increase of cell invasion. Overall, these findings suggest that down-regulation of FILIP1L associated with DNA methylation is related with the invasive phenotype in various cancers. Thus, modulation of FILIP1L expression has the potential to be a target for cancer therapy.     

An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering an interference reaction. The complete 3′ handle could be removed without loss of activity. However, manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference.     

Structural and mechanistic insights into the inhibition of type I-F CRISPR-Cas system by anti-CRISPR protein AcrIF23

Prokaryotes evolved clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins as a kind of adaptive immune defense against mobile genetic elements including harmful phages. To counteract this defense, many mobile genetic elements in turn encode anti-CRISPR proteins (Acrs) to inactivate the CRISPR-Cas system. While multiple mechanisms of Acrs have been uncovered, it remains unknown whether other mechanisms are utilized by uncharacterized Acrs. Here, we report a novel mechanism adopted by recently identified AcrIF23. We show that AcrIF23 interacts with the Cas2/3 helicase-nuclease in the type I-F CRISPR-Cas system, similar to AcrIF3. The structure of AcrIF23 demonstrated a novel fold and structure-based mutagenesis identified a surface region of AcrIF23 involved in both Cas2/3-binding and its inhibition capacity. Unlike AcrIF3, however, we found AcrIF23 only potently inhibits the DNA cleavage activity of Cas2/3 but does not hinder the recruitment of Cas2/3 to the CRISPR RNA-guided surveillance complex (the Csy complex). Also, in contrast to AcrIF3 which hinders substrate DNA recognition by Cas2/3, we show AcrIF23 promotes DNA binding to Cas2/3. Taken together, our study identifies a novel anti-CRISPR mechanism used by AcrIF23 and highlights the diverse mechanisms adopted by Acrs.     

Appl1 Is Dispensable for Mouse Development, and Loss of Appl1 Has Growth Factor-selective Effects on Akt Signaling in Murine Embryonic Fibroblasts

The adaptor protein APPL1 (adaptor protein containing pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motifs) was first identified as a binding protein of AKT2 by yeast two-hybrid screening. APPL1 was subsequently found to bind to several membrane-bound receptors and was implicated in their signal transduction through AKT and/or MAPK pathways. To determine the unambiguous role of Appl1 in vivo, we generated Appl1 knock-out mice. Here we report that Appl1 knock-out mice are viable and fertile. Appl1-null mice were born at expected Mendelian ratios, without obvious phenotypic abnormalities. Moreover, Akt activity in various fetal tissues was unchanged compared with that observed in wild-type littermates. Studies of isolated Appl1^−/− murine embryonic fibroblasts (MEFs) showed that Akt activation by epidermal growth factor, insulin, or fetal bovine serum was similar to that observed in wild-type MEFs, although Akt activation by HGF was diminished in Appl1^−/− MEFs. To rule out a possible redundant role played by the related Appl2, we used small interfering RNA to knock down Appl2 expression in Appl1^−/− MEFs. Unexpectedly, cell survival was unaffected under normal culture conditions, and activation of Akt was unaltered following epidermal growth factor stimulation, although Akt activity did decrease further after HGF stimulation. Furthermore, we found that Appl proteins are required for HGF-induced cell survival and migration via activation of Akt. Our studies suggest that Appl1 is dispensable for development and only participate in Akt signaling under certain conditions.     

In Vivo Protein Interactions and Complex Formation in the Pectobacterium atrosepticum Subtype I-F CRISPR/Cas System

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas; CRISPR associated) are a bacterial defense mechanism against extra-chromosomal elements. CRISPR/Cas systems are distinct from other known defense mechanisms insofar as they provide acquired and heritable immunity. Resistance is accomplished in multiple stages in which the Cas proteins provide the enzymatic machinery. Importantly, subtype-specific proteins have been shown to form complexes in combination with small RNAs, which enable sequence-specific targeting of foreign nucleic acids. We used Pectobacterium atrosepticum, a plant pathogen that causes soft-rot and blackleg disease in potato, to investigate protein-protein interactions and complex formation in the subtype I-F CRISPR/Cas system. The P. atrosepticum CRISPR/Cas system encodes six proteins: Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4). Using co-purification followed by mass spectrometry as well as directed co-immunoprecipitation we have demonstrated complex formation by the Csy1-3 and Cas6f proteins, and determined details about the architecture of that complex. Cas3 was also shown to co-purify all four subtype-specific proteins, consistent with its role in targeting. Furthermore, our results show that the subtype I-F Cas1 and Cas3 (a Cas2-Cas3 hybrid) proteins interact, suggesting a protein complex for adaptation and a role for subtype I-F Cas3 proteins in both the adaptation and interference steps of the CRISPR/Cas mechanism.     

HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.     

CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

Despite chronic inflammation, psoriatic lesions hardly ever progress to skin cancer. Aberrant function of the CCHCR1 gene (Coiled-Coil α-Helical Rod protein 1, HCR) within the PSORS1 locus may contribute to the onset of psoriasis. As CCHCR1 is expressed in certain cancers and regulates keratinocyte (KC) proliferation in a transgenic mouse model, we studied its relation to proliferation in cutaneous squamous cell cancer (SCC) cell lines by expression arrays and quantitative RT-PCR and in skin tumors by immunohistochemistry. CCHCR1 protein was detected in the pushing border of SCC and lining basal cell carcinoma islands. Different from psoriasis, Ki67 had a similar expression pattern as CCHCR1. The most intense CCHCR1 staining occurred in areas positive for epidermal growth factor receptor (EGFR). Expression of CCHCR1 mRNA was upregulated 30–80% in SCC lines when compared to normal KCs and correlated positively with Ki67 expression. The most aggressive and invasive tumor cell lines (RT3, FaDu) expressed CCHCR1 mRNA less than non-tumorigenic HaCaT cells. Moreover, the tumor promoters okadaic acid and menadione downregulated CCHCR1 mRNA. We conclude that both in psoriasis and the early stages of KC transformation, CCHCR1 may function as a negative regulator of proliferation, but beyond a certain point in oncogenesis cannot control this phenomenon any longer.     

A Brain-specific Grb2-associated Regulator of Extracellular Signal-regulated Kinase (Erk)/Mitogen-activated Protein Kinase (MAPK) (GAREM) Subtype,GAREM2, Contributes to Neurite Outgrowth of Neuroblastoma Cells by Regulating Erk Signaling

Grb2-associated regulator of Erk/MAPK1 (GAREM) is an adaptor molecule in the EGF-mediated signaling pathway. GAREM is expressed ubiquitously in human organs and cultured cells. Two GAREM homologues are encoded by the human genome. Therefore, previously identified GAREM is named GAREM1. Here we characterized a new subtype of GAREM, GAREM2, that is specifically expressed in the mouse, rat, and human brain. Three GAREM2 tyrosines (Tyr-102, Tyr-429, and Tyr-551) are phosphorylated upon EGF stimulation and are necessary for binding to Grb2. Furthermore, GAREM2 and Shp2 regulate Erk activity in EGF-stimulated cells. These characteristics are similar to those of GAREM1. GAREM2 is expressed in some neuroblastoma cell lines and is also tyrosine-phosphorylated and bound to Grb2 after treatment with EGF. Eventually, GAREM2 regulates Erk activation in the presence of EGF or insulin like growth factor 1. GAREM2 also regulates insulin-like growth factor 1-induced neuronal differentiation of the SH-SY5Y neuroblastoma cell line. Although the structure and function of both GAREM subtypes are similar, GAREM1 is recruited into the nucleus and GAREM2 is not. Nuclear localization of GAREM1 might be controlled by a GAREM1-specific nuclear localization sequence and 14-3-3ϵ binding. The N-terminal 20 amino acids of GAREM1 make up its nuclear localization sequence that is also a 14-3-3ϵ binding site. The GAREM family is a new class of adaptor molecules with subtype-specific biological functions.     

All Mammalian Hedgehog Proteins Interact with Cell Adhesion Molecule,Down-regulated by Oncogenes (CDO) and Brother of CDO (BOC) in a Conserved Manner

Hedgehog (Hh) signaling proteins stimulate cell proliferation, differentiation, and tissue patterning at multiple points in animal development. A single Hh homolog is present in Drosophila, but three Hh homologs, Sonic Hh, Indian Hh, and Desert Hh, are present in mammals. Distribution, movement, and reception of Hh signals are tightly regulated, and abnormal Hh signaling is associated with developmental defects and cancer. In addition to the integral membrane proteins Patched and Smoothened, members of the Drosophila Ihog family of adhesion-like molecules have recently been shown to bind Hh proteins with micromolar affinity and positively regulate Hh signaling. Cell adhesion molecule-related, down-regulated by oncogenes (CDO) and Brother of CDO (BOC) are the closest mammalian relatives of Drosophila Ihog, and CDO binds Sonic Hh with micromolar affinity and positively regulates Hh signaling. Despite these similarities, structural and biochemical studies have shown that Ihog and CDO utilize nonorthologous domains and completely different binding modes to interact with cognate Hh proteins. We report here biochemical and x-ray structural studies of Sonic, Indian, and Desert Hh proteins both alone and complexed with active domains of CDO and BOC. These results show that all mammalian Hh proteins bind CDO and BOC in the same manner. We also show that interactions between Hh proteins and CDO are weakened at low pH. Formation of Hh-mediated Hh oligomers is thought to be an important feature of normal Hh signaling, but no conserved self-interaction between Hh proteins is apparent from inspection of 14 independent Hh-containing crystal lattices.     

A Dynamic cpSRP43-Albino3 Interaction Mediates Translocase Regulation of Chloroplast Signal Recognition Particle (cpSRP)-targeting Components

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.     

Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3)

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.