Bernard Liu | Gilead - Academia.edu (original) (raw)

Papers by Bernard Liu

The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the h... more The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the heart of phosphotyrosine signaling. Since its serendipitous discovery, there has been a tremendous advancement in technologies and an array of techniques available for studying SH2 domains and phosphotyrosine signaling. In this chapter, we provide a glimpse of the history of SH2 domains and describe many of the tools and techniques that have been developed along the way and discuss future directions for SH2 domain studies. We highlight the gist of each chapter in this volume in the context of: the structural biology and phosphotyrosine binding; characterizing SH2 specificity and generating prediction models; systems biology and proteomics; SH2 domains in signal transduction; and SH2 domains in disease, diagnostics, and therapeutics. Many of the individual chapters provide an in-depth approach that will allow scientists to interrogate the function and role of SH2 domains.

Cancer Research, 2018

SGN-CD48A is a novel multiple myeloma (MM) antibody-drug conjugate (ADC) composed of a CD48-direc... more SGN-CD48A is a novel multiple myeloma (MM) antibody-drug conjugate (ADC) composed of a CD48-directed antibody conjugated to 8 molecules of a cytotoxic antimitotic drug, monomethyl auristatin E (MMAE), via a next generation β-glucuronidase-cleavable linker. Previously, we showed that SGN-CD48A has potent single agent antitumor activity against MM cells, and produces durable complete remissions in mouse xenograft models. Here, we describe the bystander effect activity and immunogenic cell death (ICD) potential of SGN-CD48A in MM models. In addition, combination studies with approved MM therapeutic antibodies were explored. ADC bystander effect is the additional cytotoxic activity on antigen-negative cells in the presence of antigen-positive tumor cells. To measure bystander effect, we generated CD48 knock-out (KO) myeloma cell lines, which also express luciferase (Luc+). SGN-CD48A demonstrated bystander effect cell killing of the CD48KO-Luc+ cells when co-cultured with parental CD48+ ...

Methods in molecular biology (Clifton, N.J.), 2017

Today there exists a rapidly expanding number of sequenced genomes. Cataloging protein interactio... more Today there exists a rapidly expanding number of sequenced genomes. Cataloging protein interaction domains such as the Src Homology 2 (SH2) domain across these various genomes can be accomplished with ease due to existing algorithms and predictions models. An evolutionary analysis of SH2 domains provides a step towards understanding how SH2 proteins integrated with existing signaling networks to position phosphotyrosine signaling as a crucial driver of robust cellular communication networks in metazoans. However organizing and tracing SH2 domain across organisms and understanding their evolutionary trajectory remains a challenge. This chapter describes several methodologies towards analyzing the evolutionary trajectory of SH2 domains including a global SH2 domain classification system, which facilitates annotation of new SH2 sequences essential for tracing the lineage of SH2 domains throughout eukaryote evolution. This classification utilizes a combination of sequence homology, prot...

Journal of Biological Chemistry, 2004

Cancer Research, 2020

The antibody-drug conjugate enfortumab vedotin (EV; AGS22C3E) targets Nectin-4 expressing tumor c... more The antibody-drug conjugate enfortumab vedotin (EV; AGS22C3E) targets Nectin-4 expressing tumor cells by delivering MMAE, a potent microtubule disrupting agent, to induce cell death. EV has demonstrated single agent activity and encouraging activity (71% ORR) when combined with pembrolizumab (anti-PD-1) in the 1L setting of cis-ineligible metastatic urothelial carcinoma (mUC) (EV-103, NCT03288545). Here we demonstrated that EV may promote multiple mechanisms of action including bystander cell killing and hallmarks of immunogenic cell death (ICD) including ER stress, immune cell recruitment and activation. Two urothelial carcinoma models, T-24 and UM-UC-3, were engineered to express Nectin-4, and both were sensitive to EV in vitro and in vivo. In these Nectin-4 expressing cell lines, EV internalized with Nectin-4, trafficked to lysosomal vesicles, and released intracellular MMAE as shown by intracellular MMAE accumulation. In addition, EV demonstrated a bystander effect by release of...

Journal of Cell Science

The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name... more The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and ...

Mutated in Colorectal Cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests... more Mutated in Colorectal Cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, and that Mcc protein is distinctly associated with the centrosome in these cells. Upon intestinal cellular differentiation, Mcc is redeployed to the non-centrosomal microtubule organizing center (ncMTOC) at the apical domain of villus cells. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by Casein Kinases 1δ/ε, which are critical modulators of WNT signaling. Together, our findings support a putative role for MCC in establishing and maintaining the cellular arc...

High-throughput analysis of peptide-binding modules

Journal of Virology, 2000

We have used coreceptor-targeted inhibitors to investigate which coreceptors are used by human im... more We have used coreceptor-targeted inhibitors to investigate which coreceptors are used by human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency viruses (SIV), and human immunodeficiency virus type 2 (HIV-2) to enter peripheral blood mononuclear cells (PBMC). The inhibitors are TAK-779, which is specific for CCR5 and CCR2, aminooxypentane-RANTES, which blocks entry via CCR5 and CCR3, and AMD3100, which targets CXCR4. We found that for all the HIV-1 isolates and all but one of the HIV-2 isolates tested, the only relevant coreceptors were CCR5 and CXCR4. However, one HIV-2 isolate replicated in human PBMC even in the presence of TAK-779 and AMD3100, suggesting that it might use an undefined, alternative coreceptor that is expressed in the cells of some individuals. SIV mac 239 and SIV mac 251 (from macaques) were also able to use an alternative coreceptor to enter PBMC from some, but not all, human and macaque donors. The replication in human PBMC of SIV rcm (from a red-c...

Cancer Research

Enfortumab vedotin (EV) is a monomethyl auristatin E (MMAE) containing antibody-drug conjugate di... more Enfortumab vedotin (EV) is a monomethyl auristatin E (MMAE) containing antibody-drug conjugate directed to Nectin-4, which is highly expressed in bladder cancers. Preclinically, EV has demonstrated tumor cell-killing by direct cytotoxicity, bystander toxicity, and induction of the hallmarks of immunogenic cell death. In EV-301, a phase 3 clinical study, EV monotherapy showed an overall survival (OS) benefit vs chemotherapy in patients with locally advanced or metastatic urothelial carcinoma (la/mUC) who had previously received platinum-based therapy and a PD-1 or PD-L1 inhibitor (Powles 2021). EV also has encouraging activity in combination with pembrolizumab in previously untreated la/mUC (73% ORR)(Friedlander 2021). Most newly diagnosed bladder cancer cases are non-muscle invasive (Chang 2016; Woldu 2017; Kates 2020; Li 2020). Standard treatment of high risk NMIBC involves transurethral resection of the bladder tumor followed by intravesical (IVES) Bacillus Calmette-Guerin (BCG) o...

Methods in molecular biology (Clifton, N.J.), 2017

The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the h... more The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the heart of phosphotyrosine signaling. Since its serendipitous discovery, there has been a tremendous advancement in technologies and an array of techniques available for studying SH2 domains and phosphotyrosine signaling. In this chapter, we provide a glimpse of the history of SH2 domains and describe many of the tools and techniques that have been developed along the way and discuss future directions for SH2 domain studies. We highlight the gist of each chapter in this volume in the context of: the structural biology and phosphotyrosine binding; characterizing SH2 specificity and generating prediction models; systems biology and proteomics; SH2 domains in signal transduction; and SH2 domains in disease, diagnostics, and therapeutics. Many of the individual chapters provide an in-depth approach that will allow scientists to interrogate the function and role of SH2 domains.

Journal of Biological Chemistry, May 25, 2004

SUMMARY Bone morphogenetic proteins (BMPs) belong to the TGFβ superfamily and play an important r... more SUMMARY Bone morphogenetic proteins (BMPs) belong to the TGFβ superfamily and play an important role in development and many cellular processes. We have found that BMP-2, BMP-6, and BMP-9 induce the most potent osteogenic differentiation of mesenchymal stem cells. Expression profiling analysis has revealed that the Inhibitor of DNA binding/differentiation (Id)-1, Id-2, and Id-3 are among the most significantly up-regulated genes upon BMP-2, BMP-6, or BMP-9 stimulation. Here, we sought to determine the ...

To reduce the errors associated with identifying false-positives, non-specific interacting peptid... more To reduce the errors associated with identifying false-positives, non-specific interacting peptides (so-called 'sticky'peptides) were identified in two ways. Peptides that bound to GST, reflected by an intensity above the mean in two out of three separate trials, were tagged as non-specific binding peptides. Alternatively, peptides that exhibited above-mean binding to the majority of individual SH2 domains were used to corroborate an assessment of “non-specific”. This latter method relies upon the data-distribution properties obtained by ...

In the past few years, a number of technologies have been described with the overall goal of prof... more In the past few years, a number of technologies have been described with the overall goal of profiling SH2 domain binding sites (Table S2). The strengths and weaknesses of the various approaches are outlined here and compared to the SH2 Rosette strategy. MacBeath’s group developed a forward-phase assay using Microarrays In Microtiter plates (MIMs) technology, in which a virtually complete set of human SH2 and PTB domains was immobilized in microarrays aligned with the wells of a 96-well plate, thereby allowing simultaneous probing of multiple microarrays (Jones et al., 2006). Using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of EGF receptor family members, they quantitatively analyzed more than 5,000 interactions with SH2 and PTB domains. Panomics has introduced a conceptually similar membrane-based SH2 domain macro-array with 34 SH2 domains, and described its use for phosphorylated peptides, recombinant proteins and cell lysates (Panomics). T...

PDF corresponds to the article as it appeared upon acceptance. Copyedited and fully formatted PDF... more PDF corresponds to the article as it appeared upon acceptance. Copyedited and fully formatted PDF and full text (HTML) versions will be made available soon. Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling

The ability of a cancer cell to avoid apoptosis is crucial to tumorigenesis and can also contribu... more The ability of a cancer cell to avoid apoptosis is crucial to tumorigenesis and can also contribute to chemoresistance. The Bcl-2 family of prosurvival proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1) plays a key role in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small-molecule inhibitor of Bcl-2, Bcl-XL, andBcl-w.While navitoclax exhibits single-agent activity in tumors dependent on Bcl-2 or Bcl-XL for survival, the expression ofMcl-1 has been shown to confer resistance to navitoclax, most notably in solid tumors. Thus, therapeutic agents that can downregulate or neutralize Mcl-1 are predicted to synergize potently with navitoclax. Here, we report the activity of navitoclax in combination with 19 clinically relevant agents across a panel of 46 human solid tumor cell lines. Navitoclax broadly enhanced the activity of multiple therapeuticagents in vitroandenhancedefficacyofbothdocetaxelanderlotinib inxenograftmodels.Theabilityof navitoclax t...

A cancer cell’s ability to avoid apoptosis is crucial to tumorigenesis and can also contribute to... more A cancer cell’s ability to avoid apoptosis is crucial to tumorigenesis and can also contribute to chemoresistance. The Bcl-2 family of pro-survival proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1) plays a key role in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small molecule inhibitor of Bcl-2, Bcl-XL and Bcl-w. While navitoclax exhibits single-agent activity in tumors dependent on Bcl-2 or Bcl-XL for survival, the expression of Mcl-1 has been shown to confer resistance to navitoclax, most notably in solid tumors. Thus, therapeutic agents that can downregulate or neutralize Mcl-1 are predicted to synergize potently with navitoclax. Here we report the activity of navitoclax in combination with 19 clinically relevant agents across a panel of 46 human solid tumor cell lines. Navitoclax broadly enhanced the activity of multiple therapeutic agents in vitro and enhanced efficacy of both docetaxel and erlotinib in xenograft models. The ability of na...

Journal for ImmunoTherapy of Cancer

BackgroundEffective cancer treatment requires durable elimination of malignant cells. Cytotoxic c... more BackgroundEffective cancer treatment requires durable elimination of malignant cells. Cytotoxic chemotherapeutic agents used to treat cancer often show initial anti-tumor efficacy, but fail to produce long-term durable responses in patients. The elicitation of durable responses and improved survival in response to cytotoxic agents may be associated with the induction of innate and adaptive immune response to the cancer. For example, tumor cells undergoing apoptosis following exposure to some cytotoxic agents emit immunostimulatory damage-associated molecular patterns (DAMPs), this form of cell death is termed immunogenic cell death (ICD). ICD can promote the recruitment and activation of both the innate and adaptive immune system, providing an additional mechanism to drive an anti-tumor response.MethodsVedotin-based antibody drug conjugates (ADCs) drive cytotoxicity in tumor cells by engaging tumor antigens on the cell surface, internalizing with the cell surface antigen, and delive...

Methods in molecular biology (Clifton, N.J.), 2017

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating sign... more The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alt...

The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the h... more The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the heart of phosphotyrosine signaling. Since its serendipitous discovery, there has been a tremendous advancement in technologies and an array of techniques available for studying SH2 domains and phosphotyrosine signaling. In this chapter, we provide a glimpse of the history of SH2 domains and describe many of the tools and techniques that have been developed along the way and discuss future directions for SH2 domain studies. We highlight the gist of each chapter in this volume in the context of: the structural biology and phosphotyrosine binding; characterizing SH2 specificity and generating prediction models; systems biology and proteomics; SH2 domains in signal transduction; and SH2 domains in disease, diagnostics, and therapeutics. Many of the individual chapters provide an in-depth approach that will allow scientists to interrogate the function and role of SH2 domains.

Cancer Research, 2018

SGN-CD48A is a novel multiple myeloma (MM) antibody-drug conjugate (ADC) composed of a CD48-direc... more SGN-CD48A is a novel multiple myeloma (MM) antibody-drug conjugate (ADC) composed of a CD48-directed antibody conjugated to 8 molecules of a cytotoxic antimitotic drug, monomethyl auristatin E (MMAE), via a next generation β-glucuronidase-cleavable linker. Previously, we showed that SGN-CD48A has potent single agent antitumor activity against MM cells, and produces durable complete remissions in mouse xenograft models. Here, we describe the bystander effect activity and immunogenic cell death (ICD) potential of SGN-CD48A in MM models. In addition, combination studies with approved MM therapeutic antibodies were explored. ADC bystander effect is the additional cytotoxic activity on antigen-negative cells in the presence of antigen-positive tumor cells. To measure bystander effect, we generated CD48 knock-out (KO) myeloma cell lines, which also express luciferase (Luc+). SGN-CD48A demonstrated bystander effect cell killing of the CD48KO-Luc+ cells when co-cultured with parental CD48+ ...

Methods in molecular biology (Clifton, N.J.), 2017

Today there exists a rapidly expanding number of sequenced genomes. Cataloging protein interactio... more Today there exists a rapidly expanding number of sequenced genomes. Cataloging protein interaction domains such as the Src Homology 2 (SH2) domain across these various genomes can be accomplished with ease due to existing algorithms and predictions models. An evolutionary analysis of SH2 domains provides a step towards understanding how SH2 proteins integrated with existing signaling networks to position phosphotyrosine signaling as a crucial driver of robust cellular communication networks in metazoans. However organizing and tracing SH2 domain across organisms and understanding their evolutionary trajectory remains a challenge. This chapter describes several methodologies towards analyzing the evolutionary trajectory of SH2 domains including a global SH2 domain classification system, which facilitates annotation of new SH2 sequences essential for tracing the lineage of SH2 domains throughout eukaryote evolution. This classification utilizes a combination of sequence homology, prot...

Journal of Biological Chemistry, 2004

Cancer Research, 2020

The antibody-drug conjugate enfortumab vedotin (EV; AGS22C3E) targets Nectin-4 expressing tumor c... more The antibody-drug conjugate enfortumab vedotin (EV; AGS22C3E) targets Nectin-4 expressing tumor cells by delivering MMAE, a potent microtubule disrupting agent, to induce cell death. EV has demonstrated single agent activity and encouraging activity (71% ORR) when combined with pembrolizumab (anti-PD-1) in the 1L setting of cis-ineligible metastatic urothelial carcinoma (mUC) (EV-103, NCT03288545). Here we demonstrated that EV may promote multiple mechanisms of action including bystander cell killing and hallmarks of immunogenic cell death (ICD) including ER stress, immune cell recruitment and activation. Two urothelial carcinoma models, T-24 and UM-UC-3, were engineered to express Nectin-4, and both were sensitive to EV in vitro and in vivo. In these Nectin-4 expressing cell lines, EV internalized with Nectin-4, trafficked to lysosomal vesicles, and released intracellular MMAE as shown by intracellular MMAE accumulation. In addition, EV demonstrated a bystander effect by release of...

Journal of Cell Science

The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name... more The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and ...

Mutated in Colorectal Cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests... more Mutated in Colorectal Cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, and that Mcc protein is distinctly associated with the centrosome in these cells. Upon intestinal cellular differentiation, Mcc is redeployed to the non-centrosomal microtubule organizing center (ncMTOC) at the apical domain of villus cells. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by Casein Kinases 1δ/ε, which are critical modulators of WNT signaling. Together, our findings support a putative role for MCC in establishing and maintaining the cellular arc...

High-throughput analysis of peptide-binding modules

Journal of Virology, 2000

We have used coreceptor-targeted inhibitors to investigate which coreceptors are used by human im... more We have used coreceptor-targeted inhibitors to investigate which coreceptors are used by human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency viruses (SIV), and human immunodeficiency virus type 2 (HIV-2) to enter peripheral blood mononuclear cells (PBMC). The inhibitors are TAK-779, which is specific for CCR5 and CCR2, aminooxypentane-RANTES, which blocks entry via CCR5 and CCR3, and AMD3100, which targets CXCR4. We found that for all the HIV-1 isolates and all but one of the HIV-2 isolates tested, the only relevant coreceptors were CCR5 and CXCR4. However, one HIV-2 isolate replicated in human PBMC even in the presence of TAK-779 and AMD3100, suggesting that it might use an undefined, alternative coreceptor that is expressed in the cells of some individuals. SIV mac 239 and SIV mac 251 (from macaques) were also able to use an alternative coreceptor to enter PBMC from some, but not all, human and macaque donors. The replication in human PBMC of SIV rcm (from a red-c...

Cancer Research

Enfortumab vedotin (EV) is a monomethyl auristatin E (MMAE) containing antibody-drug conjugate di... more Enfortumab vedotin (EV) is a monomethyl auristatin E (MMAE) containing antibody-drug conjugate directed to Nectin-4, which is highly expressed in bladder cancers. Preclinically, EV has demonstrated tumor cell-killing by direct cytotoxicity, bystander toxicity, and induction of the hallmarks of immunogenic cell death. In EV-301, a phase 3 clinical study, EV monotherapy showed an overall survival (OS) benefit vs chemotherapy in patients with locally advanced or metastatic urothelial carcinoma (la/mUC) who had previously received platinum-based therapy and a PD-1 or PD-L1 inhibitor (Powles 2021). EV also has encouraging activity in combination with pembrolizumab in previously untreated la/mUC (73% ORR)(Friedlander 2021). Most newly diagnosed bladder cancer cases are non-muscle invasive (Chang 2016; Woldu 2017; Kates 2020; Li 2020). Standard treatment of high risk NMIBC involves transurethral resection of the bladder tumor followed by intravesical (IVES) Bacillus Calmette-Guerin (BCG) o...

Methods in molecular biology (Clifton, N.J.), 2017

The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the h... more The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the heart of phosphotyrosine signaling. Since its serendipitous discovery, there has been a tremendous advancement in technologies and an array of techniques available for studying SH2 domains and phosphotyrosine signaling. In this chapter, we provide a glimpse of the history of SH2 domains and describe many of the tools and techniques that have been developed along the way and discuss future directions for SH2 domain studies. We highlight the gist of each chapter in this volume in the context of: the structural biology and phosphotyrosine binding; characterizing SH2 specificity and generating prediction models; systems biology and proteomics; SH2 domains in signal transduction; and SH2 domains in disease, diagnostics, and therapeutics. Many of the individual chapters provide an in-depth approach that will allow scientists to interrogate the function and role of SH2 domains.

Journal of Biological Chemistry, May 25, 2004

SUMMARY Bone morphogenetic proteins (BMPs) belong to the TGFβ superfamily and play an important r... more SUMMARY Bone morphogenetic proteins (BMPs) belong to the TGFβ superfamily and play an important role in development and many cellular processes. We have found that BMP-2, BMP-6, and BMP-9 induce the most potent osteogenic differentiation of mesenchymal stem cells. Expression profiling analysis has revealed that the Inhibitor of DNA binding/differentiation (Id)-1, Id-2, and Id-3 are among the most significantly up-regulated genes upon BMP-2, BMP-6, or BMP-9 stimulation. Here, we sought to determine the ...

To reduce the errors associated with identifying false-positives, non-specific interacting peptid... more To reduce the errors associated with identifying false-positives, non-specific interacting peptides (so-called 'sticky'peptides) were identified in two ways. Peptides that bound to GST, reflected by an intensity above the mean in two out of three separate trials, were tagged as non-specific binding peptides. Alternatively, peptides that exhibited above-mean binding to the majority of individual SH2 domains were used to corroborate an assessment of “non-specific”. This latter method relies upon the data-distribution properties obtained by ...

In the past few years, a number of technologies have been described with the overall goal of prof... more In the past few years, a number of technologies have been described with the overall goal of profiling SH2 domain binding sites (Table S2). The strengths and weaknesses of the various approaches are outlined here and compared to the SH2 Rosette strategy. MacBeath’s group developed a forward-phase assay using Microarrays In Microtiter plates (MIMs) technology, in which a virtually complete set of human SH2 and PTB domains was immobilized in microarrays aligned with the wells of a 96-well plate, thereby allowing simultaneous probing of multiple microarrays (Jones et al., 2006). Using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of EGF receptor family members, they quantitatively analyzed more than 5,000 interactions with SH2 and PTB domains. Panomics has introduced a conceptually similar membrane-based SH2 domain macro-array with 34 SH2 domains, and described its use for phosphorylated peptides, recombinant proteins and cell lysates (Panomics). T...

PDF corresponds to the article as it appeared upon acceptance. Copyedited and fully formatted PDF... more PDF corresponds to the article as it appeared upon acceptance. Copyedited and fully formatted PDF and full text (HTML) versions will be made available soon. Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling

The ability of a cancer cell to avoid apoptosis is crucial to tumorigenesis and can also contribu... more The ability of a cancer cell to avoid apoptosis is crucial to tumorigenesis and can also contribute to chemoresistance. The Bcl-2 family of prosurvival proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1) plays a key role in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small-molecule inhibitor of Bcl-2, Bcl-XL, andBcl-w.While navitoclax exhibits single-agent activity in tumors dependent on Bcl-2 or Bcl-XL for survival, the expression ofMcl-1 has been shown to confer resistance to navitoclax, most notably in solid tumors. Thus, therapeutic agents that can downregulate or neutralize Mcl-1 are predicted to synergize potently with navitoclax. Here, we report the activity of navitoclax in combination with 19 clinically relevant agents across a panel of 46 human solid tumor cell lines. Navitoclax broadly enhanced the activity of multiple therapeuticagents in vitroandenhancedefficacyofbothdocetaxelanderlotinib inxenograftmodels.Theabilityof navitoclax t...

A cancer cell’s ability to avoid apoptosis is crucial to tumorigenesis and can also contribute to... more A cancer cell’s ability to avoid apoptosis is crucial to tumorigenesis and can also contribute to chemoresistance. The Bcl-2 family of pro-survival proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1) plays a key role in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small molecule inhibitor of Bcl-2, Bcl-XL and Bcl-w. While navitoclax exhibits single-agent activity in tumors dependent on Bcl-2 or Bcl-XL for survival, the expression of Mcl-1 has been shown to confer resistance to navitoclax, most notably in solid tumors. Thus, therapeutic agents that can downregulate or neutralize Mcl-1 are predicted to synergize potently with navitoclax. Here we report the activity of navitoclax in combination with 19 clinically relevant agents across a panel of 46 human solid tumor cell lines. Navitoclax broadly enhanced the activity of multiple therapeutic agents in vitro and enhanced efficacy of both docetaxel and erlotinib in xenograft models. The ability of na...

Journal for ImmunoTherapy of Cancer

BackgroundEffective cancer treatment requires durable elimination of malignant cells. Cytotoxic c... more BackgroundEffective cancer treatment requires durable elimination of malignant cells. Cytotoxic chemotherapeutic agents used to treat cancer often show initial anti-tumor efficacy, but fail to produce long-term durable responses in patients. The elicitation of durable responses and improved survival in response to cytotoxic agents may be associated with the induction of innate and adaptive immune response to the cancer. For example, tumor cells undergoing apoptosis following exposure to some cytotoxic agents emit immunostimulatory damage-associated molecular patterns (DAMPs), this form of cell death is termed immunogenic cell death (ICD). ICD can promote the recruitment and activation of both the innate and adaptive immune system, providing an additional mechanism to drive an anti-tumor response.MethodsVedotin-based antibody drug conjugates (ADCs) drive cytotoxicity in tumor cells by engaging tumor antigens on the cell surface, internalizing with the cell surface antigen, and delive...

Methods in molecular biology (Clifton, N.J.), 2017

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating sign... more The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alt...

To reduce the errors associated with identifying false-positives, non-specific interacting peptid... more To reduce the errors associated with identifying false-positives, non-specific interacting peptides (so-called 'sticky'peptides) were identified in two ways. Peptides that bound to GST, reflected by an intensity above the mean in two out of three separate trials, were tagged as non-specific binding peptides. Alternatively, peptides that exhibited above-mean binding to the majority of individual SH2 domains were used to corroborate an assessment of “non-specific”.

In the past few years, a number of technologies have been described with the overall goal of prof... more In the past few years, a number of technologies have been described with the overall goal of profiling SH2 domain binding sites ). The strengths and weaknesses of the various approaches are outlined here and compared to the SH2 Rosette strategy. MacBeath's group developed a forward-phase assay using Microarrays In Microtiter plates (MIMs) technology, in which a virtually complete set of human SH2 and PTB domains was immobilized in microarrays aligned with the wells of a 96-well plate, thereby allowing simultaneous probing of multiple microarrays . Using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of EGF receptor family members, they quantitatively analyzed more than 5,000 interactions with SH2 and PTB domains. Panomics has introduced a conceptually similar membrane-based SH2 domain macro-array with 34 SH2 domains, and described its use for phosphorylated peptides, recombinant proteins and cell lysates (Panomics). Taking advantage of the Luminex fluorescent bead technology, this group also reported a forward-phase bead-based assay in which different SH2 domains are conjugated to microspheres with distinct fluorophore labels. Unlike traditional pull-down experiments, where one immobilized SH2 domain can be

Section S1. SH2 domain proteins in organisms with incomplete genomes Several recently sequenced g... more Section S1. SH2 domain proteins in organisms with incomplete genomes Several recently sequenced genomes are either incomplete or poorly annotated and, therefore, were not included in the original list of 21 organisms. However, these organisms provide additional insight into the evolution of Metazoa that afford a more complete picture of the evolution of phosphotyrosine (pTyr) signaling. To this end, 7 additional genomes were examined including the choanoflagellate Monosiga ovata, hydrazoan Hydra magnipapillata, placozoan Trichoplax adhaerens, sponges Suberites domuncula and Ephydatia fluviatilis, amphioxus (lancelet) Branchiostoma floridae, and African clawed frog Xenopus laevis. A complete list of SH2 domain proteins from these organisms and the SH2 family to which these proteins belong can be found in table S4.