Grb7 is a member of the Grb7 protein family, which also includes Grb10 and Grb14. These adaptor proteins function mainly as scaffolds in phosphorylation-dependent signaling pathways. The family is known to interact with receptor tyrosine kinases and other signaling molecules, and their activity is influenced by protein-protein interactions within their conserved five-domain structure. Grb7 has also been linked to cancer progression and is thought to form dimers and undergo intramolecular interactions that may regulate its signaling functions. In this study, we focused on understanding how the SH2 domain of Grb7 interacts with its RA-PH (RAPH) region, which may partially regulate its overall structure and activity. We introduced five single-site mutations (V45A, R46A, R46K, E47D, S48A) into the SH2 domain and studied their effects using biochemical, biophysical, and computational methods. Circular dichroism (CD) spectroscopy showed that all mutants maintained the overall fold of the SH2 domain. However, the introduction of mutations enhanced the thermal instability of SH2 from 59°C to a range of 56°C-47°C. Surface plasmon resonance (SPR) analysis revealed that mutations at R46 resulted in approximately 1.5-6 times weaker binding to the RAPH region. AlphaFold and ClusPro models and matrices scores supported the biophysical experimental findings. Docking and size exclusion experiments confirmed the RA and PH domains form a stable unit. Truncating the distorted poly-proline region of Grb7 improved its model quality. Collectively, the results provide additional insight into the regulatory mechanisms of Grb7 and may aid in the development of Grb7-signaling dependent cancer therapeutics.

Supplemental Table 2: EC50 (µm) of the drugs tested on cells dissociated from patients tumors expressing (middle column) DNAJB1::PRKACA or (right column) ATP1B1::PRKACA.

The Spatial transcriptomics data from figure 7 with the cell by cell data for the transcripts of Cyp3A, Muc13 and Col11A1

Fibrolamellar Hepatocellular carcinoma (FLC) is a very rare liver cancer which occurs in young adults and children under the age of 40. This cancer is lethal and was first recognized in 1980. The term “fibrolamellar” refers to the fibrous bands that are visible when examining tumor tissue under a microscope. In FLC, a deletion of 400,000 base pairs occurs in a chromosome 19 copy, leading to the formation of a fusion protein comprising heat shock protein DNAJB1 and the catalytic subunit of Protein Kinase A (DNAJB1-PRKACA). Patients show no symptoms in the early stage of the disease. The reported survival rate over the past five years is below 50%. Surgery remains the sole partially effective treatment for FLC, with no currently approved systemic therapies available. The primary model compound of this project is Napabucasin. It is also known as BBI608 is a naphthoquinone natural product isolated from the plants Newbouldia laevis, Ekmanianthe longiflora, and Handroanthus impetiginosus. It has shown wide spectrum anti-cancer activities and is now in trials for cancer treatment. The Napabucasin was found to be a top hit in drug screening showing potency against all FLC patient derived xenografts (PDX) models. Napabucasin is known to work in multiple signaling pathways and have been shown cytotoxic against both PDX and direct-from-patient FLC tumor cells. We anticipate that the organic synthesis and development of optimized Napabucasin analogues, along with the evaluation of their cytotoxicity and proliferation effectiveness compared to the parent compound, Napabucasin, will result in compounds with improved bioavailability and efficacy, particularly in FLC model cell lines (FLX1 and Huh7-Chimera). Shalini Rawat, Jared Alderman, Priscilla Baeza, Lianna Hartshorn, Celeste Villasenor, Jakim Brazil, Barbara Lyons. Napabucasin analogues with increased bioavailability and efficacy in fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5738.

Abstract Tumor cells of almost all patients with fibrolamellar carcinoma (FLC) have a somatic mutation, a ~400 kB deletion on one copy of chromosome 19 that results in a fusion transcript of DNAJB1 to PRKACA, the catalytic subunit of protein kinase A. The resulting fusion oncoprotein, DNAJB1::PRKACA, has kinase activity, and is required and sufficient for transformation. To understand the oncogenesis of this tumor and to help identify potential therapeutic targets, we explored the pathways of pathogenesis mediated by the aberrant kinase. We explored three mechanisms that might explain transformation by the oncogenic kinase: - A difference in the substrate specificity of DNAJB1::PRKACA kinase versus native PRKACA kinase. - A difference in the interactome of DNAJB1::PRKACA kinase versus native PRKACA kinase. - An increase of total catalytic kinase activity (DNAJB1::PRKACA + PRKACA) versus the native PRKACA condition. We found, comparing cytosolic extracts of tumor and normal liver cells, that there are differences in the substrate specificity of the DNAJB1::PRKACA kinase. Additionally, such comparisons revealed differences in the interactions between the catalytic subunit of the kinase and cytosolic components. We also demonstrated a consistent increase in total PRKACA catalytic subunit in the FLC tumor, relative to the adjacent nontransformed tissue. Further, there is an increase in free, unregulated catalytic activity in the tumor cells and a change in the localization of the catalytic subunit in the tumor cells. Additionally, we have found that this increase in free catalytic activity is sufficient to account for the changes in the transcriptome in tumor cells of FLC patients. Importantly, enhanced expression in hepatocytes of either DNAJB1::PRKACA kinase or native PRKACA kinase caused very similar changes in the transcriptome, and the resultant transcriptomes closely resembled that of tumor cells from FLC patients. This suggests that the DNAJB1 domain is dispensable and not critical for transformation. In our investigation of patient tumors we found two rare and different subclasses of FLC that are indistinguishable from “classic” FLC, based on their transcriptome and based on their drug response profiles. Importantly, both subclasses are associated with changes of PRKACA, but neither involves the DNAJB1 domain. Thus, the DNAJB1 domain is not a critical component of transformation, and the significant driver is an increase of the cellular catalytic activity. Citation Format: Mahsa Shirani, Michael Tomasini, Solomon Levin, soeren heissel, Henrik Molina, Ype De Jong, charles Rice, Philip Coffino, Barbara Lyons, Sanford Simon. Increased activity of protein kinase A is sufficient to cause fibrolamellar carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 137.

Abstract Fibrolamellar carcinoma (FLC) is a rare, usually lethal primary liver tumor that affects children, adolescents and young adults. Sequencing of FLC tumors in hepatocytes reveals that in 99% of the patients there is one recurrent genomic alteration: A deletion of 400 kB. This produces DNAJB1::PRKACA, a fusion of the first exon of DNAJB1 with the 2nd – 10th exons of PRKACA, the catalytic subunit of protein kinase A (PKA). PKA exists as a holoenzyme of two catalytic subunits and two regulatory subunits. The regulatory subunit both inhibits the catalytic and localizes it in the cell. Calibrated mass spectrometry shows that in normal liver there is always an excess of regulatory>catalytic subunits, but in the adjacent FLC tumor tissue there is an excess of catalytic>regulatory subunits. Different biochemistry assays and proximity ligation reveal both free catalytic, unbound to regulatory subunits, and an increase of kinase activity in the tumor cells. Much of the increase of catalytic subunit is in the nucleus. Transducing primary human hepatocytes (PHH) with DNAJB1::PRKACA is sufficient to recapitulate the transcriptome of FLC tumors. Transduction of PHH with just the wt PRKACA is also sufficient to recapitulate the transcriptome of FLC tumors. Thus, just increasing the level of kinase is sufficient, there is nothing special about the fusion domain. From our tissue repository we have four patients who have a tumor that looks like FLC but does not have a fusion to the catalytic subunit. The only alteration in these patients is loss of regulatory subunit. The transcriptome of these patient tumors is indistinguishable from that of classic DNAJB1::PRKACA FLC in hepatocytes. Additionally, we have a few dozen patients who have either DNAJB1::PRKACA or another fusion, ATP1B1::PRKACA in the ductal cells of their liver (producing cholangiocarcinomas) or ductal cells of their pancreas (producing IOPN, Intraductal Oncolytic Pancreatic Neoplasms). The transcriptome of these patients clusters with the transcriptome of the FLC patients with DNAJB1::PRKACA in the hepatocytes. A functional precision medicine drug repurposing screen found that the EC50 of the response of freshly resected tumors from patients to a wide panel of drugs is the same as that of FLC patients with DNAJB1::PRKACA in their hepatocytes. Based on sequencing one could conclude that patients with deletion of regulatory subunit in the hepatocytes, or expression of DNAJB1::PRKACA in the hepatocytes, or expression of DNAJB1::PRKACA or ATP1B1::PRKACA in the cholangiocytes or pancreatic ductal cells are at least three distinct diseases. An analysis of the cell biological changes reveals that they all of an increase of the ratio of catalytic:regulatory subunit, they have an increase of catalytic subunit in the nucleus, and these all result in the same changes of transcriptome and the same drug-response profile. Thus, looking beyond sequencing to the cellular changes leads to a different conclusion, that maybe they should be considered the same disease. Citation Format: Sanford Simon, Mahsa Shirani, David Requena, Denise Ng, Gadi Lalazar, Solomon Levin, Michael S. Torbenson, Aatur D. Singhi, Henrik Molina, Charles M. Rice, Philip Coffino, Barbara A. Lyons. Precision medicine for the fusion protein driven cancer, fibrolamellar carcinoma (FLC): Beyond sequencing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B022.

Abstract Fibrolamellar carcinoma (FLC) is characterized by a single genomic alteration, a 400 kB deletion resulting in the fusion transcript DNAJB1::PRKACA, which encodes a fusion oncoprotein essential for tumor initiation and maintenance. This study aims to find therapeutics that can kill FLC cells. We use three model systems: Patient tumors, fresh from resection, that we have made into organoids, that we have implanted into immune compromised mice (PDX), or we have screened directly, immediately after resection. We found lack of efficacy of agents current in the clinic. We have previously characterized the transcriptome of FLC and identified a number of oncogenic genes and pathways that are upregulated including the wnt pathway, EGF, and the wnt pathway. However, agents that blocked these had no effect on tumor survival. We switched to using three different approaches for therapy: i) A functional precision medicine screen using a drug-repurposing library; ii) antisense oligonucleotides against the RNA junction of DNAJB1::PRKACA transcript; iii) Degrader of the DNAJB1::PRKACA fusion protein. Functional precision medicine: We found a number of agents that were extremely efficacious. What they shared in common was pathways of metabolism of these drugs that were down-regulated in FLC. For example, irinotecan, a topoisomerase I inhibitor, was extremely effective. It is removed from liver cells through the addition of a sugar group by UGT1A1 which is decreased at the transcript and protein level. There were some variations in the extent to which patient tumors responded to irinotecan, but the variations could be eliminated by blocking the anti-apoptotic pathway Bcl-xL. We are currently preparing the combination of irinotecan and a PROTAC (proteolysis targeting chimeras) against Bcl-xL for a clinical trial. Antisense oligos: We created shRNA that tiled across the DNAJB1::PRKACA junction and identified some that eliminated DNAJB1::PRKACA at the RNA and protein level with no effect on DNAJB1 and no effect on PRKACA. When these were induced in FLC tumors cells grown as PDX, the tumors not only stopped growing, but shrank. This demonstrates that DNAJB1::PRKACA not only triggers FLC, but also continues to drive FLC and the FLC tumors are oncogenically addicted to DNAJB1::PRKACA. The same shRNA had no detectable effects on non-FLC liver tumors. We next tested siRNA against FLC grown as PDX. The efficacy of the siRNA were increased by conjugation to the sugar GalNAc which binds to the asialyoglycoprotein receptor on FLC cells. Degraders of the oncoprotein: We developed a degrader that selectively degraded the DNAJB1::PRKACA with no detectable effects on the wt PRKACA. This degrader effectively killed FLC tumors growing as PDX. Each of these three approaches represent emerging technologies for pediatric tumors. For each we now have a proof of principle, and our efforts are now focused on improving delivery and studies of efficacy and safety in the hope of moving these into the clinic to provide respite for this usually lethal childhood tumor. Citation Format: Mahsa Shirani, Michael Tomasini, Christoph Neumayer, Denise Ng, Gadi Lalazar, Bassem Shebl, Philip Coffino, Barbara A. Lyons, Sanford Simon. Emerging therapies for the treatment of the fusion protein driven cancer, fibrolamellar carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A068.

Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that is driven by the fusion of DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). PKA activity is controlled through regulatory proteins that both inhibit catalytic activity and control localization, and an excess of regulatory subunits ensures PRKACA activity is inhibited. Here, we found an increase in the ratio of catalytic to regulatory units in FLC patient tumors driven by DNAJB1::PRKACA using mass spectrometry, biochemistry, and immunofluorescence, with increased nuclear localization of the kinase. Overexpression of DNAJB1::PRKACA, ATP1B1::PRKACA, or PRKACA, but not catalytically inactive kinase, caused similar transcriptomic changes in primary human hepatocytes, recapitulating the changes observed in FLC. Consistently, tumors in patients missing a regulatory subunit or harboring an ATP1B1::PRKACA fusion were indistinguishable from FLC based on the histopathological, transcriptomic, and drug–response profiles. Together, these findings indicate that the DNAJB1 domain of DNAJB1::PRKACA is not required for FLC. Instead, changes in PKA activity and localization determine the FLC phenotype. Significance: Alterations leading to unconstrained protein kinase A signaling, regardless of the presence or absence of PRKACA fusions, drive the phenotypes of fibrolamellar hepatocellular carcinoma, reshaping understanding of the pathogenesis of this rare liver cancer.

Supplementary Figure 1 - 6 and Supplementary Tables 1, 2, and 4

Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a somatic dysregulation of protein kinase A. We show that the proteome of FLC tumors is distinct from that of adjacent nontransformed tissue. These changes can account for some of the cell biological and pathological alterations in FLC cells, including their drug sensitivity and glycolysis. Hyperammonemic encephalopathy is a recurrent problem in these patients, and established treatments based on the assumption of liver failure are unsuccessful. We show that many of the enzymes that produce ammonia are increased and those that consume ammonia are decreased. We also demonstrate that the metabolites of these enzymes change as expected. Thus, hyperammonemic encephalopathy in FLC may require alternative therapeutics.

List of compounds in the primary screen

In fibrolamellar hepatocellular carcinoma (FLC), hyperammonemic encephalopathy is a common occurrence and occasionally causes death. Using mass spectrometry, we quantitatively analyzed the proteomes of FLC patient’s tumor and adjacent normal, to find pathways that are changed in FLC. These data identified multiple proteins that were altered in the proteome, among these, enzymes involved in metabolism of ammonia. These results were confirmed with immunofluorescence demonstrating that these alterations occur in all tumor cells. These results suggest that FLC cells have defects in the two primary ammonia detoxification pathways in the liver, which are responsible for detoxification of 70% of the ammonia in the body: 1) consumption of ammonia by glutamine synthetase (GLUL), and 2) addition of ammonia by ornithine carbamoyltransferase (OTC) to the urea cycle. Additionally, they also generate extra ammonia because of upregulation of glutaminase (GLS). This was tested with a targeted metabolomics of the reactants and products of these enzymes. The results were consistent with both a loss of the two pathways for consumption of ammonia activation of a pathway for generating ammonia. This production of ammonia is consistent with the observation that surgical resection of fibrolamellar reduces the ammonia in patients. All FLC patients with hyperammonemic encephalopathy and documented urine test results showed increased urinary orotic acid, evidence of blockage of the OTC pathway. This study implies that hyperammonemic encephalopathy in FLC may require alternatives to commonly used therapies. Citation Format: Mahsa Shirani, Solomon Levin, Michael D. Tomasini, James Knox, Bassem Shebl, David Requena, Jackson Clark, Søren Heissel, Hanan Alwaseem, Rodrigo Surjan, Ron Lahasky, Henrik Molina, Barbara Lyons, Rachael D. Migler, Philip Coffino, Sanford M. Simon. Urea cycle metabolism is disturbed in Fibrolamellar carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6729.

Fibrolamellar hepatocellular carcinoma, FLC, is a usually lethal cancer affecting children, and young adults. We have shown that all patients have a disruption in the ecology of protein kinase A activity. Hundreds of patients tested have a fusion of the first exon of DNABJ1, a heat shock protein cofactor, to PRKACA, the catalytic subunit of protein kinase A and expression of this DNAJB1-PRKACA fusion oncokinase is sufficient to produce the tumor. One patient is missing the regulatory subunit of protein kinase A and three patients have a fusion of the first exon of a different protein, ATP1B1, to the catalytic subunit of protein kinase A. We characterized the proteome and phosphoproteome of FLC cells relative to adjacent normal tissue. The changes were sufficiently characteristic to identify cell extracts from FLC, based solely on the proteome or phosphoproteome, and distinguishable from other tumor or normal liver. By calibrating protein level we found that tumor cells have an increase of catalytic subunit to such an extent as to exceed the capacity of protein kinase A regulatory subunits. This has two implications. First, there is free catalytic subunit that is unregulated, creating a high basal level of kinase activity. Second, the catalytic subunit is no longer localized by tethering to the regulatory subunit. Thus, the catalytic subunit has access to novel substrates. As an independent confirmation, we found the free basal kinase activity was higher in FLC cells. As a further test, we showed free catalytic subunit, that is not conjugated to regulatory subunit, is higher in FLC cells. We next tested whether these changes were solely due to overexpression of the catalytic subunit or if there was some additional change in the intrinsic activity of the kinase as a result of the fusion. We purified, without using an affinity tag, either PRKACA (which is myristoylated), DNAJB1-PRKACA, and PRKACA which was not myristoylated (the DNAJB1-PRKACA is not myristoylated). As a further control we also used the PRKACAL206R mutation, which does not engage regulatory subunits and is present in some forms of Cushing’s disease. We purified cytosol from human liver, blocked activity of all the endogenous kinases, and added our purified kinase. Mass spectrometry was then used to identify all newly phosphorylated proteins. While many proteins were equally phosphorylated by all four kinases (PRKACA, DNAJB1-PRKACA, PRKACA not myristoylated, PRKACAL206R) there were some distinct differences. From an analysis of these we found alterations in the optimal recognition sequence for phosphorylation for each variant of the kinase. Significantly, when we examined the human tissue data, we found many proteins that were phosphorylated by the DNAJB1-PRKACA in vitro were also phosphorylated in patient tumor tissue, but not in adjacent non-transformed tissue. This validated these observations of altered phosphorylation from the in vitro assays. Citation Format: Solomon Levin, Mahsa Shironi, Melissa Jarmel, Michael Tomasini, Bassem Shebl, Tova Finkelstein, David Requena, Soeren Heissel, Henrik Molina, Philip Coffino, Barbara Lyons, Sanford M. Simon. Pathogenesis of fibrolamellar: Proteome and phosphome of an oncokinase driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2494.

Abstract To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. Significance: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor. This article is highlighted in the In This Issue feature, p. 2355

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization‐dependent regulation could occur downstream of the membrane‐bound kinase in RTK‐mediated signaling pathways. Extrapolation to the full‐length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine‐phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated‐tyrosine‐mimic Y492E‐FL‐Grb7 protein (Y492E‐FL‐Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild‐type FL Grb7(WT‐FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization Kd of WT‐FL‐Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT‐FL‐Grb7 in comparison the tyrosine‐phosphorylation mimic Y492E‐FL‐Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl‐1, Mec‐3 (LIM) domains isoform 2 and filamin α. These interactions are believed to play a role in regulating Grb7‐mediated cell migration function. The full‐length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the ras associating and pleckstrin homology domains, and the importance of this oligomerization in Grb7 function, is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, nuclear magnetic resonance, nuclear relaxation studies, glutaraldehyde cross linking, and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.

Growth factor receptor bound protein 7 (Grb7) is a signal‐transducing adaptor protein that mediates specific protein–protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein‐binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras‐GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti‐apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor‐treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time‐dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd.

Grb7 is an adaptor molecule mediating signal transduction from multiple cell surface receptors to diverse downstream pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase, ErbB2, are overexpressed in 20–30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, although the participants in these pathways have not been fully determined. In this study, we report the Grb7 protein interacts with Filamin‐a, an actin‐crosslinking component of the cell cytoskeleton. Additionally, we have demonstrated the interaction between Grb7 and Flna is specific to the RA‐PH domains of Grb7, and the immunoglobulin‐like repeat 16–19 domains of Flna. We demonstrate that full‐length Grb7 and Flna interact in the mammalian cellular environment, as well as in vitro. Immunofluorescent microscopy shows potential co‐localization of Grb7 and Flna in membrane ruffles upon epidermal growth factor stimulation. These studies are amongst the first to establish a clear connection between Grb7 signaling and cytoskeletal remodeling. Copyright © 2013 John Wiley & Sons, Ltd.

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor-bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7-Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic mutant (Y80E-Grb7-SH2) is largely dimerization deficient and binds a tyrosine-phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild-type SH2 domain. Another dimerization-deficient mutant (F99R-Grb7-SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E-Grb7-SH2 and F99R-Grb7-SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type-Grb7-SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7-SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways.