Tyrosine Phosphorylation Sites Research Articles

Integration of Distinct ShcA Signaling Complexes Promotes Breast Tumor Growth and Tyrosine Kinase Inhibitor Resistance.

The commonality between most phospho-tyrosine signaling networks is their shared use of adaptor proteins to transduce mitogenic signals. ShcA (SHC1) is one such adaptor protein that employs two phospho-tyrosine binding domains (PTB and SH2) and key phospho-tyrosine residues to promote mammary tumorigenesis. Receptor tyrosine kinases (RTK), such as ErbB2, bind the ShcA PTB domain to promote breast tumorigenesis by engaging Grb2 downstream of the ShcA tyrosine phosphorylation sites to activate AKT/mTOR signaling. However, breast tumors also rely on the ShcA PTB domain to bind numerous negative regulators that limit activation of secondary mitogenic signaling networks. This study examines the role of PTB-independent ShcA pools in controlling breast tumor growth and resistance to tyrosine kinase inhibitors. We demonstrate that PTB-independent ShcA complexes predominately rely on the ShcA SH2 domain to activate multiple Src family kinases (SFK), including Src and Fyn, in ErbB2-positive breast cancers. Using genetic and pharmacologic approaches, we show that PTB-independent ShcA complexes augment mammary tumorigenesis by increasing the activity of the Src and Fyn tyrosine kinases in an SH2-dependent manner. This bifurcation of signaling complexes from distinct ShcA pools transduces non-redundant signals that integrate the AKT/mTOR and SFK pathways to cooperatively increase breast tumor growth and resistance to tyrosine kinase inhibitors, including lapatinib and PP2. This study mechanistically dissects how the interplay between diverse intracellular ShcA complexes impacts the tyrosine kinome to affect breast tumorigenesis.Implications: The ShcA adaptor, within distinct signaling complexes, impacts tyrosine kinase signaling, breast tumor growth, and resistance to tyrosine kinase inhibitors. Mol Cancer Res; 16(5); 894-908. ©2018 AACR.

ECSM2, an endothelial specific VE-cadherin binding protein, has a tyrosine phosphorylation site essential to cell migration

Endothelial cell-specific molecule 2 (ECSM2) is a transmembrane protein located in cell-cell junction of endothelial cells (EC). ECSM2 was determined to play an important role in vascular development, EC migration, apoptosis and proliferation, however, no functional domains were determined in intracellular and extracellular region of ECSM2. In current work, functional domains of ECSM2, the relationship of ECSM2 with other endothelial specific protein such as VE-cadherin and the role of ECSM2 in neovascular diseases were determined. It was shown that the 54th amino acid residue of ECSM2 extracellular domain was a tyrosine phosphorylation site, whose mutation led to the loss of EGF-induced tyrosine phosphorylation and inhibition of cell migration. In primary human umbilical vein endothelial cells, ECSM2 bound with VE-cadherin and VEGF stimulation enhanced their binding. In hepatocellular carcinoma, ECSM2 expression was increased, as compared with para-cancerous tissue. This data firstly revealed the functional sites of ECSM2, the crosstalk between ECSM2 and other endothelial cell specific molecules, the expression of ECSM2 in tissues of angiogenesis diseases, thus providing understanding about ECSM2 in depth.

Dynamic Multi‐Site Phosphorylation by Fyn and Abl Drives the Interaction Between CRKL and the Novel Scaffolding Receptors DCBLD1 and DCBLD2

Discoidin, CUB, and LCCL domain containing 1 (DCBLD1) and DCBLD2 compose a family of orphan transmembrane scaffolding receptors that possess similar domain structure to that of neuropilins, critical co‐receptors for neuronal guidance cues. While DCBLD1 remains largely uncharacterized in the literature, DCBLD2 has both known and anticipated roles in vascular remodeling and neuronal positioning. Further, DCBLD2 is up‐regulated in several cancers and is required for hyperactive epidermal growth factor receptor (EGFR)‐driven tumorigenesis. While a few studies have demonstrated both positive and negative regulatory roles of DCBLD2 in growth factor receptor signaling, the conserved features of DCBLD family members that drive their molecular activities remain undefined. Previously, we identified DCBLD2 as a Src family kinase substrate that, when phosphorylated on tyrosines in intracellular YXXP motifs, would bind the Src homology 2 (SH2) domain of the signaling adaptor CRKL (CT10 regulator of kinase‐like). These intracellular YXXP motifs are highly conserved, both across vertebrates and between DCBLD family members. Similarly, we demonstrate here that DCBLD1 YXXP motifs are also required to interact with the CRKL–SH2 domain, distinguishing this motif as a conserved signaling feature of DCBLD family proteins. We show that Src family kinases (SFKs) and Abl differentially mediate the CRKL/DCBLD interaction. Although SFKs and Abl each are able to induce both DCBLD family members to bind the CRKL–SH2 domain, the effect of Abl is stronger for DCBLD1. Using LC‐MS/MS, we quantified relative changes in phosphorylation at several YXXP sites in DCBLD1 and DCBLD2 using both stable isotope labeled standards and a label free method, allowing us to map site‐specific preferences for SFKs and Abl. In addition to these published findings, we have characterized phosphorylation of additional tyrosine residues and have identified proteins differentially induced to bind DCBLD family members from a variety of cell types. We propose two mechanisms for DCBLD signaling, namely, a co‐receptor mechanism in tandem with growth factor receptors and a ligand‐induced clustering mechanism. Together, these findings provide a platform to elucidate the mechanisms the drive the biological activities of these unexplored receptors.Support or Funding InformationU.S. National Science Foundation IOS grants [1021795 and 1656510]; Vermont Genetics Network through U.S. National Institutes of Health Grant [8P20GM103449] from the INBRE program of the NIGMS; U.S. National Institutes of Health Grant [5P20RR016435] from the COBRE program of the NIGMS; University of Vermont College of Arts and Sciences Small Grant Research Award; University of Vermont College of Arts and Sciences Undergraduate APLE research AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating Sema6A‐PlxnA Signaling Mechanisms in Development: Identification of Key PlxnA Phosphorylation Sites and the Discovery of a Novel Naturally‐Released Sema

Semaphorins (Semas) are a family of secreted and transmembrane proteins that play critical roles in the developing nervous system, cardiovasculature, and immune system. Semas signal predominantly through Plexin (Plxn) receptors to regulate cellular processes such as cytoskeletal dynamics, proliferation, and differentiation. We have previously shown that the transmembrane Sema6A and its PlxnA2 receptor are critical in vertebrate eye development, as morpholino‐based knock‐down of these proteins in zebrafish results in decreased early eye field cohesion, smaller eye size, and impaired retinal lamination. Due to the transmembrane nature of Sema6A, reverse signaling can occur and it is thought to contribute to the observed phenotypes. Although several cellular players governing Sema‐Plxn signaling have been identified, the molecular mechanisms that initiate forward and reverse signaling are only partially understood. Therefore, we aimed to investigate the receptor‐proximal events of Sema‐Plxn signaling and we report here two main findings towards a better understanding of the early events of Sema‐Plxn signaling: 1) PlxnA receptor phosphorylation at two tyrosine sites is critical for zebrafish eye development and 2) Sema6A has a functional naturally‐released ectodomain, sSema6A.We used mass spectrometry‐based approaches to identify highly‐conserved, Fyn kinase‐mediated PlxnA tyrosine phosphorylation sites. Mutation of these sites to phenylalanine results in significantly decreased Fyn‐dependent PlxnA tyrosine phosphorylation. Furthermore, in contrast to wildtype human PLXNA2 mRNA, mRNA harboring these point mutations cannot rescue eye developmental defects when co‐injected with a plxnA2 morpholino in zebrafish embryos. Interestingly, while investigating whether or not these sites are phosphorylated upon Sema6A ligand binding, we serendipitously discovered that a functional soluble Sema6A, sSema6A, is spontaneously released from cells expressing the full‐length transmembrane Sema6A. Using zebrafish eye explants, we show that sSema6A promotes early eye field cohesion, a process known to be Sema6A‐dependent. While other soluble Sema ectodomains have been identified in the immune system, we describe here the first soluble ectodomain from a transmembrane Sema that has predominant roles in the nervous system. Together these data suggest that Sema6A may have long‐range effects in addition to its canonical contact‐mediated functions and that Fyn‐dependent phosphorylation is a key feature of vertebrate PlxnA signal transduction. Future work will investigate if the transmembrane and secreted forms of Sema6A can induce Fyn‐mediated PlxnA tyrosine phosphorylation.Support or Funding InformationNational Science Foundation (NSF) IOS grants 1021795 and 1625154; NSF DBI REU grant 1262786;the Vermont Genetics Network through U. S. National Institutes of Health (NIH) Grant 8P20GM103449 from the INBRE program of the NIGMS; and U.S. NIH Grant 5P20RR016435 fromthe COBRE program of the NIGMS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen.

The specificity of tyrosine kinases is attributed predominantly to localization effects dictated by non-catalytic domains. We developed a method to profile the specificities of tyrosine kinases by combining bacterial surface-display of peptide libraries with next-generation sequencing. Using this, we showed that the tyrosine kinase ZAP-70, which is critical for T cell signaling, discriminates substrates through an electrostatic selection mechanism encoded within its catalytic domain (Shah et al., 2016). Here, we expand this high-throughput platform to analyze the intrinsic specificity of any tyrosine kinase domain against thousands of peptides derived from human tyrosine phosphorylation sites. Using this approach, we find a difference in the electrostatic recognition of substrates between the closely related Src-family kinases Lck and c-Src. This divergence likely reflects the specialization of Lck to act in concert with ZAP-70 in T cell signaling. These results point to the importance of direct recognition at the kinase active site in fine-tuning specificity.

Small and large intestine express a truncated Dab1 isoform that assembles in cell-cell junctions and co-localizes with proteins involved in endocytosis

Disabled-1 (Dab1) is an essential intracellular adaptor protein in the reelin pathway. Our previous studies in mice intestine showed that Dab1 transmits the reelin signal to cytosolic signalling pathways. Here, we determine the Dab1 isoform expressed in rodent small and large intestine, its subcellular location and co-localization with clathrin, caveolin-1 and N-Wasp. PCR and sequencing analysis reveal that rodent small and large intestine express a Dab1 isoform that misses three (Y198, Y200 and Y220) of the five tyrosine phosphorylation sites present in brain Dab1 isoform (canonical) and contains nuclear localization and export signals. Western blot assays show that both, crypts, which shelter progenitor cells, and enterocytes express the same Dab1 isoform, suggesting that epithelial cell differentiation does not regulate intestinal generation of alternatively spliced Dab1 variants. They also reveal that the canonical and the intestinal Dab1 isoforms differ in their total degree of phosphorylation. Immunostaining assays show that in enterocytes Dab1 localizes at the apical and lateral membranes, apical vesicles, close to adherens junctions and desmosomes, as well as in the nucleus; co-localizes with clathrin and with N-Wasp but not with caveolin-1, and in Caco-2 cells Dab1 localizes at cell-to-cell junctions by a Ca2+-dependent process. In conclusion, the results indicate that in rodent intestine a truncated Dab1 variant transmits the reelin signal and may play a role in clathrin-mediated apical endocytosis and in the control of cell-to-cell junction assembly. A function of intestinal Dab1 variant as a nucleocytoplasmic shuttling protein is also inferred from its sequence and nuclear location.

Shp2-Mitogen-Activated Protein Kinase Signaling Drives Proliferation during Zebrafish Embryo Caudal Fin Fold Regeneration.

ABSTRACTRegeneration of the zebrafish caudal fin following amputation occurs through wound healing, followed by formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. We show that ptpn11a−/− ptpn11b−/− zebrafish embryos, lacking functional Shp2, fail to regenerate their caudal fin folds. Rescue experiments indicated that Shp2a has a functional signaling role, requiring its catalytic activity and SH2 domains but not the two C-terminal tyrosine phosphorylation sites. Surprisingly, expression of Shp2a variants with increased and reduced catalytic activity, respectively, rescued caudal fin fold regeneration to similar extents. Expression of mmp9 and junbb, indicative of formation of the wound epidermis and distal blastema, respectively, suggested that these processes occurred in ptpn11a−/− ptpn11b−/− zebrafish embryos. However, cell proliferation and MAPK phosphorylation were reduced. Pharmacological inhibition of MEK1 in wild-type zebrafish embryos phenocopied loss of Shp2. Our results suggest an essential role for Shp2a–mitogen-activated protein kinase (MAPK) signaling in promoting cell proliferation during zebrafish embryo caudal fin fold regeneration.

RELN signaling modulates glioblastoma growth and substrate-dependent migration.

Glioblastoma (GBM) represents the most common and most malignant type of primary brain tumor and significantly contributes to cancer morbidity and mortality. Invasion into the healthy brain parenchyma is a major feature of glioblastoma aggressiveness. Reelin (RELN) is a large secreted extracellular matrix glycoprotein that regulates neuronal migration and positioning in the developing brain and sustains functionality in the adult brain. We here show that both RELN and its main downstream effector DAB1 are silenced in glioblastoma as compared to non-neoplastic tissue and mRNA expression is inversely correlated with malignancy grade. Furthermore, RELN expression is positively correlated with patient survival in two large, independent clinically annotated datasets. RELN silencing occurs via promoter hypermethylation as shown by both database mining and bisulfite sequencing of the RELN promoter. Consequently, treatment with 5'-Azacytidine and trichostatin A induced RELN expression in vitro. On the functional level, we found RELN to regulate glioblastoma cell migration both in a DAB1 (tyrosine phosphorylation)-dependent and -independent fashion, depending on the substrate provided. Moreover, stimulation of RELN signaling strongly reduced proliferation in glioblastoma cells. This phenotype depends on DAB1 stimulation by RELN, as a mutant that lacks all RELN induced tyrosine phosphorylation sites (DAB1-5F) failed to induce a growth arrest. Proteomic analyzes revealed that these effects are mediated by a reduction in E2F targets and dephosphorylation of ERK1/2. Taken together, our data establish a relevance of RELN signaling in glioblastoma pathology and thereby might unearth novel, yet unrecognized treatment options.

Intrathecal delivery of a palmitoylated peptide targeting Y382-384 within the P2X7 receptor alleviates neuropathic pain.

Pain hypersensitivity resulting from peripheral nerve injury depends on pathological microglial activation in the dorsal horn of the spinal cord. This microglial activity is critically modulated by P2X7 receptors (P2X7R) and ATP stimulation of these receptors produces mechanical allodynia, a defining feature of neuropathic pain. Peripheral nerve injury increases P2X7R expression and potentiates its cation channel function in spinal microglia. Here, we report a means to preferentially block the potentiation of P2X7R function by delivering a membrane permeant small interfering peptide that targets Y382-384, a putative tyrosine phosphorylation site within the P2X7R intracellular C-terminal domain. Intrathecal administration of this palmitoylated peptide (P2X7R379-389) transiently reversed mechanical allodynia caused by peripheral nerve injury in both male and female rats. Furthermore, targeting Y382-384 suppressed P2X7R-mediated release of cytokine tumor necrosis factor alpha and blocked the adoptive transfer of mechanical allodynia caused by intrathecal injection of P2X7R-stimulated microglia. Thus, Y382-384 site-specific modulation of P2X7R is an important microglial mechanism in neuropathic pain.

Dynamic multi-site phosphorylation by Fyn and Abl drives the interaction between CRKL and the novel scaffolding receptors DCBLD1 and DCBLD2.

Discoidin, CUB, and LCCL Domain-containing (DCBLD) 2 is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also upregulated in several cancers and can drive glioblastomas downstream of activated Epidermal Growth Factor Receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-Like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL-SH2 binding. We report Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL-SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promotes DCBLD1 and DCBLD2 binding to the CRKL-SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.

Fyn-dependent phosphorylation of PlexinA1 and PlexinA2 at conserved tyrosines is essential for zebrafish eye development.

Plexins (Plxns) are semaphorin (Sema) receptors that play important signaling roles, particularly in the developing nervous system and vasculature. Sema-Plxn signaling regulates cellular processes such as cytoskeletal dynamics, proliferation, and differentiation. However, the receptor-proximal signaling mechanisms driving Sema-Plxn signal transduction are only partially understood. Plxn tyrosine phosphorylation is thought to play an important role in these signaling events as receptor and nonreceptor tyrosine kinases have been shown to interact with Plxn receptors. The Src family kinase Fyn can induce the tyrosine phosphorylation of PlxnA1 and PlxnA2. However, the Fyn-dependent phosphorylation sites on these receptors have not been identified. Here, using mass spectrometry-based approaches, we have identified highly conserved, Fyn-induced PlexinA (PlxnA) tyrosine phosphorylation sites. Mutation of these sites to phenylalanine results in significantly decreased Fyn-dependent PlxnA tyrosine phosphorylation. Furthermore, in contrast to wild-type human PLXNA2 mRNA, mRNA harboring these point mutations cannot rescue eye developmental defects when coinjected with a plxnA2 morpholino in zebrafish embryos. Together these data suggest that Fyn-dependent phosphorylation at two critical tyrosines is a key feature of vertebrate PlxnA1 and PlxnA2 signal transduction.

Nucleotide variants of the Nramp1 gene in pigs and their effects on disease resistance

The Nramp1 gene is an important factor associated with susceptibility to infectious diseases caused by intracellular pathogens. The objective of this study was to determine the structures and sequences of Nramp1 in Tibetan, Gansu Black, Large White, Yorkshire, and Duroc pigs. Sequence analysis of the Nramp1 gene in the five pig breeds revealed the presence of 11 nucleotide variants in intronic regions, 2 nucleotide variants in the control region, 10 nucleotide variants and one deletion in the 3’ non-coding region, and 15 nucleotide variants in the exons. However, only 4 nucleotide variants resulted in amino acid changes. Further analysis predicted that the Tibetan pig Nramp1 protein harbors 10 transmembrane domains. The analysis also predicted 10 serine phosphorylation sites, 3 threonine phosphorylation sites, and 4 tyrosine phosphorylation sites in the Nramp1 protein of Tibetan pigs. Predictions of the Tibetan pig Nramp1 tertiary structure revealed 7 putative a -helices and 5 putative b-sheets. Predictions of the Yorkshire pig Nramp1 protein revealed significant differences compared with the Tibetan pig Nramp1 protein. The results indicated that differences in transmembrane domains and tertiary structures of the Nramp1 protein of the Tibetan and Yorkshire pig breeds could explain differences in the disease resistance of these two breeds

Identification of STS-1 as a novel ShcA-binding protein

ShcA is a cytoplasmic signaling protein that supports signal transduction by receptor protein-tyrosine kinases by providing auxiliary tyrosine phosphorylation sites that engage additional signaling proteins. The principal binding partner for tyrosine phosphorylation sites on ShcA is Grb2. In the current study, we have used phosphotyrosine-containing peptides to isolate and identify STS-1 as a novel ShcA-binding protein. Our results further show that the interaction between STS-1 and ShcA is regulated in response to EGF receptor activation.

Abstract 322: Distinct pools of ShcA coupled tyrosine kinase signaling influences breast tumor heterogeneity and therapeutic responsiveness

Abstract Phospho-tyrosine (pTyr) signaling networks are frequently activated in breast cancers (BrCa) and are considered to be major oncogenic drivers of tumor progression. Therapeutic interventions, such as the tyrosine kinase (TK) inhibitor Trastuzumab, focus on targeting TK activity and downstream effectors. Although successful for early stage tumors, a subset of patients experience relapse due to intrinsic or acquired resistance. This includes activation of alternative receptor tyrosine kinase (RTK) and/or cytoplasmic TKs, many of which require recruitment of the adaptor protein, ShcA. ShcA is a key convergence point downstream of RTKs and serves to integrate multiple signal transduction pathways dysregulated in BrCa. Specifically, ShcA contains two pTyr binding motifs including an amino-terminal PTB domain and a carboxy-terminal SH2 domain which facilitate its interactions with TKs including ErbB2 and Src Family Kinases (SFK), respectively. The CH-1 domain houses three tyrosine phosphorylation sites at residues 239/240 and 317 which transduce Ras-dependent and independent signals. Using a well characterized transgenic mouse model of BrCa where ShcA can no longer engage the transforming oncogene through its PTB domain, we demonstrate that loss of PTB-driven ShcA (ShcA-PTBMut) signaling delays mammary tumor onset. However, once formed, the growth and angiogenic potential of these tumors is significantly increased relative to control mice. Increased growth potential of ShcA-PTBMut tumors is associated with the hyper-activation of the c-Src tyrosine kinase. Deletion of c-Src in ShcA-PTBMut breast tumor cells significantly delays tumor onset but is dispensable for the growth of tumors that retain an intact ShcA PTB domain. These data suggest that the ShcA PTB domain can recruit negative regulators that limit the activation of downstream tumorigenic signaling networks. Interestingly, tumors expressing ShcA-PTBMut debilitated in SH2 driven pTyr interactions (SH2Mut), are significantly delayed in tumor onset relative to ShcA-PTBMut controls. Paradoxically, deletion of c-Src in the context of ShcA-PTBMut-SH2Mut further accelerates tumor growth which is attributed to increased levels of Fyn and Lyn. These observations support the high dependence of intracellular ShcA pools on other SFK family members to retain tumorigenic potential when adapting to low levels and/or activity of c-Src. We demonstrate that uncoupling of PTB-driven ShcA signaling from upstream RTKs can potentiate ShcA signaling from intracellular pools to hyper-activate SFKs. This data is clinically relevant as c-Src is frequently hyper-activated in Trastuzumab-resistant BrCa. This is the first study to identify a tumor suppressive role of the ShcA PTB domain and to characterize an intrinsic ShcA SH2 domain-SFK dependent resistance mechanism downstream of activated RTKs in mammary tumorigenesis. Citation Format: Jacqueline R. Ha, Ryuhjin Ahn, Young Kyuen Im, Valerie Sabourin, Harvey W. Smith, Ivan Topisirovic, Tony Pawson, Peter Siegel, William J. Muller, Josie Ursini-Siegel. Distinct pools of ShcA coupled tyrosine kinase signaling influences breast tumor heterogeneity and therapeutic responsiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 322. doi:10.1158/1538-7445.AM2017-322

Abstract 2371: Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease of the blood. About 30 % of AML patients carry an oncogenic mutant of the type III receptor tyrosine kinase FLT3. Among the various mutations in FLT3, the internal tandem duplication (ITD) mutations in the juxtamembrane domain are the most common type of mutation, while point mutations in the kinase domain also occur. Although oncogenic FLT3 mutants are constitutively active, the wild-type receptor needs its ligand (FLT3 ligand, FL) for activation. Ligand binding to the receptor induces dimerization, auto-phosphorylation, and activation of the receptor. Tyrosine phosphorylation sites on FLT3 constitute docking sites for interacting proteins that transduce pro-survival and proliferative signals. The so-called activation loop tyrosine residue (in FLT3, Y842) is well conserved in all receptor tyrosine kinases and long been known essential for the activity of some but not all receptor tyrosine kinases. Recently we have shown that activation loop tyrosine residue in KIT is not essential for its activation but plays an important role in receptor stabilization and downstream signaling. However, so far the role of FLT3 activation loop tyrosine residue remains unknown. We generated 32D cell lines carrying a Y-to-F mutant (Y842F) of FLT3 in either the wild-type or ITD background to study the role of this tyrosine residue. We show that the Y842 residue is not necessary for FLT3 activation or stability but is required for oncogenic transformation. We observed that Y842F mutation results in reduced cell viability, proliferation and in vitro colony formation in semi-solid medium. In addition, cells expressing the Y842F mutant in FLT3-ITD background display a significant reduction in tumor volume and weight in a xenograft model in nude mice. Gene set enrichment analysis (GESA) shows that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we demonstrated that phosphorylated Y842 is one of the binding sites of the protein tyrosine phosphataseSHP2, which is required for activation of signaling through the RAS/ERK pathway. Taken together, our data suggest that activation loop tyrosine residue in FLT3 plays an important role in the cellular transformation which is regulated by RAS/ERK signaling. Citation Format: Julhash U. Kazi, Lars Rönnstrand. Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2371. doi:10.1158/1538-7445.AM2017-2371

Unphosphorylated STAT3 regulates the antiproliferative, antiviral, and gene-inducing actions of type I interferons

Type I interferon (IFNα/β) induces antiviral and antiproliferative responses in cells through the induction of IFN-stimulated genes (ISGs). Although the roles of IFN-activated STAT1 and STAT2 in the IFN response are well described, the function of STAT3 is poorly characterized. We investigated the role of STAT3 in the biological response to IFNα/β in mouse embryonic fibroblasts (MEFs) with a germ line deletion of STAT3. These STAT3 knockout (STAT3-KO) MEFs were reconstituted with STAT3 or the F705-STAT3 mutant (unphosphorylated STAT3) where the canonical Y705 tyrosine phosphorylation site was mutated. We show that both STAT3 and unphosphorylated STAT3 expression enhance the sensitivity of MEFs to the antiviral, antiproliferative and gene-inducing actions of IFN. By chromatin immunoprecipitation assays, unphosphorylated STAT3 appears to bind, albeit weakly, to select gene promoters to enhance their expression. These results suggest that unphosphorylated STAT3 plays an important role in the IFN response pathway.

The Guanine-Nucleotide Exchange Factor Caldag Gefi Fine-Tunes Functional Properties of Regulatory T Cells.

Using quantitative phosphopeptide sequencing of unstimulated versus stimulated primary murine Foxp3+ regulatory and Foxp3– conventional T cells (Tregs and Tconv, respectively), we detected a novel and differentially regulated tyrosine phosphorylation site within the C1 domain of the guanine-nucleotide exchange factor CalDAG GEFI. We hypothesized that the Treg-specific and activation-dependent reduced phosphorylation at Y523 allows binding of CalDAG GEFI to diacylglycerol, thereby impacting the formation of a Treg-specific immunological synapse. However, diacylglycerol binding assays of phosphomutant C1 domains of CalDAG GEFI could not confirm this hypothesis. Moreover, CalDAG GEFI–/– mice displayed normal Treg numbers in thymus and secondary lymphoid organs, and CalDAG GEFI–/– Tregs showed unaltered in vitro suppressive capacity when compared to CalDAG GEFI+/+ Tregs. Interestingly, when tested in vivo, CalDAG GEFI–/– Tregs displayed a slightly reduced suppressive ability in the transfer colitis model when compared to CalDAG GEFI+/+ Tregs. Additionally, CRISPR-Cas9-generated CalDAG GEFI–/– Jurkat T cell clones showed reduced adhesion to ICAM-1 and fibronectin when compared to CalDAG GEFI-competent Jurkat T cells. Therefore, we speculate that deficiency in CalDAG GEFI impairs adherence of Tregs to antigen-presenting cells, thereby impeding formation of a fully functional immunological synapse, which finally results in a reduced suppressive potential.

Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response

Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747LREA750, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238/Y1239 and MET-Y1252/1253. This study provides unique insight into the TKI-mediated modulation of mutant EGFR signaling, which can be applied to the development of biomarkers of EGFR TKI response.

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity.

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long-term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multisite phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity, and reduced GEF activity.

Exogenous spermine preserves mitochondrial bioenergetics via regulating Src kinase signaling in the spinal cord.

Regulation of mitochondrial metabolism is becoming an important target in inhibiting necrosis and apoptosis following secondary spinal cord injury, and physiological compounds that reduce mitochondrial dysfunction are regarded as efficient protective reagents following injury. It has been demonstrated that spermine, a polyamine composed of four primary amines, may be taken up by a mitochondria‑specific uniporter and may preserve mitochondrial bioenergetics, suggesting that it may be important in the pathophysiology of mitochondria. However, the protective mechanism has not yet been definitively clarified. In the present study, isolated spinal cord mitochondria were incubated with spermine to evaluate its physiological functions and Src kinase activities. The results revealed that spermine increased oxidative phosphorylation, attenuated mitochondrial swelling and maintained the membrane potential. An inhibitor of Src kinases, amino‑5-(4‑chlorophenyl)‑7‑(t‑butyl)pyrazolo[3,4‑d]pyrimidine (PP2), markedly reduced the effects of spermine. However, inhibition of tyrosine phosphatases by vanadate led to marginal increases in the effects of spermine. Therefore, the present study hypothesized that tyrosine phosphorylation sites are present in the subunits of respiratory chains and mitochondrial permeability transition pore proteins, which may be modified via phosphorylation and dephosphorylation. Furthermore, spermine may upregulate the phosphorylation of Src kinases, and PP2 and vanadate conversely regulate Src phosphorylation. The results of the present study suggest that spermine is a strategic regulator within mitochondria that may activate Src kinases in the spinal cord, and tyrosine phosphorylation signaling is a primary regulatory pathway of mitochondrial metabolism.