Src Family Kinase Fyn Research Articles

BLNK negatively regulates innate antifungal immunity through inhibiting c-Cbl-mediated macrophage migration

B cell linker protein (BLNK) is crucial for orchestrating B cell receptor-associated spleen tyrosine kinase (Syk) signaling. However, the role of BLNK in Syk-coupled C-type lectin receptor (CLR) signaling in macrophages remains unclear. Here, we delineate that CLRs govern the Syk-mediated activation of BLNK, thereby impeding macrophage migration by disrupting podosome ring formation upon stimulation with fungal β-glucans or α-mannans. Mechanistically, BLNK instigates its association with casitas B-lineage lymphoma (c-Cbl), competitively impeding the interaction between c-Cbl and Src-family kinase Fyn. This interference disrupts Fyn-mediated phosphorylation of c-Cbl and subsequent c-Cbl-associated F-actin assembly. Consequently, BLNK deficiency intensifies CLR-mediated recruitment of the c-Cbl/phosphatidylinositol 3-kinase complex to the F-actin cytoskeleton, thereby enhancing macrophage migration. Notably, mice with monocyte-specific BLNK deficiency exhibit heightened resistance to infection with Candida albicans, a prominent human fungal pathogen. This resistance is attributed to the increased infiltration of Ly6C+ macrophages into renal tissue. These findings unveil a previously unrecognized role of BLNK for the negative regulation of macrophage migration through inhibiting CLR-mediated podosome ring formation during fungal infections.

SRC family kinase inhibition rescues molecular and behavioral phenotypes, but not protein interaction network dynamics, in a mouse model of Fragile X syndrome.

Glutamatergic synapses encode information from extracellular inputs using dynamic protein interaction networks (PINs) that undergo widespread reorganization following synaptic activity, allowing cells to distinguish between signaling inputs and generate coordinated cellular responses. Here, we investigate how Fragile X Messenger Ribonucleoprotein (FMRP) deficiency disrupts signal transduction through a glutamatergic synapse PIN downstream of NMDA receptor or metabotropic glutamate receptor (mGluR) stimulation. In cultured cortical neurons or acute cortical slices from P7, P17 and P60 FMR1-/y mice, the unstimulated protein interaction network state resembled that of wildtype littermates stimulated with mGluR agonists, demonstrating resting state pre-activation of mGluR signaling networks. In contrast, interactions downstream of NMDAR stimulation were similar to WT. We identified the Src family kinase (SFK) Fyn as a network hub, because many interactions involving Fyn were pre-activated in FMR1-/y animals. We tested whether targeting SFKs in FMR1-/y mice could modify disease phenotypes, and found that Saracatinib (SCB), an SFK inhibitor, normalized elevated basal protein synthesis, novel object recognition memory and social behavior in FMR1-/y mice. However, SCB treatment did not normalize the PIN to a wild-type-like state in vitro or in vivo, but rather induced extensive changes to protein complexes containing Shank3, NMDARs and Fyn. We conclude that targeting abnormal nodes of a PIN can identify potential disease-modifying drugs, but behavioral rescue does not correlate with PIN normalization.

Abstract 1102: Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC

Abstract ALK/ROS1 TKIs have significantly improved outcomes for patients with ALK/ROS1+ lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately 1/3 of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in most patients remains unknown/poorly characterized. To model and characterize acquired resistance to crizotinib, we used ROS1+ and ALK+ primary, patient tumor-derived non-small cell lung cancer (NSCLC) cell lines to generate acquired resistance models to the ALK/ROS1 inhibitor crizotinib. We used several techniques to probe the mechanisms driving resistance: DNA and RNA sequencing (seq), fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. The crizotinib-resistant line initially driven by a TPM3-ROS1 fusion (CUTO28-CR) displayed exquisite sensitivity to Src family kinase (SFK) TKIs in proliferation assays, which was modestly improved by addition of ERBB TKI afatinib. Bulk RNA seq revealed upregulation of SFKs Fyn and Src transcripts (141- and 2.31-fold, respectively). Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells vs parental (EGFR:CEP7 ratio 2.1 vs 1.0), and RNA seq revealed upregulation of EGFR and HER2 transcripts (1.79- and 2.45-fold, respectively). Western blot analysis confirmed upregulation of Fyn at the protein level. CUTO28-CR cells treated with a SFK TKI revealed dose-dependent deactivation of Akt signaling, and addition of afatinib caused inhibition of MAPK signaling. Stimulation with epidermal growth factor (EGF) enhanced MAPK signaling in CUTO28-CR cells more than in parental cells, consistent with EGFR/HER2 dependence. Because of a noted propensity for the EML4-ALK line (H3122) to upregulate ERBB kinase activity under sustained ALK TKI treatment, we maintained these cells in low dose (25nM) afatinib while driving crizotinib resistance to identify additional bypass signaling pathways. These H3122-CAR cells were partially resensitized to crizotinib with the addition of SFK TKIs, and the addition of afatinib with SFK TKIs further resensitized these resistant cells to crizotinib. Bulk RNA seq of H3122-CAR cells revealed upregulation of Fyn and Src (2.64- and 3.63-fold, respectively), and western blot analysis confirmed upregulation at the protein level. In conclusion, we demonstrated increased SFK (particularly Fyn and/or Src) expression and/or activation is a mechanism utilized to drive bypass signaling, in cooperation with ERBB family kinases, to circumvent treatment with ALK/ROS1 TKI crizotinib. To our knowledge, this is the first report of SFK-driven bypass signaling as a mechanism of acquired resistance to an ALK/ROS1 TKI in NSCLC. The upregulation of Src and/or Fyn in NSCLC biopsies may guide the exploration of combination SFK TKIs with ALK/ROS1 TKIs. Citation Format: Logan C. Tyler, Nan Chen, Anh T. Le, Hala Nijmeh, Liming Bao, Robert C. Doebele. Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC [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 1102.

Fyn and TOM1L1 are recruited to clathrin-coated pits and regulate Akt signaling.

The epidermal growth factor (EGF) receptor (EGFR) controls many aspects of cell physiology. EGF binding to EGFR elicits the membrane recruitment and activation of phosphatidylinositol-3-kinase, leading to Akt phosphorylation and activation. Concomitantly, EGFR is recruited to clathrin-coated pits (CCPs), eventually leading to receptor endocytosis. Previous work uncovered that clathrin, but not receptor endocytosis, is required for EGF-stimulated Akt activation, and that some EGFR signals are enriched in CCPs. Here, we examine how CCPs control EGFR signaling. The signaling adaptor TOM1L1 and the Src-family kinase Fyn are enriched within a subset of CCPs with unique lifetimes and protein composition. Perturbation of TOM1L1 or Fyn impairs EGF-stimulated phosphorylation of Akt2 but not Akt1. EGF stimulation also triggered the TOM1L1- and Fyn-dependent recruitment of the phosphoinositide 5-phosphatase SHIP2 to CCPs. Thus, the recruitment of TOM1L1 and Fyn to a subset of CCPs underlies a role for these structures in the support of EGFR signaling leading to Akt activation.

PI3K/AKT Activation Mediates B-Cell Transformation By the “T” Splice-Variant of Fyn Kinase

The SRC-family kinase FYN has key roles downstream of growth-factor receptor activation and is implicated in various cancers. Activating FYNutations were reported in angioimmunoblastic T-cell lymphoma (AITL), but its role in transforming lymphocytes is poorly characterized. FYN also may play a role in signaling downstream of the B cell Receptor (BCR) receptor in Diffuse Large B-Cell Lymphoma (DLBCL), and laboratory studies show inhibition of FYN and other SRC family kinases (SFKs) can block BCR-mediated lymphomagenesis. To investigate FYN's role in oncogenic singling, we introduced FYN alleles to IL3-dependent FL5.12 murine pro-B cells and tested for selective advantage upon cytokine withdrawal and ability to transform the cells to cytokine independence. FYN B and T variants result from alternate splicing of exon 7, with B originally detected in brain and T in hematopoietic cells, but both are expressed and may be deregulated associated with disease states in diffuse tissues. We employed wildtype (WT) alleles for both spliceoforms along with mutations detected recurrently in AITL (L174R and R176C), kinase active (Y528H), and kinase dead (K299A). No FYN B alleles showed any selective advantage in the prolymphocyte cells subjected to cytokine withdrawal. In contrast, all FYN T alleles other than kinase dead were strongly enriched through multiple rounds of IL3 withdrawal and rescue. Indeed, complete withdrawal of cytokine resulted in transformation to IL3-independent growth, generating stable cell lines addicted to FYN kinase activity for survival. The SFK inhibitor PP2 was toxic to all FYN-T transformed cells but not to baseline FL5.12 cells growing in cytokine. WT- and L147R-transformed cells were similarly sensitive to PP2, while the other AITL-derived mutant R176C demonstrated reduced sensitivity. Immunoblotting demonstrated PP2-sensitive phosphorylation of AKT and the MTORC1 target 4EBP1, suggesting PI3K/AKT activation as a key survival pathway downstream from transformation by FYN T alleles. Consistently, the AKT inhibitor ipatasertib and PDK1 inhibitor GSK2334470 were highly active against all FYN-transformed lines, and AKT phosphorylation was sensitive to PDK1 inhibition, showing convergence on AKT activation in all transformants. Pan-PI3K inhibition was active against FYN transformants, while the delta isoform-specific inhibitor idelalisib showed only partial activity, suggesting activation of multiple PI3K isoforms downstream of FYN activity. These results suggest that AKT, PI3K, and PDK1 are important targets downstream of FYN, and that the PI3K/AKT pathway is the key survival mechanism in lymphomagenesis mediated by FYN. We show that WT and activating mutants of the FYN T spliceoform specifically, but not FYN B, induce drug-sensitive B lymphocyte malignant transformation by activating PI3K/AKT/MTORC1 signaling. DisclosuresNo relevant conflicts of interest to declare.

Phosphoproteomic Characterization of Primary AML Samples and Relevance for Response Toward FLT3-inhibitors.

Phosphoproteomic Characterization of Primary AML Samples and Relevance for Response Toward FLT3-inhibitors.

Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation.

During embryonic development and adulthood, Reelin exerts several important functions in the brain including the regulation of neuronal migration, dendritic growth and branching, dendritic spine formation, synaptogenesis and synaptic plasticity. As a consequence, the Reelin signaling pathway has been associated with several human brain disorders such as lissencephaly, autism, schizophrenia, bipolar disorder, depression, mental retardation, Alzheimer’s disease and epilepsy. Several elements of the signaling pathway are known. Core components, such as the Reelin receptors very low-density lipoprotein receptor (VLDLR) and Apolipoprotein E receptor 2 (ApoER2), Src family kinases Src and Fyn, and the intracellular adaptor Disabled-1 (Dab1), are common to most but not all Reelin functions. Other downstream effectors are, on the other hand, more specific to defined tasks. Reelin is a large extracellular protein, and some aspects of the signal are regulated by its processing into smaller fragments. Rather than being inhibitory, the processing at two major sites seems to be fulfilling important physiological functions. In this review, I describe the various cellular events regulated by Reelin and attempt to explain the current knowledge on the mechanisms of action. After discussing the shared and distinct elements of the Reelin signaling pathway involved in neuronal migration, dendritic growth, spine development and synaptic plasticity, I briefly outline the data revealing the importance of Reelin in human brain disorders.

Interplay between the tyrosine kinases Chk and Csk and phosphatase PTPRJ is critical for regulating platelets in mice

AbstractThe Src family kinases (SFKs) Src, Lyn, and Fyn are essential for platelet activation and also involved in megakaryocyte (MK) development and platelet production. Platelet SFKs are inhibited by C-terminal Src kinase (Csk), which phosphorylates a conserved tyrosine in their C-terminal tail, and are activated by the receptor-type tyrosine phosphatase PTPRJ (CD148, DEP-1), which dephosphorylates the same residue. Deletion of Csk and PTPRJ in the MK lineage in mice results in increased SFK activity, but paradoxically hypoactive platelets resulting from negative feedback mechanisms, including upregulation of Csk homologous kinase (Chk) expression. Here, we investigate the role of Chk in platelets, functional redundancy with Csk, and the physiological consequences of ablating Chk, Csk, and PTPRJ in mice. Platelet count was normal in Chk knockout (KO) mice, reduced by 92% in Chk;Csk double KO (DKO) mice, and partially rescued in Chk;Csk;Ptprj triple KO (TKO) mice. Megakaryocyte numbers were significantly increased in both DKO and TKO mice. Phosphorylation of the inhibitory tyrosine of SFKs was almost completely abolished in DKO platelets, which was partially rescued in Src and Fyn in TKO platelets. This residual phosphorylation was abolished by Src inhibitors, revealing an unexpected mechanism in which SFKs autoinhibit their activity by phosphorylating their C-terminal tyrosine residues. We demonstrate that reduced inhibitory phosphorylation of SFKs leads to thrombocytopenia, with Csk being the dominant inhibitor in platelets and Chk having an auxiliary role. PTPRJ deletion in addition to Chk and Csk ameliorates the extent of thrombocytopenia, suggesting targeting it may have therapeutic benefits in such conditions.

Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation.

The plasma membrane consists of a variety of discrete domains differing from the surrounding membrane in composition and properties. Selective partitioning of protein to these microdomains is essential for membrane functioning and integrity. Studying the nanoscale size and dynamic nature of the membrane microdomains requires advanced imaging approaches with a high spatiotemporal resolution and, consequently, expensive and specialized equipment, unavailable for most researchers and unsuited for large-scale studies. Thus, understanding of protein partitioning to the membrane microdomains in health and disease is still hampered by the lack of inexpensive live-cell approaches with an appropriate spatial resolution. Here, we have developed a novel approach based on Gaussia princeps luciferase protein-fragment complementation assay to quantitively investigate protein partitioning to cholesterol and sphingomyelin-rich domains, sometimes called ‘lipid rafts’, in intact living cells with a high-spatial resolution. In the assay, the reporter construct, carrying one half of the luciferase protein, is targeted to lipid microdomains through the fused acetylation motif from Src-family kinase Fyn. A protein of interest carries the second half of the luciferase protein. Together, this serves as a reversible real-time sensor of raft recruitment for the studied protein. We demonstrated that the assay can efficiently detect the dynamic alterations in raft localization of two disease-associated proteins: Akt and APP. Importantly, this method can be used in high-throughput screenings and other large-scale studies in living cells. This inexpensive, and easy to implement raft localization assay will benefit all researchers interested in protein partitioning in rafts.

Targeted enhancement of flotillin-dependent endocytosis augments cellular uptake and impact of cytotoxic drugs

Cellular uptake is limiting for the efficacy of many cytotoxic drugs used to treat cancer. Identifying endocytic mechanisms that can be modulated with targeted, clinically-relevant interventions is important to enhance the efficacy of various cancer drugs. We identify that flotillin-dependent endocytosis can be targeted and upregulated by ultrasound and microbubble (USMB) treatments to enhance uptake and efficacy of cancer drugs such as cisplatin. USMB involves targeted ultrasound following administration of encapsulated microbubbles, used clinically for enhanced ultrasound image contrast. USMB treatments robustly enhanced internalization of the molecular scaffold protein flotillin, as well as flotillin-dependent fluid-phase internalization, a phenomenon dependent on the protein palmitoyltransferase DHHC5 and the Src-family kinase Fyn. USMB treatment enhanced DNA damage and cell killing elicited by the cytotoxic agent cisplatin in a flotillin-dependent manner. Thus, flotillin-dependent endocytosis can be modulated by clinically-relevant USMB treatments to enhance drug uptake and efficacy, revealing an important new strategy for targeted drug delivery for cancer treatment.

A\u03b2 oligomers promote oligodendrocyte differentiation and maturation via integrin \u03b21 and Fyn kinase signaling

Alzheimer´s disease (AD) is characterized by a progressive cognitive decline that correlates with the levels of amyloid β-peptide (Aβ) oligomers. Strong evidences connect changes of oligodendrocyte function with the onset of neurodegeneration in AD. However, the mechanisms controlling oligodendrocyte responses to Aβ are still elusive. Here, we tested the role of Aβ in oligodendrocyte differentiation, maturation, and survival in isolated oligodendrocytes and in organotypic cerebellar slices. We found that Aβ peptides specifically induced local translation of 18.5-kDa myelin basic protein (MBP) isoform in distal cell processes concomitant with an increase of process complexity of MBP-expressing oligodendrocytes. Aβ oligomers required integrin β1 receptor, Src-family kinase Fyn and Ca2+/CaMKII as effectors to modulate MBP protein expression. The pharmacological inhibition of Fyn kinase also attenuated oligodendrocyte differentiation and survival induced by Aβ oligomers. Similarly, using ex vivo organotypic cerebellar slices Aβ promoted MBP upregulation through Fyn kinase, and modulated oligodendrocyte population dynamics by inducing cell proliferation and differentiation. Importantly, application of Aβ to cerebellar organotypic slices enhanced remyelination and oligodendrocyte lineage recovery in lysolecithin (LPC)-induced demyelination. These data reveal an important role of Aβ in oligodendrocyte lineage function and maturation, which may be relevant to AD pathogenesis.

Tyrosine phosphorylation activates 6-phosphogluconate dehydrogenase and promotes tumor growth and radiation resistance

6-Phosphogluconate dehydrogenase (6PGD) is a key enzyme that converts 6-phosphogluconate into ribulose-5-phosphate with NADP+ as cofactor in the pentose phosphate pathway (PPP). 6PGD is commonly upregulated and plays important roles in many human cancers, while the mechanism underlying such roles of 6PGD remains elusive. Here we show that upon EGFR activation, 6PGD is phosphorylated at tyrosine (Y) 481 by Src family kinase Fyn. This phosphorylation enhances 6PGD activity by increasing its binding affinity to NADP+ and therefore activates the PPP for NADPH and ribose-5-phosphate, which consequently detoxifies intracellular reactive oxygen species (ROS) and accelerates DNA synthesis. Abrogating 6PGD Y481 phosphorylation (pY481) dramatically attenuates EGF-promoted glioma cell proliferation, tumor growth and resistance to ionizing radiation. In addition, 6PGD pY481 is associated with Fyn expression, the malignancy and prognosis of human glioblastoma. These findings establish a critical role of Fyn-dependent 6PGD phosphorylation in EGF-promoted tumor growth and radiation resistance.

Fyn Regulates Binding Partners of Cyclic-AMP Dependent Protein Kinase A.

The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif. This led to the hypothesis that Fyn could affect proteins in complex with PKA. To test this, we employed a quantitative mass spectrometry approach to identify Fyn-dependent binding partners in complex with PKA-C. We found Fyn enhanced the binding of PKA-C to several cytoskeletal regulators that localize to the centrosome and Golgi apparatus. Three of these Fyn-induced PKA interactors, AKAP9, PDE4DIP, and CDK5RAP2, were validated biochemically and were shown to exist in complex with Fyn and PKA in a glioblastoma cell line. Intriguingly, the complexes formed between PKA-C and these known AKAPs were dependent upon Fyn catalytic activity and expression levels. In addition, we identified Fyn-regulated phosphorylation sites on proteins in complex with PKA-C. We also identified and biochemically validated a novel PKA-C interactor, LARP4, which complexed with PKA in the absence of Fyn. These results demonstrate the ability of Fyn to influence the docking of PKA to specific cellular scaffolds and suggest that Fyn may affect the downstream substrates targeted by PKA.

Ginsenoside-Rp3 inhibits platelet activation and thrombus formation by regulating MAPK and cyclic nucleotide signaling

Background & purposeGinseng (Panax ginseng C.A. Mayer) contains saponin fractions called ginsenosides, which are thought to be the main components responsible for its various pharmacological activities. Ginsenosides have cardioprotective and antiplatelet effects. In the present study, we evaluated the effects of ginsenoside Rp3 (G-Rp3) on platelet function. MethodsThe in vitro effects of G-Rp3 were evaluated on agonist-induced human and rat platelet aggregation, while [Ca2+]i mobilization, granule secretion, integrin αIIbβ3 activation, and clot retraction were assessed in rat platelets. Its effects on vasodilator-stimulated phosphoprotein (VASP) expression, phosphorylation of MAPK signaling molecules, and PI3K/Akt activation were also studied. Moreover, the tyrosine phosphorylation of components of the P2Y12 receptor downstream signaling pathway was also examined. The in vivo effects of G-Rp3 were studied using an acute pulmonary thromboembolism model and lung histopathology. Key resultsG-Rp3 significantly inhibited collagen, ADP, and thrombin-induced platelet aggregation. G-Rp3 elevated cAMP levels and VASP phosphorylation and suppressed agonist-induced [Ca2+]i mobilization, ATP release, and P-selectin expression along with fibrinogen binding to integrin αIIbβ3, fibronectin adhesion, and clot retraction. G-Rp3 also attenuated the phosphorylation of MAPK, Src, and PLCγ2 as well as PI3K/Akt activation. Furthermore, it inhibited tyrosine phosphorylation of the Src family kinases (Src, Fyn, and Lyn) and PLCγ2 and protected mice from thrombosis. Conclusion and implicationG-Rp3 modulates agonist-induced platelet activation and thrombus formation by inhibiting granule secretion, integrin αIIbβ3 activation, MAPK signaling, and Src, PLCγ2, and PI3K/Akt activation, and VASP stimulation. Our data suggest that G-Rp3 has therapeutic potential as a treatment for platelet-related cardiovascular disorders.

Identification of Proteins Interacting with Fyn Kinase at Fertilization in Patiria miniata

Release of Ca2+ from the endoplasmic reticulum is the integral molecular mechanism responsible for initiating development at fertilization. The components of the signaling pathway controlling this Ca2+ release are not fully known in any organism. Eggs and sperm of the starfish, Patiria miniata, present an attractive model to study the molecular aspects of fertilization because of the ability to obtain millions of viable sperm and eggs, along with the ease of in vitro fertilization. Two Src family kinases (SFKs) and the lipase PLCγ have been shown to be involved in this signaling pathway and are necessary for Ca2+ release. However, additional interacting proteins and the receptor(s) initiating this pathway have not been identified or characterized. In this study, GST fusion protein constructs of the SH2 domains of Fyn (a SFK) were prepared and affinity interactions were performed at different time points before and after sperm addition to eggs. The affinity complexes were subjected to Western blotting to visualize the interacting proteins. Using an anti‐phosphotyrosine antibody, 5 distinct bands indicate that tyrosine phosphorylated egg proteins of 250, 150, 100, ~80, and ~65kDa bind to the fusion proteins as fast as 1 minute post sperm addition, while the same 5 bands plus two additional bands at ~200kDa and ~125kDa are seen in eggs at 2, 3, 5, 10, and 20 minutes post sperm addition, with varying intensities. Furthermore, tyrosine phosphorylated proteins from ~70kDa to ~45kDa are seen to bind with varying intensity at 5, 10, and 20 minutes post sperm addition. These findings indicate that multiple proteins are interacting with the SFK Fyn in a fertilization‐specific manner. In future experiments, mass spectrometry and mRNA knock down strategies will be used to identify these proteins and to discover their function in regulating Ca2+ release at fertilization.Support or Funding InformationThis work was funded by the NSF and Florida Institute of Technology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel Role for Hedgehog Acyltransferase in the Uptake of Palmitoyl‐CoA into the Endoplasmic Reticulum

Post‐translational attachment of fatty acids to proteins plays a fundamental role in signaling pathways in normal and cancer cells. Most fatty acylated proteins are localized within cells, but a subset are secreted, including Hedgehog and Wnt proteins. Hedgehog proteins, of which Sonic Hedgehog (Shh) is the best studied, are modified by attachment of the 16‐carbon fatty acid palmitate during passage through the secretory pathway. Palmitoylation of Shh is mediated by Hedgehog Acyltransferase (Hhat) and is essential for short and long range Shh signaling in development and tumorigenesis. Hhat is localized to the endoplasmic reticulum (ER) and is a member of the membrane bound‐O‐acyltransferase (MBOAT) family of enzymes. The palmitoylation reaction occurs in the lumen of the ER, and thus palmitoyl‐CoA must be present in the ER lumen for Hhat to serve as a substrate. However, how palmitoyl‐CoA, which is not permeable across membranes, enters the ER lumen remains unknown. In mitochondria, a carnitine palmitoyltransferase (CPT1/2) system shuttles palmitoyl‐CoA across both the inner and outer mitochondrial membranes. We were interested in determining if a similar system exists in the ER or if the CPT1/2 system is shared at ER‐mitochondrial contact sites. Using cell‐based radiolabeling experiments, we showed that Etomoxir, a CPT1 inhibitor, strongly reduced palmitoylation of Shh but had no effect on palmitoylation of the Src family kinase Fyn, which is palmitoylated by cytoplasmic palmitoyl‐CoA. Etomoxir treatment had no effect on the ability of purified Hhat to palmitoylate Shh in vitro. We then developed in vitro assays to monitor palmitoyl‐CoA uptake into the lumen of microsomal membranes and to determine if Hhat plays a role in promoting this process. Using either fluorescent (NBD) or radiolabeled (125I) analogs of palmitoyl‐CoA, we observed a 3‐fold increase in uptake of palmitoyl‐CoA into the lumen of microsomal membranes prepared from cells overexpressing Hhat compared to membranes from cells transfected with empty vector. Increased palmitoyl‐CoA uptake was dependent on Hhat catalytic activity, as it was inhibited by a small molecule Hhat inhibitor and was not observed with H379A, an inactive Hhat mutant. Palmitoyl‐CoA uptake was also reduced after treatment of membranes with Etomoxir, and addition of the CPT1 substrate carnitine counteracted this inhibition. Microsomal membranes from Hhat −/− cells exhibited 2‐fold lower palmitoyl‐CoA uptake, compared to membranes from wild type cells, which supports the hypothesis that endogenous Hhat enhances uptake of this fatty acyl CoA. Taken together, this data suggests that a CPT1/2‐like system exists in the ER, or as part of an ER‐mitochondrial contact site. This system, along with Hhat, promotes uptake of palmitoyl CoA into the ER lumen to enable Hhat to catalyze fatty acylation of Shh. Understanding the mechanism by which fatty acids enter the ER lumen and are attached to proteins involved in cell signaling pathways can potentially reveal novel therapeutic targets in cancers characterized by aberrant signaling pathways.‐‐‐‐‐Support or Funding InformationNIH GM116860This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ubiquitinated CD36 sustains insulin-stimulated Akt activation by stabilizing insulin receptor substrate 1 in myotubes

Both the magnitude and duration of insulin signaling are important in executing its cellular functions. Insulin-induced degradation of insulin receptor substrate 1 (IRS1) represents a key negative feedback loop that restricts insulin signaling. Moreover, high concentrations of fatty acids (FAs) and glucose involved in the etiology of obesity-associated insulin resistance also contribute to the regulation of IRS1 degradation. The scavenger receptor CD36 binds many lipid ligands, and its contribution to insulin resistance has been extensively studied, but the exact regulation of insulin sensitivity by CD36 is highly controversial. Herein, we found that CD36 knockdown in C2C12 myotubes accelerated insulin-stimulated Akt activation, but the activated signaling was sustained for a much shorter period of time as compared with WT cells, leading to exacerbated insulin-induced insulin resistance. This was likely due to enhanced insulin-induced IRS1 degradation after CD36 knockdown. Overexpression of WT CD36, but not a ubiquitination-defective CD36 mutant, delayed IRS1 degradation. We also found that CD36 functioned through ubiquitination-dependent binding to IRS1 and inhibiting its interaction with cullin 7, a key component of the multisubunit cullin-RING E3 ubiquitin ligase complex. Moreover, dissociation of the Src family kinase Fyn from CD36 by free FAs or Fyn knockdown/inhibition accelerated insulin-induced IRS1 degradation, likely due to disrupted IRS1 interaction with CD36 and thus enhanced binding to cullin 7. In summary, we identified a CD36-dependent FA-sensing pathway that plays an important role in negative feedback regulation of insulin activation and may open up strategies for preventing or managing type 2 diabetes mellitus.

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.

Suppression of protein tyrosine phosphatase N23 predisposes to breast tumorigenesis via activation of FYN kinase.

Disruption of the balanced modulation of reversible tyrosine phosphorylation has been implicated in the etiology of various human cancers, including breast cancer. Protein Tyrosine Phosphatase N23 (PTPN23) resides in chromosomal region 3p21.3, which is hemizygously or homozygously lost in some breast cancer patients. In a loss-of-function PTPome screen, our laboratory identified PTPN23 as a suppressor of cell motility and invasion in mammary epithelial and breast cancer cells. Now, our TCGA (The Cancer Genome Atlas) database analyses illustrate a correlation between low PTPN23 expression and poor survival in breast cancers of various subtypes. Therefore, we investigated the tumor-suppressive function of PTPN23 in an orthotopic transplantation mouse model. Suppression of PTPN23 in Comma 1Dβ cells induced breast tumors within 56 wk. In PTPN23-depleted tumors, we detected hyperphosphorylation of the autophosphorylation site tyrosine in the SRC family kinase (SFK) FYN as well as Tyr142 in β-catenin. We validated the underlying mechanism of PTPN23 function in breast tumorigenesis as that of a key phosphatase that normally suppresses the activity of FYN in two different models. We demonstrated that tumor outgrowth from PTPN23-deficient BT474 cells was suppressed in a xenograft model in vivo upon treatment with AZD0530, an SFK inhibitor. Furthermore, double knockout of FYN and PTPN23 via CRISPR/CAS9 also attenuated tumor outgrowth from PTPN23 knockout Cal51 cells. Overall, this mechanistic analysis of the tumor-suppressive function of PTPN23 in breast cancer supports the identification of FYN as a therapeutic target for breast tumors with heterozygous or homozygous loss of PTPN23.

Computational-experimental approach to drug-target interaction mapping: A case study on kinase inhibitors.

Due to relatively high costs and labor required for experimental profiling of the full target space of chemical compounds, various machine learning models have been proposed as cost-effective means to advance this process in terms of predicting the most potent compound-target interactions for subsequent verification. However, most of the model predictions lack direct experimental validation in the laboratory, making their practical benefits for drug discovery or repurposing applications largely unknown. Here, we therefore introduce and carefully test a systematic computational-experimental framework for the prediction and pre-clinical verification of drug-target interactions using a well-established kernel-based regression algorithm as the prediction model. To evaluate its performance, we first predicted unmeasured binding affinities in a large-scale kinase inhibitor profiling study, and then experimentally tested 100 compound-kinase pairs. The relatively high correlation of 0.77 (p < 0.0001) between the predicted and measured bioactivities supports the potential of the model for filling the experimental gaps in existing compound-target interaction maps. Further, we subjected the model to a more challenging task of predicting target interactions for such a new candidate drug compound that lacks prior binding profile information. As a specific case study, we used tivozanib, an investigational VEGF receptor inhibitor with currently unknown off-target profile. Among 7 kinases with high predicted affinity, we experimentally validated 4 new off-targets of tivozanib, namely the Src-family kinases FRK and FYN A, the non-receptor tyrosine kinase ABL1, and the serine/threonine kinase SLK. Our sub-sequent experimental validation protocol effectively avoids any possible information leakage between the training and validation data, and therefore enables rigorous model validation for practical applications. These results demonstrate that the kernel-based modeling approach offers practical benefits for probing novel insights into the mode of action of investigational compounds, and for the identification of new target selectivities for drug repurposing applications.