c-Src Kinase Research Articles

Phosphorylation of mammalian mitochondrial EF-Tu by Fyn and c-Src kinases

Src Family Kinases (SFKs) are tyrosine kinases known to regulate glucose and fatty acid metabolism as well as oxidative phosphorylation (OXPHOS) in mammalian mitochondria. We and others discovered the association of the SFK kinases Fyn and c-Src with mitochondrial translation components. This translational system is responsible for the synthesis of 13 mitochondrial (mt)-encoded subunits of the OXPHOS complexes and is, thus, essential for energy generation. Mitochondrial ribosomal proteins and various translation elongation factors including Tu (EF-Tumt) have been identified as possible Fyn and c-Src kinase targets. However, the phosphorylation of specific residues in EF-Tumt by these kinases and their roles in the regulation of protein synthesis are yet to be explored. In this study, we report the association of EF-Tumt with cSrc kinase and mapping of phosphorylated Tyr (pTyr) residues by these kinases. We determined that a specific Tyr residue in EF-Tumt at position 266 (EF-Tumt-Y266), located in a highly conserved c-Src consensus motif is one of the major phosphorylation sites. The potential role of EF-Tumt-Y266 phosphorylation in regulation of mitochondrial translation investigated by site-directed mutagenesis. Its phosphomimetic to Glu residue (EF-Tumt-E266) inhibited ternary complex (EF-Tumt•GTP•aatRNA) formation and translation in vitro. Our findings along with data mining analysis of the c-Src knock out (KO) mice proteome suggest that the SFKs have possible roles for regulation of mitochondrial protein synthesis and oxidative energy metabolism in animals.

Abstract 14954: Mitochondrial C-Src Regulates Impaired Mitochondrial Bioenergetics in Right Ventricles in Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) has poor prognosis with presence of right ventricular (RV) dysfunction. However, no RV targeted therapies are available. Mitochondrial (Mito) energy deficiency is detrimental on RV function in PH, but the underlying mechanism remains unclear. Tyrosine kinase phosphorylation (P-Tyr) has been a critical regulatory mechanism in mito. While mito localized c-Src is recognized as the major tyrosine kinase, little is known regarding its role in RV in PH. Hypothesis: We hypothesized that mito c-Src regulates impaired mito bioenergetics in RV in PH. Methods: PH was induced in adult Sprague-Dawley (SD) rats by injecting 20 mg/kg SU5416, followed by 3 wks of hypoxia (10% FiO 2 ) and then 4 wks of normoxia exposure. Control rats injected with vehicle were kept in normoxia. After echocardiography and hemodynamics, RV tissues were collected for mito respiration using high resolution O2K-FluoRespirometer, mito morphology using electron microscopy, and biochemical assays. Cardiomyocytes and H9c2 cells were used for in vitro mechanistic studies. Results: Compared to controls, PH rats show increased RV systolic pressure and RV dysfunction. Mito complex I-coupled oxidative phosphorylation and electron transfer capacity are significantly reduced in RV of PH rats (O 2 flux per mass: 216±21 vs. 90±19 and 209±22 vs. 91±22 pmol·s-1·mg-1, respectively, p <0.01, n=6 each), which is associated with decreased ATP but no changes in mito membrane integrity, mito DNA content, or mito size. Mito c-Src kinase activity and expression are significantly reduced in RV of PH rats and in dysfunctional RV of human patients. Chemical or molecular inhibition of mito c-Src results in a similar reduction of mito respiration in culture as found in RV of PH rats and is associated with decreased ATP production. Mito c-Src phosphorylates NADH dehydrogenase [ubiquinone] flavoprotein 2 (NDUFV2), a critical protein for complex I, and P-Tyr of NDUFV2 is reduced in RV of PH rats. Conclusions: Impaired mito bioenergetics is associated with progression of RV dysfunction. Basal mito c-Src kinase activity is required for mito bioenergetics and mito c-Src-dependent P-Tyr of NDUFV2 may serve as a regulatory mechanism in impaired RV mito bioenergetics in settings of PH.

SNAP23-Mediated Perturbation of Cholesterol-Enriched Membrane Microdomain Promotes Extracellular Vesicle Production in Src-Activated Cancer Cells.

Extracellular vesicles (EVs) originating from intraluminal vesicles (ILVs) formed within multivesicular bodies (MVBs), often referred to as small EV (sEV) or exosomes, are aberrantly produced by cancer cells and regulate the tumor microenvironment. The tyrosine kinase c-Src is upregulated in a wide variety of human cancers and is involved in promoting sEV secretion, suggesting its role in malignant progression. In this study, we found that activated Src liberated synaptosomal-associated protein 23 (SNAP23), a SNARE molecule, from lipid rafts to non-rafts on cellular membrane. We also demonstrated that SNAP23 localized in non-rafts induced cholesterol downregulation and ILV formation, resulting in the upregulation of sEV production in c-Src-transformed cells. Furthermore, the contribution of the SNAP23-cholesterol axis on sEV upregulation was confirmed in pancreatic cancer cells. High SNAP23 expression is associated with poor prognosis in patients with pancreatic cancer. These findings suggest a unique mechanism for the upregulation of sEV production via SNAP23-mediated cholesterol downregulation in Src-activated cancer cells.

Acacetin Inhibits Cell Proliferation and Induces Apoptosis in Human Hepatocellular Carcinoma Cell Lines.

Human hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of death across the world. Recent evidence suggests that STAT3 regulates proliferative, survival, metastasis, and angiogenesis genes in HCC. Novel agents that suppress STAT3 activation can be used to prevent or treat HCC. We used a functional proteomics tumor pathway technology platform and multiple HCC cell lines to investigate the effects of acacetin (ACN) on STAT3 activation, protein kinases, phosphatases, products of STAT3-regulated genes, and apoptosis. ACN was found to inhibit STAT3 activation in a dose- and time-dependent manner in HCC cells. Upstream kinases c-Src, Janus-activated kinase 1, and Janus-activated kinase 2 were also inhibited. The ACN inhibition of STAT3 was abolished by vanadate treatment, suggesting the involvement of tyrosine phosphatase activity. ACN was found to suppress the protein expression of genes involved in proliferation, survival, and angiogenesis via STAT3 inhibition. ACN appears to be a novel STAT3 inhibitor and may be a promising therapeutic compound for application in the treatment of HCC and other cancers.

Activation of GPR75 Signaling Pathway Contributes to the Effect of a 20-HETE Mimetic, 5,14-HEDGE, to Prevent Hypotensive and Tachycardic Responses to Lipopolysaccharide in a Rat Model of Septic Shock.

The orphan receptor, G protein-coupled receptor (GPR) 75, which has been shown to mediate various effects of 20-hydroxyeicosatetraenoic acid (20-HETE), is considered as a therapeutic target in the treatment of cardiovascular diseases in which changes in the production of 20-HETE play a key role in their pathogenesis. Our previous studies showed that 20-HETE mimetic, N -(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (5,14-HEDGE), protects against vascular hyporeactivity, hypotension, tachycardia, and arterial inflammation induced by lipopolysaccharide (LPS) in rats. This study tested the hypothesis that the GPR75 signaling pathway mediates these effects of 5,14-HEDGE in response to systemic exposure to LPS. Mean arterial pressure reduced by 33 mm Hg, and heart rate increased by 102 beats/min at 4 hours following LPS injection. Coimmunoprecipitation studies demonstrated that (1) the dissociation of GPR75/Gα q/11 and GPR kinase interactor 1 (GIT1)/protein kinase C (PKC) α, the association of GPR75/GIT1, large conductance voltage and calcium-activated potassium subunit β (MaxiKβ)/PKCα, MaxiKβ/proto-oncogene tyrosine-protein kinase (c-Src), and epidermal growth factor receptor (EGFR)/c-Src, MaxiKβ, and EGFR tyrosine phosphorylation were decreased, and (2) the association of GIT1/c-Src was increased in the arterial tissues of rats treated with LPS. The LPS-induced changes were prevented by 5,14-HEDGE. N -[20-Hydroxyeicosa-6( Z ),15( Z )-dienoyl]glycine, a 20-HETE antagonist, reversed the effects of 5,14-HEDGE in the arterial tissues of LPS-treated rats. Thus, similar to 20-HETE, by binding to GPR75 and activating the Gα q/11 /PKCα/MaxiKβ, GIT1/PKCα/MaxiKβ, GIT1/c-Src/MaxiKβ, and GIT1/c-Src/EGFR signaling pathways, 5,14-HEDGE may exert its protective effects against LPS-induced hypotension and tachycardia associated with vascular hyporeactivity and arterial inflammation.

Structure-Activity Relationship (SAR) of Flavones on Their Anti-Inflammatory Activity in Murine Macrophages in Culture through the NF-κB Pathway and c-Src Kinase Receptor.

Flavones benefit human health through their anti-inflammatory activity; however, their structure-activity relationship is unclear. Herein, we selected 15 flavones with the same backbone but different substituents and systematically assessed their anti-inflammatory activities in RAW 264.7 regarding cellular-Src kinase (c-Src) affinity, suppression of IκBα phosphorylation, inhibition of nitric oxide (NO) and inducible nitric oxidase (iNOS) production, and downregulation of genes of proinflammatory cytokines interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α). Overall, our results showed that the double bond between C2-C3 and C3'- and C4'-OH promoted anti-inflammatory activity, while C8- and C5'-OH and the methoxy group on C4' attenuated the overall anti-inflammatory and antioxidant activities. The hydroxyl groups at other positions exhibited more complicated functions. The two most effective flavones are 3',4'-dihydroxyflavone and luteolin with inhibitory concentration (IC50) values for inhibiting the LPS-induced nitric oxide level are 9.61 ± 1.36 and 16.90 ± 0.74 μM, respectively. Furthermore, they suppressed the production of iNOS by approximately 90% and inhibited IL-1β and IL-6 by more than 95%. Taken together, our results established a relationship between the flavone structure and anti-inflammatory activity in vitro.

P998: REGULATION OF BCL-X SPLICING BY SRC KINASES INHIBITORS IN MYELOPROLIFERATIVE NEOPLASMS

Background: Myeloproliferative neoplasms (MPN) are a group of incurable diseases that include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Both ET and PV are associated with prolonged clinical courses, with symptoms that affect their quality of life, and risk of progression to MF and acute leukemia. MF is the MPN subtype with the most adverse prognosis and is associated with a high risk of leukemic transformation. The FDA approved the use of ruxolitinib, a JAK1/2 kinase inhibitor, that proved to be effective in reducing symptoms and spleen volume, but not in inducing complete molecular remission. Although hematopoietic stem cell transplantation involves a high risk of mortality, it is nowadays the only curative treatment for MF. Therefore, it is relevant to identify new therapeutic strategies to improve the clinical outcome of MPN. Recently, there has been a growing interest in Bcl‐xL, the long isoform encoded by alternative splicing of the Bcl‐x gene, that acts as an anti‐apoptotic regulator. Bcl-xL appears to be up-regulated in MPN patients and we previously showed that its modulation correlates with the clinical severity of MPN subgroups. We also demonstrated that ABT-737, a specific Bcl-xL inhibitor, has a synergistic effect in combination with Ruxolitinib in HEL cell lines and MPN patients’ cells, suggesting that Bcl-xL inhibition could increase the power of treatment (Petiti et al. J Cell Mol Med. 2020). Unfortunately, ABT-737 is characterized by poor bioavailability, so a new approach to target Bcl-xL must be investigated. Paronetto et al. found that tyrosine phosphorylation of Sam68 in cancer cells may protect them from apoptosis by altering the Bcl-xS/L ratio in favor of Bcl-xL. Intriguingly, c-Src is one of the tyrosine kinases that phosphorylate Sam68 and several papers indicated its involvement in the MPN pathogenesis, without explaining the molecular mechanism. Aims: We aimed to molecular investigate the mechanism linking c-Src kinase activity with the deregulation of Bcl-x splicing in MPN, intending to consider c-Src kinase inhibitors, widely bioavailable, as indirect modulators of Bcl-xL to improve MPNs outcome. Methods: HEL cell lines, JAK2 V617F-mutated in homozygosis, has been incubated with different concentrations of ruxolitinib (JAK1/2 inhibitor) and dasatinib (SRC kinases inhibitor) at different concentrations and time points. Then, proliferation, and apoptosis were evaluated. Subsequently, the c-Src pathway, Sam68, and Bcl-xL/S ratio were investigated at both RNA and protein levels. Results: Performing in vitro study on HEL cell lines, we identified that dasatinib induced apoptosis and inhibition of proliferation in HEL cell lines, with a synergistic effect in combination with ruxolitinib. We also showed that dasatinib alone inhibited c-Src and Sam68 phosphorylation and down-modulated Bclx-L protein expression. Combination treatment with ruxolitinib and dasatinib increased both pro-apoptotic Cleaved Caspase 3 and BAX protein expression. Furthermore, we noted that treatment with ruxolitinib alone decreases the mRNA levels of Bclx-L, but also of the Bcl-xS isoform. On the contrary, combined drug therapy modulated the Bcl-xL/S mRNA ratio in favor of the pro-apoptotic Bcl-xS isoform. Summary/Conclusion: Although further studies will be necessary to better understand the c-Src role in Bcl-x splicing regulation in MPN, our preliminary data suggest that indirect downmodulation of Bcl-xL through SRC kinases inhibitors in combination with standard therapy could be useful in the future for the treatment of MPN patients.

Identifying a Feasible Transition Pathway between Two Conformational States for a Protein.

Proteins usually need to transit between different conformational states to fulfill their biological functions. In the mechanistic study of such transition processes by molecular dynamics simulations, identification of the minimum free energy path (MFEP) can substantially reduce the sampling space, thus enabling rigorous thermodynamic evaluation of the process. Conventionally, the MFEP is derived by iterative local optimization from an initial path, which is typically generated by simple brute force techniques like the targeted molecular dynamics (tMD). Therefore, the quality of the initial path determines the successfulness of MFEP estimation. In this work, we propose a method to improve derivation of the initial path. Through iterative relaxation-biasing simulations in a bidirectional manner, this method can construct a feasible transition pathway connecting two known states for a protein. Evaluation on small, fast-folding proteins against long equilibrium trajectories supports the good sampling efficiency of our method. When applied to larger proteins including the catalytic domain of human c-Src kinase as well as the converter domain of myosin VI, the paths generated by our method deviate significantly from those computed with the generic tMD approach. More importantly, free energy profiles and intermediate states obtained from our paths exhibit remarkable improvements over those from tMD paths with respect to both physical rationality and consistency with a priori knowledge.

Allopregnanolone Promotes Migration and Invasion of Human Glioblastoma Cells through the Protein Tyrosine Kinase c-Src Activation.

Glioblastomas (GBs) are the most aggressive and common primary malignant brain tumors. Steroid hormone progesterone (P4) and its neuroactive metabolites, such as allopregnanolone (3α-THP) are synthesized by neural, glial, and malignant GB cells. P4 promotes cellular proliferation, migration, and invasion of human GB cells at physiological concentrations. It has been reported that 3α-THP promotes GB cell proliferation. Here we investigated the effects of 3α-THP on GB cell migration and invasion, the participation of the enzymes involved in its metabolism (AKR1C1-4), and the role of the c-Src kinase in 3α-THP effects in GBs. 3α-THP 100 nM promoted migration and invasion of U251, U87, and LN229 human-derived GB cell lines. We observed that U251, LN229, and T98G cell lines exhibited a higher protein content of AKR1C1-4 than normal human astrocytes. AKR1C1-4 silencing did not modify 3α-THP effects on migration and invasion. 3α-THP activated c-Src protein at 10 min (U251 cells) and 15 min (U87 and LN229 cells). Interestingly, the pharmacological inhibition of c-Src decreases the promoting effects of 3α-THP on cell migration and invasion. Together, these data indicate that 3α-THP promotes GB migration and invasion through c-Src activation.

Practical Protocols for Efficient Sampling of Kinase-Inhibitor Binding Pathways Using Two-Dimensional Replica-Exchange Molecular Dynamics.

Molecular dynamics (MD) simulations are increasingly used to study various biological processes such as protein folding, conformational changes, and ligand binding. These processes generally involve slow dynamics that occur on the millisecond or longer timescale, which are difficult to simulate by conventional atomistic MD. Recently, we applied a two-dimensional (2D) replica-exchange MD (REMD) method, which combines the generalized replica exchange with solute tempering (gREST) with the replica-exchange umbrella sampling (REUS) in kinase-inhibitor binding simulations, and successfully observed multiple ligand binding/unbinding events. To efficiently apply the gREST/REUS method to other kinase-inhibitor systems, we establish modified, practical protocols with non-trivial simulation parameter tuning. The current gREST/REUS simulation protocols are tested for three kinase-inhibitor systems: c-Src kinase with PP1, c-Src kinase with Dasatinib, and c-Abl kinase with Imatinib. We optimized the definition of kinase-ligand distance as a collective variable (CV), the solute temperatures in gREST, and replica distributions and umbrella forces in the REUS simulations. Also, the initial structures of each replica in the 2D replica space were prepared carefully by pulling each ligand from and toward the protein binding sites for keeping stable kinase conformations. These optimizations were carried out individually in multiple short MD simulations. The current gREST/REUS simulation protocol ensures good random walks in 2D replica spaces, which are required for enhanced sampling of inhibitor dynamics around a target kinase.

SRC Kinase-Mediated Tyrosine Phosphorylation of TUBB3 Regulates Its Stability and Mitotic Spindle Dynamics in Prostate Cancer Cells.

Tubulin is an integral part of the cytoskeleton and plays a pivotal role in cellular signaling, maintenance, and division. β-tubulin is also the molecular target for taxane compounds such as docetaxel (DTX) and cabazitaxel (CTX), both first-line treatments for several solid cancers. Increased expression of Class III β-tubulin (TUBB3), a primarily neural isoform of β-tubulin, correlates with taxane resistance and poor prognosis. Although tyrosine kinase c-Src has been implicated to phosphorylate β-tubulins during both hematopoietic and neural differentiation, the mechanisms by which Src modulates tubulins functions are still poorly understood. Here, we report, for the first time, that TUBB3 is phosphorylated at Tyrosine 340 (Y340) by c-SRC in prostate cancer cells. We also showed that Y340 phosphorylation regulates TUBB3 protein stability and subcellular localization. Furthermore, we demonstrated that inhibition of SRC kinase activity compromises spindle stability in mitotic cells, at least partly due to the lack of TUBB3 Y340 phosphorylation. Given the importance of TUBB3 as a clinical biomarker of poor prognosis and drug resistance, characterization of TUBB3 posttranslational regulation could potentially serve as new biomarkers for disease recurrence and/or treatment failure.

Onc201 reduces osteoclastogenesis and prevents ovariectomy-induced bone loss via inhibiting RANKL-induced NFATc1 activation and the integrin signaling pathway

Osteoporosis is an osteolytic disease with a disrupted balance between the resorption and formation of bone as well as bone microstructure degeneration, leading to bone loss and increased fracture risk, which greatly affects patients’ quality of life. Currently, inhibition of osteoclast bone resorption remains the mainstream treatment for osteoporosis. Onc201, a new compound, induces the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and has an efficient anticancer effect in clinical trials. However, its effects on osteolytic disease and the mechanism of action are unclear. We examined the effect of Onc201 on nuclear factor κB ligand-receptor activator (RANKL)-induced osteoclasts via Cell Counting Kit-8, bone resorption assay, luciferase reporter assay, immunofluorescence staining, calcium ion intensity assay and employed an ovariectomy model to investigate the effect of Onc201 on osteoporosis in the mice. Results showed that Onc201 inhibited the function and formation of osteoclasts induced by RANKL in a manner that was dependent on time and concentration, and did not cause cytotoxicity. Mechanistically, Onc201 inhibited osteoclast-relevant genes and NFATc1 expression, the main transcriptional regulatory factor of the formation of osteoclasts induced by RANKL; meanwhile, downregulating the expressions of the osteoclast cytoskeleton key signal molecules integrin αvβ3, focal adhesion kinase (FAK), c-Src, and spleen-associated tyrosine kinase (SYK). In addition, Onc201 had a protective effect on the mouse model of bone loss caused by ovariectomy-induced estrogen deficiency, which is consistent with the in vitro results. Our findings suggest that the new small-molecular compound Onc201 has the potential to prevent osteoclast-related osteolytic diseases.

Effects of Electrical Stimulation on the Signal Transduction-Related Proteins, c-Src and Focal Adhesion Kinase, in Fibroblasts.

Electrical stimulation of the skin and muscles, e.g., in the fields of rehabilitation medicine and acupuncture, is known to locally increase blood flow and metabolism, and thus have beneficial health effects. However, little is known about the changes in cellular morphology or regulation of the localization of specific proteins in response to electrical stimuli. The present study was performed to examine the effects of electrical stimulation on the cytoskeletal system of cultured fibroblasts. Following application of electrical stimulation to cultured fibroblastic cells for a period of about 2 h, the stress fibers in the cells became thicker and the cells showed a contracted appearance. Cells were subjected to periodic electrical stimulation for 0 (unstimulated control), 2, 5, or 20 h. The stress fibers showed an increase in thickness within 2 h, and became gradually thicker until 20 h. In addition, the focal adhesions and stress fibers were enlarged after 2 h of continuous stimulation, and both stress fibers and focal adhesions became larger and thicker after 20 h of periodic stimulation. Cells showed increased staining of focal adhesions with anti-phosphotyrosine antibody (PY-20) after electrical stimulation. Cells also showed increased staining of tyrosine-phosphorylated focal adhesion kinase (FAK) (pY397) and tyrosine-phosphorylated c-Src (pY418), indicating that electrical stimulation affected signal transduction-related proteins.

A FRET-Based Biosensor for the Src N-Terminal Regulatory Element.

In signaling proteins, intrinsically disordered regions often represent regulatory elements, which are sensitive to environmental effects, ligand binding, and post-translational modifications. The conformational space sampled by disordered regions can be affected by environmental stimuli and these changes trigger, vis a vis effector domain, downstream processes. The disordered nature of these regulatory elements enables signal integration and graded responses but prevents the application of classical approaches for drug screening based on the existence of a fixed three-dimensional structure. We have designed a genetically encodable biosensor for the N-terminal regulatory element of the c-Src kinase, the first discovered protooncogene and lead representative of the Src family of kinases. The biosensor is formed by two fluorescent proteins forming a FRET pair fused at the two extremes of a construct including the SH4, unique and SH3 domains of Src. An internal control is provided by an engineered proteolytic site allowing the generation of an identical mixture of the disconnected fluorophores. We show FRET variations induced by ligand binding. The biosensor has been used for a high-throughput screening of a library of 1669 compounds with seven hits confirmed by NMR.

Mechanical force can enhance c-Src kinase activity by impairing autoinhibition

Cellular mechanosensing is pivotal for virtually all biological processes, and many molecular mechano-sensors and their way of function are being uncovered. In this work, we suggest that c-Src kinase acts as a direct mechano-sensor. c-Src is responsible for, among others, cell proliferation, and shows increased activity in stretched cells. In its native state, c-Src has little basal activity, because its kinase domain binds to an SH2 and SH3 domain. However, it is known that c-Src can bind to p130Cas, through which force can be transmitted to the membrane. Using molecular dynamics simulations, we show that force acting between the membrane-bound N-terminus of the SH3 domain and p130Cas induces partial SH3 unfolding, thereby impeding rebinding of the kinase domain onto SH2/SH3 and effectively enhancing kinase activity. Forces involved in this process are slightly lower or similar to the forces required to pull out c-Src from the membrane through the myristoyl linker, and key interactions involved in this anchoring are salt bridges between negative lipids and nearby basic residues in c-Src. Thus, c-Src appears to be a candidate for an intriguing mechanosensing mechanism of impaired kinase inhibition, which can be potentially tuned by membrane composition and other environmental factors.

Domain camoflouge of c-Src: segmental labeling highlights oncoprotein domains for neutron scattering

c-Src kinase is a non-receptor tyrosine kinase that aberrantly phosphorylates several signaling proteins in different cancers. It is a multidomain protein comprising N-terminal disordered SH4 and globular SH3, SH2, and kinase (SH1) domains. The regulatory domain SH3 has been shown to form an intramolecular fuzzy complex with the disordered SH4 domain during c-Src activity and upregulation. However, studying the conformations of individual domains, especially disordered regions, during such interactions remains a technical challenge.

Relevance of c-Src and protein phosphatase 2A to aromatase activity: Evidence of an acute self-regulating oestrogenic signalling complex in rat central nervous system.

We previously reported that aromatase protein levels do not parallel aromatase enzyme activity. This suggests that oestrogenic signalling may be modulated via post-translational modification of aromatase protein. The tyrosine and serine phosphorylation state of aromatase are known to influence its activity. To investigate the possible relevance of aromatase phosphorylation to the incongruity observed between aromatase protein and its activity, we explored interactions between aromatase and the tyrosine kinase c-Src and the serine protein phosphatases 2A and 5 (PP2A and PP5), as well as the relationship between levels of tyrosine-phosphorylated aromatase and the extrapolated aromatase activity. We found that (a) hypothalamic aromatase was significantly more heavily tyrosine-phosphorylated than spinal aromatase; (b) aromatase was oligomerized with c-Src and PP2A/PP5, potentially activating aromatase via tyrosine-phosphorylation and serine-dephosphorylation; (c) the associations of c-Src and PP2A/PP5 with hypothalamic aromatase were substantially greater than with spinal aromatase; and (d) aromatase, oestrogen receptor α, PP2A, and c-Src were present in a common membrane oligomer. The existence of c-Src and PP2A in an oligomer that also contains aromatase and membrane oestrogen receptor α (and presumably other signalling molecules) indicates the presence in the CNS of a potentially self-regulating oestrogenic signalling unit. The degree to which such a complex operates autonomously and the regulatory factors thereof are likely to have substantial physiological implications and clinical relevance.

Progesterone and its Metabolite Allopregnanolone Promote Invasion of Human Glioblastoma Cells Through Metalloproteinase-9 and cSrc Kinase

Progesterone and its Metabolite Allopregnanolone Promote Invasion of Human Glioblastoma Cells Through Metalloproteinase-9 and cSrc Kinase

FABP4 activates the JAK2/STAT2 pathway via Rap1a in the homocysteine-induced macrophage inflammatory response in ApoE−/− mice atherosclerosis

AbstractAtherosclerosis is a chronic inflammatory vascular disease, and inflammation plays a critical role in its formation and progression. Elevated serum homocysteine (Hcy) is an independent risk factor for atherosclerosis. Previous studies have shown that fatty acid binding protein 4 (FABP4) plays an important role in macrophage inflammation and lipid metabolism in atherosclerosis induced by Hcy. However, the underlying molecular mechanism of FABP4 in Hcy-induced macrophage inflammation remains unknown. In this study, we found that FABP4 activated the Janus kinase 2/signal transducer and activator of transcription 2 (JAK2/STAT2) pathway in macrophage inflammation induced by Hcy. Of note, we further observed that ras-related protein Rap-1a (Rap1a) induced the Tyr416 phosphorylation and membrane translocation of non-receptor tyrosine kinase (c-Src) to activate the JAK2/STAT2 pathway. In addition, the suppressor of cytokine signaling 1 (SOCS1)—a transcriptional target of signal transducer and activator of transcription (STATs) inhibited the JAK2/STAT2 pathway and Rap1a expression via a negative feedback loop. In summary, these results demonstrated that FABP4 promotes c-Src phosphorylation and membrane translocation via Rap1a to activate the JAK2/STAT2 pathway, contributing to Hcy-accelerated macrophage inflammation in ApoE−/− mice.

Conformation-tunable ATP-competitive kinase inhibitors.

Small molecule kinase inhibitors have shown immense clinical utility for diverse indications. While >60 kinase inhibitors have been approved (and many more in clinical trials), it remains unclear whether the clinical efficacy of a kinase inhibitor is solely dependent on enzymatic inhibition, or whether non-catalytic functions play a role in the efficacy of some kinase inhibitors. Here, we designed and synthesized a series of pyrazolopyrimidine kinase inhibitors that modulate the global kinase conformation of c-Src kinase. Expanding upon our findings from the pyrazolopyrimidine inhibitor series, we designed, synthesized, and evaluated three pair of conformation-selective kinase inhibitors, each with a unique hinge-binding scaffold. We profiled each pair of kinase inhibitors across 468 kinases and identified 38 kinases that could be studied using these pair of conformation-selective inhibitors. We also explore the binding of conformation-selective kinase inhibitors to mutant kinases of EGFR, FLT3, and KIT. Together, these studies yield important insight into the design of conformation-tunable kinase inhibitors and provide a toolset of compounds to study the role of protein conformation on kinase signaling.