Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca²⁺ signal generated in cells by different effectors, where the Ca²⁺-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca²⁺-dependent and Ca²⁺-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca²⁺/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca²⁺-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca²⁺/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca²⁺ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.     

Cigarette smoke particle-phase extract induces HO-1 expression in human tracheal smooth muscle cells: role of the c-Src/NADPH oxidase/MAPK/Nrf2 signaling pathway

Heme oxygenase-1 (HO-1) is known as an oxidative stress protein that is up-regulated by various stimuli. HO-1 has been shown to protect cells against oxidative damage. Cigarette smoke is a potential inflammatory mediator that causes chronic obstructive pulmonary disease and asthma. In this study, we report that cigarette smoke particle-phase extract (CSPE) is an inducer of HO-1 expression mediated through various signaling pathways in human tracheal smooth muscle cells (HTSMCs). CSPE-induced HO-1 protein, mRNA expression, and promoter activity were attenuated by pretreatment with a ROS scavenger (N-acetyl-l-cysteine) and inhibitors of c-Src (PP1), NADPH oxidase [diphenylene iodonium chloride (DPI) and apocynin (APO)], MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs for Src, p47^phox, NOX2, p42, p38, JNK2, or NF-E2-related factor 2 (Nrf2). CSPE-stimulated translocation of p47^phox and Nrf2, ROS production, and NADPH oxidase activity was attenuated by transfection with siRNAs for Src, p47^phox, and NOX2 or pretreatment with PP1, DPI, or APO. Furthermore, CSPE-induced NOX2, c-Src, and p47^phox complex formation was revealed by immunoprecipitation using an anti-NOX2, anti-p47^phox, or anti-c-Src Ab followed by Western blot against anti-NOX2, anti-p47^phox, or anti-c-Src Abs. These results demonstrate that CSPE-induced ROS generation is mediated through a c-Src/NADPH oxidase/MAPK pathway and in turn initiates the activation of Nrf2 and ultimately induces HO-1 expression in HTSMCs.     

c-Src tyrosine kinase mediates high glucose-induced endothelin-1 expression

Endothelin-1 (ET-1) plays an important role in the pathophysiology of diabetes-associated cardiovascular disorders. The molecular mechanisms leading to ET-1 upregulation in diabetes are not entirely defined. c-Src tyrosine kinase regulates important pathophysiological aspects of vascular response to insults. In this study, we aimed to elucidate whether high glucose-activated c-Src signaling plays a role in the regulation of ET-1 expression. Human endothelial cells EAhy926 (ECs) were exposed to normal or high levels of glucose for 24 h. Male C57BL/6J mice were rendered diabetic with streptozotocin and then treated with a specific c-Src inhibitor (Src I1) or c-Src siRNA. Real-time PCR, Western blot, and ELISA, were used to investigate ET-1 regulation. The c-Src activity and expression were selectively downregulated by pharmacological inhibition and siRNA-mediated gene silencing, respectively. High glucose dose-dependently up-regulated c-Src phosphorylation and ET-1 gene and protein expression levels in human ECs. Chemical inhibition or silencing of c-Src significantly decreased the high-glucose augmented ET-1 expression in cultured ECs. In vivo studies showed significant elevations in the aortic ET-1 mRNA expression and plasma ET-1 concentration in diabetic mice compared to non-diabetic animals. Treatment with Src I1, as well as in vivo silencing of c-Src, significantly reduced the upregulated ET-1 expression in diabetic mice. These data provide new insights into the regulation of ET-1 expression in endothelial cells in diabetes. Pharmacological targeting of c-Src activity and/or expression may represent a potential therapeutic strategy to reduce ET-1 level and to counteract diabetes-induced deleterious vascular effects.     

Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation

Timeless was originally identified in Drosophila as an essential component of circadian cycle regulation, where its function is tightly controlled at the protein level by tyrosine phosphorylation and subsequent degradation. In mammals, Timeless has also been implicated in circadian rhythms as well as cell cycle control and embryonic development. Here we report that mammalian Timeless is an SH3 domain-binding protein and substrate for several members of the Src protein–tyrosine kinase family, including Fyn, Hck, c-Src and c-Yes. Co-expression of Tim with Fyn or Hck was followed by ubiquitylation and subsequent degradation in human 293 T cells. While c-Src and c-Yes also promoted Tim ubiquitylation, in this case ubiquitylation correlated with Tim protein accumulation rather than degradation. Both c-Src and c-Yes selectively promoted modification of Tim through Lys63-linked polyubiquitin, which may explain the differential effects on Tim protein turnover. These data show distinct and opposing roles for individual Src-family members in the regulation of Tim protein levels, suggesting a unique mechanism for the regulation of Tim function in mammals.     

pp60cSrc Is a Caspase-3 Substrate and Is Essential for the Transformed Phenotype of A431 Cells

The protein tyrosine kinase c-Src is a major signal transduction element in many growth factor receptor signals for proliferation and transformation. We showed recently that c-Src is a mediator of antiapoptotic signals through regulation of the antiapoptotic gene Bcl-X_L. A431 cells overexpress the EGF receptor (EGFR) and possess high Src activity. In A431 cells, Src is activated by the EGFR, and inhibition of the EGF receptor results in c-Src inhibition. In this study we show that (i) inhibition of the EGFR kinase or Src kinase by specific inhibitors results in growth inhibition and inhibition of colony formation in soft agar. The relative efficacies of the EGFR kinase inhibitor and of the Src kinase inhibitor are similar suggesting the major role src plays in the oncogenic signaling of EGFR in A431 cells. (ii) The Src kinase inhibitor PP1 sensitizes A431 cells to CDDP-induced apoptosis. (iii) CDDP induces caspase-3-dependent cleavage of the c-Src C-terminal portion and a concomitant reduction in Bcl-X_L levels. We conclude that c-Src is an important antiapoptotic signaling molecule downstream of the EGF receptor that contributes to the transformed phenotype of A431 cells.     

Involvement of Reactive Oxygen Species in a Feed-forward Mechanism of Na/K-ATPase-mediated Signaling Transduction

Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-l-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial ²²Na⁺ transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of the Na/K-ATPase α1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport.     

Mercuric chloride mediates a protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase which is independent of the phosphorylation / dephosphorylation of a carboxyl terminal tyrosine

Little is known about the regulatory mechanism of c-Src kinase in cells except the suggested regulation through phosphorylation and dephosphorylation of its carboxyl terminal tyrosine residue (Y527). We here demonstrated that exposure of NIH3T3 cells to mercuric chloride (HgCl₂) induces both aggregation and activation of Src kinase protein through a redox-linked mechanism. The aggregation of Src proteins was suggested to be induced by the sulfhydryl groups-to-Hg²⁺ reaction-mediated polymerization of cell membrane proteins to which the Src proteins associate noncovalently. The possibility was ruled out that the aggregation occurred secondarily to the promotion of protein tyrosine phosphorylation. Further study revealed that the Src kinase was activated by HgCl₂ at least in part independent of the known Csk kinase-linked or Y527-phosphorylation/dephosphorylation-mediated control. Correspondingly, CNBr cleavage mapping of phosphopeptides for autophosphorylated c-Src protein demonstrated selective promotion of phosphorylation at Y416 in HgCl₂-treated cells without obvious change in the phosphorylation level at Y527. These results suggest a unique protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase in NIH3T3 cells. © 1996 Wiley-Liss, Inc.     

Propofol Inhibits SIRT2 Deacetylase through a Conformation-specific,Allosteric Site

meta-Azi-propofol (AziPm) is a photoactive analog of the general anesthetic propofol. We photolabeled a myelin-enriched fraction from rat brain with [³H]AziPm and identified the sirtuin deacetylase SIRT2 as a target of the anesthetic. AziPm photolabeled three SIRT2 residues (Tyr¹³⁹, Phe¹⁹⁰, and Met²⁰⁶) that are located in a single allosteric protein site, and propofol inhibited [³H]AziPm photolabeling of this site in myelin SIRT2. Structural modeling and in vitro experiments with recombinant human SIRT2 determined that propofol and [³H]AziPm only bind specifically and competitively to the enzyme when co-equilibrated with other substrates, which suggests that the anesthetic site is either created or stabilized in enzymatic conformations that are induced by substrate binding. In contrast to SIRT2, specific binding of [³H]AziPm or propofol to recombinant human SIRT1 was not observed. Residues that line the propofol binding site on SIRT2 contact the sirtuin co-substrate NAD⁺ during enzymatic catalysis, and assays that measured SIRT2 deacetylation of acetylated α-tubulin revealed that propofol inhibits enzymatic function. We conclude that propofol inhibits the mammalian deacetylase SIRT2 through a conformation-specific, allosteric protein site that is unique from the previously described binding sites of other inhibitors. This suggests that propofol might influence cellular events that are regulated by protein acetylation state.     

The Role of the Focal Adhesion Protein PINCH1 for the Radiosensitivity of Adhesion and Suspension Cell Cultures

Focal adhesion (FA) signaling mediated by adhesion to extracellular matrix and growth factor receptors contributes to the regulation of the cellular stress response to external stimuli. Critical to focal adhesion assembly and signaling is the adapter protein PINCH1. To evaluate whether the prosurvival function of PINCH1 in radiation cell survival depends on cell adhesion, we examined PINCH1 ^fl/fl and PINCH1 ^−/− mouse embryonic fibroblasts and human cancer cell lines. Here, we found that the enhanced cellular radiosensitivity mediated by PINCH1 depletion observed under adhesion conditions is conserved when cells are irradiated under suspension conditions. This unsuspected finding could not be explained by the observed modification of adhesion and growth factor associated signaling involving FAK, Paxillin, p130^CAS, Src, AKT, GSK3β and ERK1/2 under suspension and serum withdrawal relative to adhesion conditions with serum. Our data suggest that the adapter protein PINCH1 critically participates in the regulation of the cellular radiosensitivity of normal and malignant cells similarly under adhesion and suspension conditions.     

SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD⁺-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.     

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

The daily phagocytosis of shed photoreceptor outer segments by pigment epithelial cells is critical for the maintenance of the retina. In a subtractive polymerase chain reaction analysis, we found that functional differentiation of human ARPE19 retinal pigment epithelial (RPE) cells is accompanied by up-regulation of annexin (anx) A2, a major Src substrate and regulator of membrane–cytoskeleton dynamics. Here, we show that anx A2 is recruited to the nascent phagocytic cup in vitro and in vivo and that it fully dissociates once the phagosome is internalized. In ARPE19 cells depleted of anx A2 by using small interfering RNA and in ANX A2^−/− mice the phagocytosis of outer segments was impaired, and in ANX A2^−/− mice there was an accumulation of phagocytosed outer segments in the RPE apical processes, indicative of retarded phagosome transport. We show that anx A2 is tyrosine phosphorylated at the onset of phagocytosis and that the synchronized activation of focal adhesion kinase and c-Src is abnormal in ANX A2^−/− mice. These findings reveal that anx A2 is involved in the circadian regulation of outer segment phagocytosis, and they provide new insight into the protein machinery that regulates phagocytic function in RPE cells.     

A novel role of vascular endothelial cadherin in modulating c-Src activation and downstream signaling of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and vascular permeability, in which c-Src tyrosine kinase plays an essential role. However, the mechanisms by which VEGF stimulates c-Src activation have remained unclear. Here, we demonstrate that vascular endothelial cadherin (VE-cadherin) plays a critical role in regulating c-Src activation in response to VEGF. In vascular endothelial cells, VE-cadherin was basally associated with c-Src and Csk (C-terminal Src kinase), a negative regulator of Src activation. VEGF stimulated Csk release from VE-cadherin by recruiting the protein tyrosine phosphatase SHP2 to VE-cadherin signaling complex, leading to an increase in c-Src activation. Silencing VE-cadherin with small interference RNA significantly reduced VEGF-stimulated c-Src activation. Disrupting the association of VE-cadherin and Csk through the reconstitution of Csk binding-defective mutant of VE-cadherin also diminished Src activation. Moreover, inhibiting SHP2 by small interference RNA and adenovirus-mediated expression of a catalytically inactive mutant of SHP2 attenuated c-Src activation by blocking the disassociation of Csk from VE-cadherin. Furthermore, VE-cadherin and SHP2 differentially regulates VEGF downstream signaling. The inhibition of c-Src, VE-cadherin, and SHP2 diminished VEGF-mediated activation of Akt and endothelial nitric-oxide synthase. In contrast, inhibiting VE-cadherin and SHP2 enhanced ERK1/2 activation in response to VEGF. These findings reveal a novel role for VE-cadherin in modulating c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function.     

5-Azacytidine-induced Protein 2 (AZI2) Regulates Bone Mass by Fine-tuning Osteoclast Survival

5-Azacytidine-induced protein 2 (AZI2) is a TNF receptor (TNFR)-associated factor family member-associated NF-κB activator-binding kinase 1-binding protein that regulates the production of IFNs. A previous in vitro study showed that AZI2 is involved in dendritic cell differentiation. However, the roles of AZI2 in immunity and its pleiotropic functions are unknown in vivo. Here we report that AZI2 knock-out mice exhibit normal dendritic cell differentiation in vivo. However, we found that adult AZI2 knock-out mice have severe osteoporosis due to increased osteoclast longevity. We revealed that the higher longevity of AZI2-deficient osteoclasts is due to an augmented activation of proto-oncogene tyrosine-protein kinase Src (c-Src), which is a critical player in osteoclast survival. We found that AZI2 inhibits c-Src activity by regulating the activation of heat shock protein 90 (Hsp90), a chaperone involved in c-Src dephosphorylation. Furthermore, we demonstrated that AZI2 indirectly inhibits c-Src by interacting with the Hsp90 co-chaperone Cdc37. Strikingly, administration of a c-Src inhibitor markedly prevented bone loss in AZI2 knock-out mice. Together, these findings indicate that AZI2 regulates bone mass by fine-tuning osteoclast survival.     

The dioxin receptor controls β1 integrin activation in fibroblasts through a Cbp–Csk–Src pathway

Recent studies have suggested a regulatory role for the dioxin receptor (AhR) in cell adhesion and migration. Following our previous work, we report here that the C-terminal Src kinase-binding protein (Cbp) signaling pathway controls β1 integrin activation and that this mechanism is AhR dependent. T-FGM AhR ?/? fibroblasts displayed higher integrin β1 activation, revealed by the increased binding of the activation reporter 9EG7 anti-β1 mAb and of a soluble fibronectin fragment, as well as by enhanced talin-β1 association. AhR ?/? fibroblasts also showed increased fibronectin secretion and impaired directional migration. Notably, interfering Cbp expression in AhR ?/? fibroblasts reduced β1 integrin activation, improved cell migration and rescued wild-type cell morphology. Cbp over-expression in T-FGM AhR ?/? cells enhanced the formation of inhibitory Csk–Cbp complexes which in turn reduced c-Src p-Tyr⁴¹⁶ activation and focal adhesion kinase (FAK) phosphorylation at the c-Src-responsive residues p-Tyr⁵⁷⁶ and p-Tyr⁵⁷⁷. The c-Src target and migration-related protein Cav1 was also hypophosphorylated at p-Tyr¹⁴ in AhR ?/? cells, and such effect was rescued by down-modulating Cbp levels. Thus, AhR regulates fibroblast migration by modulating β1 integrin activation via Cbp-dependent, Src-mediated signaling.     

Targeted disruption of the tyrosine phosphatase PTPα leads to constitutive downregulation of the kinases Src and Fyn

A role for the receptor-like protein tyrosine phosphatase α (PTPα) in regulating the kinase activity of Src family members has been proposed because ectopic expression of PTPα enhances the dephosphorylation and activation of Src and Fyn [1], [2], [3]. We have generated mice lacking catalytically active PTPα to address the question of whether PTPα is a physiological activator of Src and Fyn, and to investigate its other potential functions in the context of the whole animal. Mice homozygous for the targeted PTPα allele (PTPα^−/−) and lacking detectable PTPα protein exhibited no gross phenotypic defects. The kinase activities of Src and Fyn were significantly reduced in PTPα^−/− mouse brain and primary embryonic fibroblasts, and this correlated with enhanced phosphorylation of the carboxy-terminal regulatory Tyr527 of Src in PTPα^−/− mice. Thus, PTPα is a physiological positive regulator of the tyrosine kinases Src and Fyn. Increased tyrosine phosphorylation of several unidentified proteins was also apparent in PTPα^−/− mouse brain lysates. These may be PTPα substrates or downstream signaling proteins. Taken together, the results indicate that PTPα has a dual function as a positive and negative regulator of tyrosine phosphorylation events, increasing phosphotyrosyl proteins through activation of Src and Fyn, and directly or indirectly removing tyrosine phosphate from other unidentified proteins.     

A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling

The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or Src?K, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src^?/? mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism.     

Involvement of c-Src Tyrosine Kinase Upstream of Class I Phosphatidylinositol (PI) 3-Kinases in Salmonella Enteritidis Rck Protein-mediated Invasion

The Salmonella outer membrane protein Rck mediates a Zipper entry mechanism controlled by tyrosine phosphorylation and class I phosphatidylinositol 3-kinase (PI 3-kinase). However, the underlying mechanism leading to this signaling cascade remains unclear. The present study showed that using Rck-coated beads or Rck-overexpressing Escherichia coli, Rck-mediated actin polymerization and invasion were blocked by PP2, a Src family tyrosine kinase inhibitor. In addition, phosphorylation of Src family kinases significantly increased after stimulation with Rck. The specific contribution of c-Src, one member of the Src family kinases, was demonstrated using c-Src-deficient fibroblasts or c-Src siRNA transfected epithelial cells. We also observed that Rck-mediated internalization led to the formation of a complex between c-Src and at least one tyrosine-phosphorylated protein. Furthermore, our results revealed that the c-Src signal molecule was upstream of PI 3-kinase during the Rck-mediated signaling pathway as Rck-mediated PI 3-kinase activation was blocked by PP2, and PI 3-kinase inhibitor had no effect on the Src phosphorylation. These results demonstrate the involvement of c-Src upstream of the PI 3-kinase in the Zipper entry process mediated by Rck.