The regulation of N-methyl-D-aspartate receptors by Src kinase

Src family kinases (SFKs) play critical roles in the regulation of many cellular functions by growth factors, G-protein-coupled receptors and ligand-gated ion channels. Recent data have shown that SFKs serve as a convergent point of multiple signaling pathways regulating N-methyl-d-aspartate (NMDA) receptors in the central nervous system. Multiple SFK molecules, such as Src and Fyn, closely associate with their substrate, NMDA receptors, via indirect and direct binding mechanisms. The NMDA receptor is associated with an SFK signaling complex consisting of SFKs; the SFK-activating phosphatase, protein tyrosine phosphatase α; and the SFK-inactivating kinase, C-terminal Src kinase. Early studies have demonstrated that intramolecular interactions with the SH2 or SH3 domain lock SFKs in a closed conformation. Disruption of the interdomain interactions can induce the activation of SFKs with multiple signaling pathways involved in regulation of this process. The enzyme activity of SFKs appears 'graded', exhibiting different levels coinciding with activation states. It has also been proposed that the SH2 and SH3 domains may stimulate catalytic activity of protein tyrosine kinases, such as Abl. Recently, it has been found that the enzyme activity of neuronal Src protein is associated with its stability, and that the SH2 and SH3 domain interactions may act not only to constrain the activation of neuronal Src, but also to regulate the enzyme activity of active neuronal Src. Collectively, these findings demonstrate novel mechanisms underlying the regulation of SFKs.     

Angiotensin II Stimulation of Renal Epithelial Cell Na/HCO3 Cotransport Activity: A Central Role for Src Family Kinase/Classic MAPK Pathway Coupling

Angiotensin II (AII) plays an important role in renal proximal tubular acidification via the costimulation of basolateral Na/HCO3 cotransporter (NBC) and apical Na/H exchanger (NHE) activities. These effects are mediated by specific G protein-coupled AII receptors, but their corresponding downstream effectors are incompletely defined. Src family tyrosine kinases (SFKs) contribute to the regulation of both transport activities by a variety of stimuli and are coupled to classic mitogen-activated protein kinase (MAPK) pathway activation in this cell type. We therefore examined these signaling intermediates for involvement in AII-stimulated NBC activity in cultured proximal tubule cells. Subpressor concentrations of AII (0.1 nM) increased NBC activity within minutes, and this effect was abrogated by selective antagonism of AT1 angiotensin receptors, SFKs, or the classic MAPK pathway. AII directly activated Src, as well as the proximal (Raf) and distal (ERK) elements of the classic MAPK module, and the activation of Src was prevented by AT1 receptor antagonism. An associated increase in basolateral membrane NBC1 content is compatible with the involvement of this proximal tubule isoform in these changes. We conclude that AII stimulation of the AT1 receptor increases NBC activity via sequential activation of SFKs and the classic MAPK pathway. Similar requirements for SFK/MAPK coupling in both cholinergic and acidotic costimulation of NBC and NHE activities suggest a central role for these effectors in the coordinated regulation of epithelial transport by diverse stimuli.     

SRC family kinases and receptors: analysis of three activation mechanisms by dynamic systems modeling

Src family kinases (SFKs) interact with a number of cellular receptors. They participate in diverse signaling pathways and cellular functions. Most of the receptors involved in SFK signaling are characterized by similar modes of regulation. This computational study discusses a general kinetic model of SFK-receptor interaction. The analysis of the model reveals three major ways of SFK activation: release of inhibition by C-terminal Src kinase, weakening of the inhibitory intramolecular phosphotyrosine-SH2 interaction, and amplification of a stimulating kinase activity. The SFK model was then extended to simulate interaction with growth factor and T-cell receptors. The modular SFK signaling system was shown to adapt to the requirements of specific signaling contexts and yield qualitatively different responses in the different simulated environments. The model also provides a systematic overview of the major interactions between SFKs and various cellular signaling systems and identifies their common properties.     

The role of c-Src in platelet-derived growth factor alpha receptor internalization

We used two approaches to investigate the role of Src family kinases (SFKs) in ligand-stimulated internalization of the platelet-derived growth factor alpha receptor (alphaPDGFR). In cells that normally express SFKs, the internalization rate of the F72/74 mutant alphaPDGFR (which is unable to recruit or activate SFKs) was slower than that of the wild-type (WT) alphaPDGFR. When expressed in cells lacking SFKs (SYF cells), internalization of the WT and mutant receptors was indistinguishable, which indicated that there was not an inherent defect in the mutant receptor's ability to undergo ligand-driven internalization. The internalization difference between the WT and mutant receptors was seen again when c-Src was expressed in the SYF cells. Surprisingly, c-Src slowed the internalization of the mutant receptor but had little effect on WT receptor. We propose the following working hypothesis to explain these findings. In resting cells SFKs suppress internalization of the alphaPDGFR by phosphorylating a hypothetical protein X. This suppression is relieved when cells are exposed to PDGF because SFKs are recruited to the activated WT alphaPDGFR and thereby no longer actively phosphorylate protein X. The internalization of the mutant receptor is slow because it is unable to recruit SFKs and thereby fails to relieve the suppression of internalization. Our findings suggest a role for SFKs in regulating the permissiveness for internalization of the alphaPDGFR.     

Interaction between the SH3 domain of Src family kinases and the proline-rich motif of HTLV-1 p13: a novel mechanism underlying delivery of Src family kinases to mitochondria

The association of the SH3 (Src homology 3) domain of SFKs (Src family kinases) with protein partners bearing proline-rich motifs has been implicated in the regulation of SFK activity, and has been described as a possible mechanism of relocalization of SFKs to subcellular compartments. We demonstrate in the present study for the first time that p13, an accessory protein encoded by the HTLV-1 (human T-cell leukaemia virus type?1), binds the SH3 domain of SFKs via its C-terminal proline-rich motif, forming a stable heterodimer that translocates to mitochondria by virtue of its N-terminal mitochondrial localization signal. As a result, the activity of SFKs is dramatically enhanced, with a subsequent increase in mitochondrial tyrosine phosphorylation, and the recognized ability of p13 to insert itself into the inner mitochondrial membrane and to perturb the mitochondrial membrane potential is abolished. Overall, the present study, in addition to confirming that the catalytic activity of SFKs is modulated by interactors of their SH3 domain, leads us to hypothesize a general mechanism by which proteins bearing a proline-rich motif and a mitochondrial localization signal at the same time may act as carriers of SFKs into mitochondria, thus contributing to the regulation of mitochondrial functions under various pathophysiological conditions.     

Reelin activates SRC family tyrosine kinases in neurons

BACKGROUND: Reelin is a large signaling molecule that regulates the positioning of neurons in the mammalian brain. Transmission of the Reelin signal to migrating embryonic neurons requires binding to the very-low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor-2 (apoER2). This induces tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1), which interacts with a shared sequence motif in the cytoplasmic tails of both receptors. However, the kinases that mediate Dab1 tyrosine phosphorylation and the intracellular pathways that are triggered by this event remain unknown. RESULTS: We show that Reelin activates members of the Src family of non-receptor tyrosine kinases (SFKs) and that this activation is dependent on the Reelin receptors apoER2 and VLDLR and the adaptor protein Dab1. Dab1 is tyrosine phosphorylated by SFKs, and the kinases themselves can be further activated by phosphorylated Dab1. Increased Dab1 protein expression in fyn-deficient mice implies a response to impaired Reelin signaling that is also observed in mice lacking Reelin or its receptors. However, fyn deficiency alone does not compound the neuronal positioning defect of vldlr- or apoer2-deficient mice, and this finding suggests functional compensation by other SFKs. CONCLUSIONS: Our results show that Dab1 is a physiological substrate as well as an activator of SFKs in neurons. Based on genetic evidence gained from multiple strains of mutant mice with defects in Reelin signaling, we conclude that activation of SFKs is a normal part of the cellular Reelin response.     

A mechanism for SRC kinase-dependent signaling by noncatalytic receptors

A fundamental issue in cell biology is how signals are transmitted across membranes. A variety of transmembrane receptors, including multichain immune recognition receptors, lack catalytic activity and require Src family kinases (SFKs) for signal transduction. However, many receptors only bind and activate SFKs after ligand-induced receptor dimerization. This presents a conundrum: How do SFKs sense the dimerization of receptors to which they are not already bound? Most proposals for resolving this enigma invoke additional players, such as lipid rafts or receptor conformational changes. Here we used simple thermodynamics to show that SFK activation is a natural outcome of clustering of receptors with SFK phosphorylation sites, provided that there is phosphorylation-dependent receptor-SFK association and an SFK bound to one receptor can phosphorylate the second receptor or its associated SFK in a dimer. A simple system of receptor, SFK, and an unregulated protein tyrosine phosphatase (PTP) can account for ligand-induced changes in phosphorylation observed in cells. We suggest that a core signaling system comprising a receptor with SFK phosphorylation sites, an SFK, and an unregulated PTP provides a robust mechanism for transmembrane signal transduction. Other events that regulate signaling in specific cases may have evolved for fine-tuning of this basic mechanism.     

Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases

Src family kinases (SFKs) are abundant in chromaffin cells that reside in the adrenal medulla and respond to cholinergic stimulation by secreting catecholamines. Our previous work indicated that SFKs regulate acetylcholine- or nicotine-induced secretion, but the site of modulatory action was unclear. Using whole cell recordings, we found that inhibition of SFK tyrosine kinase activity by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine) treatment or expression of a kinase-defective c-Src reduced the peak amplitude of nicotine-induced currents in chromaffin cells or in human embryonic kidney cells ectopically expressing functional neuronal alpha3beta4alpha5 acetylcholine receptors (AChRs). Conversely, the phosphotyrosine phosphatase inhibitor, sodium vanadate, or expression of mutationally activated c-Src resulted in enhanced current amplitudes. These results suggest that SFKs and putative phosphotyrosine phosphatases regulate the activity of AChRs by opposing actions. This proposed model was supported further by the findings that SFKs physically associate with the receptor and that the AChR is tyrosine-phosphorylated.     

Src family tyrosine kinases participate in insulin-like growth factor I mitogenic signaling in 3T3-L1 cells.

Insulin-like growth factor-I (IGF-I) stimulates proliferation and differentiation of many cell types, including preadipocytes. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of the extracellular regulated kinase (ERK)-1 and -2 mitogen-activated protein kinase (MAPK) pathway, and that IGF-I-stimulated MAPK is predominantly downstream of Shc, not IRS-1 phosphorylation. The Src family of nonreceptor tyrosine kinases has been shown to mediate the mitogenic effects of other growth factors that also activate Shc and the ERK-1 and -2 MAPKs. Although Src family kinases (SFK) have been implicated in IGF-I action, no specific role for SFKs in IGF-I regulation of mitogenesis has been previously demonstrated. We studied the role of SFKs in IGF-I mitogenic signaling in 3T3-L1 preadipocytes. The SFK-selective inhibitor PP1 completely inhibited both IGF-I-stimulated DNA synthesis and MAPK activation in proliferating 3T3-L1 cells. PP1 inhibited IGF-I phosphorylation of Shc but not of IRS-1. In addition, IGF-I activation of MAPK was inhibited in proliferating cells transiently transfected with a dominant-negative c-Src. Finally, the kinetics of SFK and MAPK activation by IGF-I suggest that SFKs may act upstream of MAPK. IGF-I activation of SFK members c-Src and Fyn occurred within 1 min of treatment, and activity was back to baseline by 10 min. Our previous studies found that IGF-I activation of MAPK peaked at 5 min and was also back to baseline by 10 min. Our results are the first to demonstrate that SFKs mediate IGF-I mitogenic signaling in 3T3-L1 cells and add to the growing body of evidence that SFKs play a crucial role in IGF-I action.     

Src family kinases as mediators of endothelial permeability: effects on inflammation and metastasis

Src family kinases (SFKs) are signaling enzymes that have long been recognized to regulate critical cellular processes such as proliferation, survival, migration, and metastasis. Recently, considerable work has elucidated mechanisms by which SFKs regulate normal and pathologic processes in vascular biology, including endothelial cell proliferation and permeability. Further, when inappropriately activated, SFKs promote pathologic inflammatory processes and tumor metastasis, in part through their effects on the regulation of endothelial monolayer permeability. In this review, we discuss the roles of aberrantly activated SFKs in mediating endothelial permeability in the context of inflammatory states and tumor cell metastasis. We further summarize recent efforts to translate Src-specific inhibitors into therapy for systemic inflammatory conditions and numerous solid organ cancers. The authors’ own research was supported in part by NIH U54 CA 090810 and P20 CA101936 (G.E.G) and NIH T32 CA 09599 (M.P.K.)     

Mutual Regulation of Src Family Kinases and the Neurotrophin Receptor TrkB

The neurotrophin receptor tyrosine kinase TrkB is critical to diverse biological processes. We investigated the interplay of Src family kinases (SFKs) and TrkB to better understand mechanisms of TrkB signaling in physiological and pathological conditions. We compared and contrasted the role of SFKs in TrkB signaling following activation of TrkB by two mechanisms, its transactivation by zinc, and its activation by its prototypic neurotrophin ligand, brain-derived neurotrophic factor (BDNF). Using biochemical, pharmacological, and chemical genetic studies of cultured rodent neurons, we found that zinc promotes preferential phosphorylation of Tyr-705/Tyr-706 of TrkB by a SFK-dependent but TrkB kinase-independent mechanism, a signaling event critical for transactivation of TrkB by zinc. By contrast, SFK activity is not essential for BDNF-mediated activation of TrkB, yet SFK activity is increased as a consequence of TrkB activation by BDNF. Moreover, BDNF-induced phosphorylation of Tyr-705/Tyr-706 of TrkB was inhibited by SFK inhibitors, implicating a regulatory role of SFKs in TrkB activation by BDNF. In sum, SFKs are activated by TrkB and, in turn, SFKs can promote TrkB activation. We propose models depicting the mutual regulation of SFKs and TrkB following activation of TrkB by zinc and BDNF.     

Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor

Wounding usually causes considerable cell damage, and released ATP promotes migration of nearby epithelium. ATP binds to purinergic receptors on the cell surface and induces transactivation of the EGF receptor through signaling by the Src family kinases (SFKs). Here we tested whether ATP activates these kinases through Pyk2, a member of the focal adhesion kinase family. Pyk2 was rapidly and potently activated by treating corneal epithelial cells with ATP, and physical interaction of Pyk2 with the SFKs was enhanced. Disruption of Pyk2 signaling either by siRNA or by expression of a dominant-negative mutant led to inhibition of ATP-induced activation of the SFKs and the EGF receptor. Inhibiting Pyk2 activity also blocked ATP stimulation of healing of wounds in epithelial cell sheets. These data suggest that ATP stimulates sequential activation of Pyk2, SFKs, and the EGF receptor to induce cell migration.     

The Src kinase Yes is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters, but not pancreatic growth factors, which stimulate its association with numerous other signaling molecules

For growth factors, cytokines, G-protein-coupled receptors and numerous other stimuli, the Src Family of kinases (SFK) play a central signaling role. SFKs also play an important role in pancreatic acinar cell function including metabolism, secretion, endocytosis, growth and cytoskeletal integrity, although the specific SFKs involved are not fully known. In the present study we used specific antibodies for the SFK, Yes, to determine its presence, activation by pancreatic secretagogues or growth factors, and interaction with cellular signaling cascades mediated by CCK in which Yes participates in to cause acinar cell responses. Yes was identified in acini and secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post-receptor stimulants activating PKC [TPA] or mobilizing cellular calcium [thapsigargin/calcium ionophore (A23187)] each activated Yes. Secretin, which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK(1)-receptor states. TPA-/CCK-stimulated Yes activation was completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130(Cas), PI3K and PTEN. This study demonstrates that in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCK's effect in acinar cell function our results suggest that it is one of the important pancreatic SFKs mediating these effects.     

Regulation of the Src Family Kinases by Csk

The non-receptor tyrosine kinase Csk serves as an indispensable negative regulator of the Src family tyrosine kinases (SFKs) by specifically phosphorylating the negative regulatory site of SFKs, thereby suppressing their oncogenic potential. Csk is primarily regulated through its SH2 domain, which is required for membrane translocation of Csk via binding to scaffold proteins such as Cbp/PAG1. The binding of scaffolds to the SH2 domain can also upregulate Csk kinase activity. These regulatory features have been elucidated by analyses of Csk structure at the atomic levels. Although Csk itself may not be mutated in human cancers, perturbation of the regulatory system consisting of Csk, Cbp/PAG1, or other scaffolds, and certain tyrosine phosphatases may explain the upregulation of SFKs frequently observed in human cancers. This review focuses on the molecular bases for the function, structure, and regulation of Csk as a unique regulatory tyrosine kinase for SFKs.     

Impairment in a negative regulatory system for TCR signaling in CD4+ T cells from old mice

To examine the involvement of lipid rafts in an age-associated decline in T cell function, we analyzed the effect of aging on the constituents of lipid rafts in resting mouse CD4(+) T cells. We found a pronounced, age-dependent reduction in PAG/Cbp, which is involved in the regulation of Src family kinases (SFKs) by recruiting Csk (a negative regulator of SFKs) to lipid rafts. This reduction is specific for T cells and is attributed, at least in part, to the reduction in its mRNA level. The reduction of PAG accompanies marked impairment in recruiting Csk to lipid rafts and a concomitant decrease in the inactive forms of SFKs. These findings indicate that old mouse CD4(+) T cells have a defect in a negative SFK regulatory system.     

Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase

Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways.     

Drosophila ASPP regulates C-terminal Src kinase activity

Src-family kinases (SFKs) control a variety of biological processes, from cell proliferation and differentiation to cytoskeletal rearrangements. Abnormal activation of SFKs has been implicated in a wide variety of cancers and is associated with metastatic behavior (Yeatman, 2004). SFKs are maintained in an inactive state by inhibitory phosphorylation of their C-terminal region by C-terminal Src kinase (Csk). We have identified Drosophila Ankyrin-repeat, SH3-domain, and Proline-rich-region containing Protein (dASPP) as a regulator of Drosophila Csk (dCsk) activity. dASPP is the homolog of the mammalian ASPP proteins, which are known to bind to and stimulate the proapoptotic function of p53. We show that dASPP is a positive regulator of dCsk. First, dASPP loss-of-function strongly enhances the specific phenotypes of dCsk mutants in wing epithelial cells. Second, dASPP interacts physically with dCsk to potentiate the inhibitory phosphorylation of Drosophila Src (dSrc). Our results suggest a role for dASPP in maintaining epithelial integrity through dCsk regulation.     

Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.     

C-terminal Src kinase-homologous kinase (CHK), a unique inhibitor inactivating multiple active conformations of Src family tyrosine kinases

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.