Activation of Src family tyrosine kinases by ferric ions

The Src-family tyrosine kinases (SFKs) are oncogenic enzymes that contribute to the initiation and progression of many types of cancer. In normal cells, SFKs are kept in an inactive state mainly by phosphorylation of a consensus regulatory tyrosine near the C-terminus (Tyr⁵³⁰ in the SFK c-Src). As recent data indicate that tyrosine modification enhances binding of metal ions, the hypothesis that SFKs might be regulated by metal ions was investigated. The c-Src C-terminal peptide bound two Fe^3 + ions with affinities at pH 4.0 of 33 and 252 μM, and phosphorylation increased the affinities at least 10-fold to 1.4 and 23 μM, as measured by absorbance spectroscopy. The corresponding phosphorylated peptide from the SFK Lyn bound two Fe^3 + ions with much higher affinities (1.2 pM and 160 nM) than the Src C-terminal peptide. Furthermore, when Lyn or Hck kinases, which had been stabilised in the inactive state by phosphorylation of the C-terminal regulatory tyrosine, were incubated with Fe^3 + ions, a significant enhancement of kinase activity was observed. In contrast Lyn or Hck kinases in the unphosphorylated active state were significantly inhibited by Fe^3 + ions. These results suggest that Fe^3 + ions can regulate SFK activity by binding to the phosphorylated C-terminal regulatory tyrosine.     

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.     

Reconstitution of Btk signaling by the atypical tec family tyrosine kinases Bmx and Txk

Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis.     

The Src family of tyrosine protein kinases in hemopoietic signal transduction.

The Src family of tyrosine protein kinases represent an expanding class of closely related intracellular enzymes that participate in the signal transduction pathways of a variety of surface receptors. One of the more surprising aspects of the information relating Src protein kinases to receptor signaling is the apparent diversity of receptor types with which the Src-related enzymes are reported to interact physically or functionally. Traditional biochemical and genetic approaches have yielded much information regarding the interactions between the Src tyrosine protein kinases and other cellular proteins in defined cell types, and emerging technologies, most notably homologous recombination in embryonal stem cells to achieve gene "knockouts," are providing new insights into the participation of the Src-related gene products in signal transduction and development.     

Structure-function relationships in Src family and related protein tyrosine kinases

There is increasing evidence to suggest that cytoplasmic tyrosine kinases of the Src family have a pivotal role in the regulation of a number of cellular processes. Members of this family have been implicated in cellular responses to a variety of extracellular signals, such as those arising from growth factors and cell-cell interactions, as well as in differentiative and developmental processes in both vertebrates and invertebrates. A better understanding of the regulation and of the structure-function relationships of these enzymes might aid in the development of specific ways to interfere with their action, as well as serving as a paradigm for regulation of other protein tyrosine kinases that have SH2 and SH3 domains. In this review we will first discuss the regulation of Src family protein tyrosine kinases, with particular emphasis on their SH2 and SH3 domains. We will then briefly review other non-receptor protein tyrosine kinases that have SH2 and SH3 domains.     

Soluble E-selectin induces monocyte chemotaxis through Src family tyrosine kinases

Cellular adhesion molecules such as E-selectin function to recruit leukocytes into the inflammatory lesions of diseases such as rheumatoid arthritis (RA) and atherosclerosis. Monocytes are the key components of the cellular infiltrates present in these disorders. We hypothesized that soluble E-selectin (sE-selectin) might mediate the chemotaxis of monocytes. In this report, we show that sE-selectin induced normal human peripheral blood monocyte migration in the nanomolar range in a concentration-dependent manner. Neutralization studies using RA human joint synovial fluids and anti-E-selectin antibody showed a mean 31% reduction in RA synovial fluid-mediated monocyte chemotaxis (p < 0.05), indicating that sE-selectin is a major monocyte recruiter in RA. Next, we investigated the role of tyrosine phosphorylation pathways in sE-selectin-induced monocyte chemotaxis. Human peripheral blood monocytes stimulated with sE-selectin showed a time-dependent increase in the tyrosine phosphorylation of a broad range of cellular proteins, predominantly in the molecular size range of Src family kinases (50-60 kDa) and mitogen-activated protein kinases (MAPKs). Western blot analysis of Src family kinases showed a time-dependent increase in Src, Hck, and Lyn phosphorylation. The pretreatment of monocytes with the Src inhibitor AG1879: 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) prior to stimulation with sE-selectin markedly inhibited Hck and Lyn phosphorylation, whereas the phosphorylation of Src was partially inhibited. In addition, the sE-selectin stimulation of monocytes resulted in the increased phosphorylation of extracellular signal-related kinase (ERK1/2) and p38 MAPK. The pretreatment of monocytes with PP2 showed 89 and 83% inhibition of ERK1/2 and p38 MAPK phosphorylation, respectively. sE-selectin also showed a time-dependent activation of Ras kinase. Furthermore, the pretreatment of monocytes with PP2 completely inhibited sE-selectin-mediated monocyte chemotaxis. Taken together, our data demonstrate a novel function for sE-selectin as a monocyte chemotactic agent and suggest that sE-selectin might be mediating its biological functions through the Src-MAPK pathway.     

Increased tyrosine phosphorylation of PSD-95 by Src family kinases after brain ischaemia

PSD (postsynaptic density)-95, a scaffold protein that tethers NMDA (N-methyl-D-aspartate) receptors to signal molecules, is implicated in pathological events resulting from excitotoxicity. The present study demonstrates that brain ischaemia and reperfusion increase the tyrosine phosphorylation of PSD-95 in the rat hippocampus. PP2, a specific inhibitor of SrcPTKs (Src family protein tyrosine kinases), prevents the ischaemia-induced increases not only in the tyrosine phosphorylation of PSD-95, but also in the interaction between PSD-95 and Src kinases. PSD-95 is phosphorylated either by purified Src/Fyn kinases in vitro or by co-expression of constitutively active Src/Fyn in COS7 cells. The results suggest that SrcPTKs are involved in PSD-95 phosphorylation. The single Tyr(523) mutation to phenylalanine (Y523F) reduces the Src/Fyn-mediated phosphorylation of PSD-95 in COS7 cells and in vitro. As shown with a rabbit polyclonal antibody against phospho-PSD-95 (Tyr(523)), Tyr(523) phosphorylation is responsible for the increased tyrosine phosphorylation of PSD-95 induced by ischaemia in the rat hippocampus. In cultured hippocampal neurons, overexpression of PSD-95 Y523F, but not PSD-95 Y533F, abolishes the facilitating effect of PSD-95 on the glutamate- or NMDA-mediated currents, implying that PSD-95 Tyr(523) phosphorylation contributes to the post-ischaemic over-activation of NMDA receptors. Thus the present study reveals an additional mechanism for the regulation of PSD-95 by tyrosine phosphorylation. This mechanism may be of pathological significance since it is associated with excitotoxicity in the ischaemic brain.     

Etk, a Btk family tyrosine kinase, mediates cellular transformation by linking Src to STAT3 activation

Etk (also called Bmx) is a member of the Btk tyrosine kinase family and is expressed in a variety of hematopoietic, epithelial, and endothelial cells. We have explored biological functions, regulators, and effectors of Etk. Coexpression of v-Src and Etk led to a transphosphorylation on tyrosine 566 of Etk and subsequent autophosphorylation. These events correlated with a substantial increase in the kinase activity of Etk. STAT3, which was previously shown to be activated by Etk, associated with Etk in vivo. To investigate whether Etk could mediate v-Src-induced activation of STAT3 and cell transformation, we overexpressed a dominant-negative mutant of Etk in an immortalized, untransformed rat liver epithelial cell line, WB, which contains endogenous Etk. Dominant-negative inactivation of Etk not only blocked v-Src-induced tyrosine phosphorylation and activation of STAT3 but also caused a great reduction in the transforming activity of v-Src. In NIH3T3 cells, although Etk did not itself induce transformation, it effectively enhanced the transforming ability of a partially active c-Src mutant (c-Src378G). Furthermore, Etk activated STAT3-mediated gene expression in synergy with this Src mutant. Our findings thus indicate that Etk is a critical mediator of Src-induced cell transformation and STAT3 activation. The role of STAT3 in Etk-mediated transformation was also examined. Expression of Etk in a human hepatoma cell line Hep3B resulted in a significant increase in its transforming ability, and this effect was abrogated by dominant-negative inhibition of STAT3. These data strongly suggest that Etk links Src to STAT3 activation. Furthermore, Src-Etk-STAT3 is an important pathway in cellular transformation.     

Phosphorylation of cyclin dependent kinase 4 on tyrosine 17 is mediated by Src family kinases

Cyclin dependent kinase 4 is a key regulator of the cell cycle and its activity is frequently deregulated in cancer. The activity of cyclin dependent kinase 4 is controlled by multiple mechanisms, including phosphorylation of tyrosine 17. This site is equivalent to tyrosine 15 of cyclin dependent kinase 1, which undergoes inhibitory phosphorylation by WEE1 and MYT1; however, the kinases that phosphorylate cyclin dependent kinase 4 on tyrosine 17 are still unknown. In the present study, we generated a phosphospecific antibody to the tyrosine 17-phosphorylated form of cyclin dependent kinase 4, and showed that this site is phosphorylated to a low level in asynchronously proliferating HCT116 cells. We purified tyrosine 17 kinases from HeLa cells and found that the Src family non-receptor tyrosine kinase C-YES contributes a large fraction of the tyrosine 17 kinase activity in HeLa lysates. C-YES also phosphorylated cyclin dependent kinase 4 when transfected into HCT116 cells, and treatment of cells with Src family kinase inhibitors blocked the tyrosine 17 phosphorylation of cyclin dependent kinase 4. Taken together, the results obtained in the present study provide the first evidence that Src family kinases, but not WEE1 or MYT1, phosphorylate cyclin dependent kinase 4 on tyrosine 17, and help to resolve how the phosphorylation of this site is regulated.     

DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases.

The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.     

Src family tyrosine kinases differentially modulate exocytosis from rat brain nerve terminals

We have studied the role of src family tyrosine kinases in regulating synaptic transmitter release from rat brain synaptosomes by using two assays that measure different aspects of synaptic vesicle exocytosis: glutamate release (that directly measures exocytosis of vesicle contents) and release of FM 2-10 styryl dye (that is proportional to the time the synaptic vesicle is fused to the plasma membrane). Depolarisation was induced by KCl (30 mM) or 4-aminopyridine (4AP: 0.3mM) to induce release by full fusion (FF) exocytosis, or by 1 mM 4AP to induce release by both FF and kiss-and-run (KR)-like exocytosis. The src family selective inhibitor, PP1 (10 microM), increased KCl and 0.3 mM 4AP-evoked Ca2+ -dependent release of glutamate, but had little effect upon exocytosis evoked by 1mM 4AP. PP1 did not affect the release of FM 2-10 under any of the depolarisation conditions used. PP1 also had no effect on overall intracellular calcium levels, as measured by FURA2, suggesting that the effects of the inhibitor are downstream of calcium entry. At the same concentration the inactive analogue of this compound, PP3, had no effect on any measure. Immunoblotting with an antibody to phosphotyrosine revealed that phosphorylation of many synaptosomal proteins was reduced by PP1. The immunoreactivity of three protein bands increased upon depolarisation and this increase was blocked by PP1. Phosphorylation of src at tyrosine-416 was reduced by PP1 but changes in its phosphorylation did not correlate with the effects of PP1 on release. These results suggest one or more members of the src family of tyrosine kinases is a negative regulator of the KR mode of exocytosis in synaptosomes, perhaps by tonically inhibiting KR under normal stimulation conditions.     

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 protein tyrosine kinases modulate L-type calcium current in human atrial myocytes

In the heart, L-type voltage dependent calcium channels (L-VDCC) provide Ca²⁺ for the activation of contractile apparatus. The best described pathway for L-type Ca²⁺ current (I_Ca,L) modulation is the phosphorylation of calcium channels by cAMP-dependent protein kinase A (PKA), the activity of which is predominantly regulated in opposite manner by β-adrenergic (β-ARs) and muscarinic receptors. The role of other kinases is controversial and often depends on tissues and species used in the studies. In different studies the inhibitors of tyrosine kinases have been shown either to stimulate or inhibit, or even have a biphasic effect on I_Ca,L. Moreover, there is no clear picture about the route of activation and the site of action of cardiac Src family nonreceptor tyrosine kinases (Src-nPTKs). In the present study we used PP1, a selective inhibitor of Src-nPTKs, alone and together with different activators of I_Ca,L, and demonstrated that in human atrial myocytes (HAMs): (i) Src-nPTKs are activated concomitantly with activation of cAMP-signaling cascade; (ii) Src-nPTKs attenuate PKA-dependent stimulation of I_Ca,L by inhibiting PKA activity; (iii) Gα_s are not involved in the direct activation of Src-nPTKs. In this way, Src-nPTKs may provide a protecting mechanism against myocardial overload under conditions of increased sympathetic activity.     

Regulation of phospholipase D by tyrosine kinases

Activation of phospholipase D (PLD) represents part of an important signalling pathway in mammalian cells, Phospholipase D catalyzed hydrolysis of phospholipids generates phosphatidic acid (PA) which is subsequently metabolized to lyso-PA (LPA) or diacylglycerol (DAG). While DAG is an endogenous activator of protein kinase C (PKC), PA and LPA have been recognized as second messengers as well, Activation of PLD in response to an external stimulus may involve PKC, Ca²⁺, G-proteins and/or tyrosine kinases. In this review, we will address the role of protein tyrosine phosphorylation in growth factor-, agonist- and oxidant-mediated activation of PLD. Furthermore, a possible link between PKC, Ca²⁺, G-proteins and tyrosine kinases is discussed to indicate the complexity involved in the regulation of PLD in mammalian cells.     

Mechanism of Csk-mediated down-regulation of Src family tyrosine kinases in epidermal growth factor signaling

The Src family tyrosine kinases (SFKs) play pivotal roles as molecular switches that link a variety of extracellular cues to intracellular signaling pathway. The function of SFK is regulated by phosphorylation at the C-terminal regulatory site mediated by Csk. Recently a novel SFK target Cbp (or PAG) was identified as a membrane-anchored scaffold protein for Csk. To establish the mechanism of Csk/Cbp-mediated regulation of SFK in vivo, we observed dynamic changes in the interaction of Csk with Cbp by utilizing fusion proteins with modified green fluorescent proteins: cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). Upon SFK activation induced by epidermal growth factor stimulation, fluorescent resonance energy transfer (FRET) response was detected transiently at membrane ruffles in COS1 cells co-expressing CFP-Csk and Cbp-YFP and in cells expressing a single-molecule FRET indicator consisting of CskSH2 and Cbp. Suppression of SFK by PP2 or use of a mutant Cbp that lacks the Csk binding site abolished the FRET response, although a dominant-negative form of Csk enhanced and sustained the FRET response, demonstrating that the FRET response is dependent upon the SFK activity. These observations show that Csk/Cbp-mediated down-regulation of SFK takes place at membrane ruffles in an early stage of epidermal growth factor signaling and suggest that the Csk/Cbp-based FRET indicators are useful for monitoring the status of SFK in living cells.