Csk-dependent and -independent control of Src family kinases directs neuronal migration in the developing cerebral cortex

The epithelial Na(+) channel (ENaC) is implicated in the pathogenesis of salt-sensitive hypertension. Recent evidence from animal models suggests that the vasoactive peptide, endothelin (ET-1), may be an important negative regulator of ENaC in vivo. We investigated the signaling pathway involved in endothelin-mediated ENaC inhibition. Experiments were performed in NIH 3T3 cells stably expressing genes for the three (alpha, beta, and gamma) ENaC subunits. In whole cell patch clamp experiments, we found that ET-1 treatment induced a dose-dependent decrease in amiloride-sensitive currents. Using receptor-specific antagonists, we determined that the effects of ET-1 were attributed to activation of the ET(B) receptor. Moreover, the inhibitory effect of ET-1 on ENaC could be completely blocked when cells were pretreated with the selective Src family kinase inhibitor, PP2. Further studies revealed that basal Src family kinase activity strongly regulates ENaC whole cell currents and single channel gating. These results suggest that Src family kinases lie in a signaling pathway activated by ET-1 and are components of a novel negative regulatory cascade resulting in ENaC inhibition.

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca(2+), followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca(2+) into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus.

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.

Cell migration is a complex biological process playing a key role in physiological and pathological conditions. During central nervous system development, positioning and function of cortical neurons is tightly regulated by cell migration. Recently, signaling events involving the urokinase-type plasminogen activator receptor, which is a key regulator for the activation of hepatocyte growth factor (HGF), have been implicated in modulating cortical neuron migration. However, the intracellular pathways controlling neuronal migration triggered by the HGF receptor Met have not been elucidated. By combining pharmacological and genetic approaches, we show here that the Ras/ERK pathway and phosphatidylinositol 3-kinase (PI3K) are both required for cortical neuron migration. By dissecting the downstream signals necessary for this event, we found that Rac1/p38 and Akt are required, whereas the c-Jun N-terminal kinase (JNK) and mTOR/p70(s6k) pathways are dispensable. This study demonstrates that concomitant activation of the Ras/ERK, PI3K/Akt, and Rac1/p38 pathways is required to achieve full capacity of cortical neurons to migrate upon HGF stimulation.

The tyrosine kinases Syk and Lyn are activated in B lymphocytes following antibody induced cross-linking of the B cell receptor for antigen (BCR). It has been suggested that activation of Syk is dependent on Lyn. We tested this hypothesis by comparing the phosphorylation and activation of several downstream effector molecules in parental DT40, DT40Syk- and DT40Lyn- B cells. The phosphorylation and activation of p90Rsk was ablated in Syk-deficient B cells but unaffected in Lyn-deficient B cells while the phosphorylation/activation of Ras GTPase activating protein (Ras GAP) and mitogen activated protein (MAP) kinase required both Syk and Lyn. Thus, these data indicate that Syk can be activated in the absence of Lyn after BCR cross-linking and results in the activation of p90Rsk via a MAP kinase-independent pathway in DT40Lyn- cells. We also demonstrated that BCR mediates the activation of p70S6k. However, activation of p70S6k in DT40Syk- and DT40Lyn- cells was comparable with that observed in parental cells. Thus, either Syk or Lyn may be sufficient for activation of p70S6k, or activation of p70S6k occurs independently of both Syk and Lyn. The kinase activity of Syk was required for the phosphorylation/activation of each of these downstream effector molecules but only the phosphorylation of Ras GAP was affected in cells expressing a mutant of Syk in which tyrosines 525 and 526 were substituted to phenlyalanines.

The spinal cord has the ability to regenerate but the microenvironment generated after trauma reduces that capacity. An increase in Src family kinase (SFK) activity has been implicated in neuropathological conditions associated with central nervous system trauma. Therefore, we hypothesized that a decrease in SFK activation by a long-term treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2), a selective SFK inhibitor, after spinal cord contusion with the New York University (NYU) impactor device would generate a permissive environment that improves axonal sprouting and/or behavioral activity. Results demonstrated that long-term blockade of SFK activation with PP2 increases locomotor activity at 7, 14, 21 and 28 days post-injury in the Basso, Beattie, and Bresnahan open field test, round and square beam crossing tests. In addition, an increase in white matter spared tissue and serotonin fiber density was observed in animals treated with PP2. However, blockade of SFK activity did not change the astrocytic response or infiltration of cells from the immune system at 28 days post-injury. Moreover, a reduced SFK activity with PP2 diminished Ephexin (a guanine nucleotide exchange factor) phosphorylation in the acute phase (4 days post-injury) after trauma. Together, these findings suggest a potential role of SFK in the regulation of spared tissue and/or axonal outgrowth that may result in functional locomotor recovery during the pathophysiology generated after spinal cord injury. Our study also points out that ephexin1 phosphorylation (activation) by SFK action may be involved in the repulsive microenvironment generated after spinal cord injury.

C-terminal Src kinase (Csk) targets Src family kinases (SFKs) and thereby inactivates them. We have previously shown that Csk binds to phosphorylated tyrosine 685 of VE-cadherin, an adhesion molecule of major importance for the regulation of endothelial junctions. This tyrosine residue is an SFK target, and its mutation (VE-cadherin-Y685F) inhibits the induction of vascular permeability in various inflammation models. Nevertheless, surprisingly, it increases leukocyte extravasation. Here, we investigated whether endothelial Csk is involved in these effects. We found that the deficiency of Csk in endothelial cells augments SFK activation and the phosphorylation of VE-cadherin-Y685 but had no net effect on vascular leak formation. In contrast, the lack of endothelial Csk enhanced leukocyte adhesion and transmigration in vitro and in vivo. Furthermore, the silencing of Csk increased tyrosine phosphorylation of the SFK substrate cortactin. Importantly, the effects of Csk silencing on the increase in SFK activation, cortactin phosphorylation, and neutrophil diapedesis were all dependent on Y685 of VE-cadherin. Deletion of cortactin, in turn, erased the supporting effect of Csk silencing on leukocyte transmigration. We have previously shown that leukocyte transmigration is regulated by endothelial cortactin in an ICAM-1-dependent manner. In line with this, blocking of ICAM-1 erased the supporting effect of Csk silencing on leukocyte transmigration. Collectively, our results establish a negative feedback loop that depends on the phosphorylation of VE-cadherin-Y685, which recruits Csk, which in turn dampens the activation of SFK and cortactin and thereby the clustering of ICAM-1 and the extravasation of neutrophils.

Signal transduction by T and B cell Ag receptors and certain receptors for Ig Fc regions (Fc γ RI, hFc γ RIIA, Fc γ RIII, Fc α R, and Fc ε RI) involves a conserved sequence motif, termed an immunoreceptor tyrosine-based activation motif (ITAM) and found in multiple receptor chains. Phosphorylation of the two ITAM tyrosines is a critical event in signal transduction. To address the function of this phosphorylation, we assessed the ability of nonphosphorylated and biphosphorylated ((p)2ITAM) ITAM peptides to bind and modify the activity of src and syk family kinases in vivo and in vitro. All (p)2ITAMs, but not their nonphosphorylated counterparts, induced extensive protein tyrosine phosphorylation in permeabilized cells. However, the patterns of proteins phosphorylated differed among (p)2ITAMs. This phosphorylation was found to reflect activation of the src family kinase Lyn, but not Syk. In vitro studies using purified Lyn showed that src family kinase activation resulted from a direct interaction with (p)2ITAM. Binding studies demonstrated clear differences in binding specificity of (p)2ITAMs. Most strikingly, Ig α (p)2ITAM and TCR-zeta c and CD3 ε (p)2ITAMs exhibit inverse binding preferences for src and syk family kinases. Taken together, these findings demonstrate a novel mechanism by which src family tyrosine kinases are activated, and are consistent with the possibility that different ITAMs may preferentially activate distinct signaling pathways as a consequence of distinct effector Src homology 2 domain (SH2) binding preference.

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.

Interdiction of Src Family Kinase Activity as a Therapeutic Option for Aberrant JAK-STAT Signaling: Potential Targeted Therapy of JAK2 V617F in Myeloproliferative Disorders with Currently Available Kinase Inhibitors.

Cbp, a C-terminal Src kinase (Csk)-binding protein, is a transmembrane phosphoprotein that has been implicated in the regulation of the Src family kinase (SFK) through recruiting Csk, a negative regulator of SFK, to a membrane microdomain of lipid rafts. To examine the contribution of Cbp to cell adhesion signaling mediated by SFK, we investigated the kinase responsible for phosphorylating Cbp and the mode of phosphorylation during the cell adhesion process. The results obtained by using mutant mice or cells that lack Csk and/or a member of SFK, Fyn, reveal that Cbp is phosphorylated predominantly by raft-localized Fyn in vivo. Upon cell adhesion onto fibronectin, Cbp becomes transiently phosphorylated (consistent with SFK activation) and recruits Csk to lipid rafts. These events are completed before the full activation of focal adhesion kinase, indicating that the transient activation and down-regulation of SFK in lipid rafts are earlier events in cell adhesion signaling. In Csk-deficient cells, continuous hyperactivation of SFK leads to continuous hyperphosphorylation of Cbp, accompanied by impaired cell spreading and migration. Silencing of Cbp by RNA interference also induced impaired cell spreading. These findings suggest that Cbp could serve as a sensor of SFK activity in early stages of cell adhesion signaling, and that Csk-mediated down-regulation of SFK is essential to allow dynamic cellular events involved in the regulation of cell spreading and migration.

The effects of membrane compartmentalization of csk on TCR signaling

Csk: C-terminal Src kinase (vertebrates)

Endogenous and synthetic inhibitors of the Src-family protein tyrosine kinases

Voltage-gated sodium (Na+) and potassium (K+)channels have been found to be regulated by Src family kinases(SFKs).However, how these channels are regulated by SFKs in cochlear spiral ganglion neurons (SGNs) remains unknown.Here, we report that altering the activity of endogenous SFKs modulated voltage-gated Na+, but not K+, currents recorded in embryonic SGNs in culture. Voltage-gated Na+ current was suppressed by inhibition of endogenous SFKs or just Src and potentiated by the activation of these enzymes. Detailed investigations showed that under basal conditions, SFK inhibitor application did not significantly affect the voltage-dependent activation, but shifted the steady-state inactivation curves of Na+ currents and delayed the recovery of Na+ currents from inactivation. Application of Src specific inhibitor, Src40–58,not only shifted the inactivation curve but also delayed the recovery of Na+ currents and moved the voltage-dependent activation curve towards the left. The pre-inhibition of SFKs occluded all the effects induced by Src40–58 application, except the left shift of the activation curve. The activation of SFKs did not change either steady-state inactivation or recovery of Na+ currents, but caused the left shift of the activation curve.SFK inhibitor application effectively prevented all the effects induced by SFK activation, suggesting that both the voltage-dependent activation and steady-state inactivation of Na+ current are subjects of SFK regulation. The different effects induced by activation versus inhibition of SFKs implied that under basal conditions, endogenously active and inactive SFKs might be differentially involved in the regulation of voltage-gated Na+ channels in SGNs.