Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase

Summary The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angle restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time ¹H–¹³C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally ¹³C-labelled sample was exploited for the stereo-specific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C, C, O) among the 25 conformers is 0.42 Å for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel -strands arranged to form two perpendicular -sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core -strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764–769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short -strands in the regions Ser¹⁵ to Glu¹⁷ and Gly²⁵ to Glu²⁷ flanking the so-called RT-Src loop. This loop (residues Glu¹⁷ to Leu²⁴), together with the n-Src loop (residues Gln³⁷ to Ser⁴⁶) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His¹⁴, Tyr¹⁶, Trp⁴¹, Phe⁵⁴ and Phe⁵⁹). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear ¹⁵N,¹H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.Abbreviations CT constant time - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - SH2 Src homology domain 2 - SH3 Src homology domain 3     

Fibroblast Growth Factors Stimulate Protein Tyrosine Phosphorylation and Mitogen-Activated Protein Kinase Activity in Primary Cultures of Hippocampal Neurons

Abstract: Fibroblast growth factors (FGFs) are not only mitogens, but they also promote the differentiation of various cell types. For instance, basic FGF (bFGF) provides a critical trophic support for hippocampal neurons in culture. To elicit their biological effects, FGFs interact with high-affinity receptors that are transmembrane proteins with a cytoplasmic portion containing a tyrosine kinase activity. The tyrosine phosphorylation pattern was examined in primary cultures of hippocampal neurons derived from rat embryos. In these cultures grown for 3 days in the absence of serum, the addition of bFGF causes a rapid increase of tyrosine phosphorylation for various proteins with an optimal level after 5 min of bFGF exposure. Concomitantly, bFGF activates mitogen-activated protein kinase (MAP kinase) activity measured with a selective MAP kinase peptide. The activity increased rapidly after the addition of bFGF and remained elevated even when cultures were treated for 1 h with bFGF. Both acidic and basic FGF were able to enhance protein tyrosine phosphorylation and MAP kinase activity, whereas nerve growth factor and epidermal growth factor did not elicit any of these responses. These data indicate that some of the transduction signals (i.e., tyrosine phosphorylation and activation of MAP kinase) that have been described for the proliferative effect of FGFs are also involved when FGFs act as trophic factors for postmitotic neurons in culture.     

Characterization of TrkB Receptor-Mediated Signaling Pathways in Rat Cerebellar Granule Neurons: Involvement of Protein Kinase C in Neuronal Survival

Abstract: TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Here, we report that both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-γ (PLC-γ) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-γ, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival-promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c- ras in a concentration-dependent manner. These results suggest that two different pathways, the c- ras and the PLC-γ pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.     

Lysophosphatidylcholine Potentiates BDNF-Induced TrkB Phosphorylation and Downstream Signals in Cerebellar Granule Neurons

We found that brain-derived neurotrophic factor (BDNF)-induced phosphorylation of mitogen-activated protein kinase (MAPK) and Akt in cerebellar granule neurons was specifically potentiated by LPC. LPC also augmented the BDNF-induced phosphorylation of TrkB, the receptor for BDNF. In TrkB-transfected CHO-K1 cells, LPC potentiated BDNF-induced MAPK phosphorylation. These results suggest that LPC plays a role in BDNF-TrkB signaling by regulating the activation of TrkB.     

Expression of the p56lck Y505F Mutation in CD45-Deficient Mice Rescues Thymocyte Development

Mice deficient in the transmembrane protein tyrosine phosphatase CD45 exhibit a block in thymocyte development. To determine whether the block in thymocyte development was due to the inability to dephosphorylate the inhibitory phosphorylation site (Y505) in p56(lck) (Lck), we generated CD45-deficient mice that express transgenes for the Lck Y505F mutation and the DO11.10 T-cell antigen receptor (TCR). CD4 single-positive T cells developed and accumulated in the periphery. Treatment with antigen resulted in thymocyte apoptosis and the loss of transgenic-TCR-bearing cells. Peripheral CD45-deficient T cells from the mice expressing both transgenes responded to antigen by increasing CD69 expression, interleukin-2 production, and proliferation. These results indicate that thymocyte development requires the dephosphorylation of the inhibitory site in Lck by CD45.     

Two-Photon Analysis of Lead Accumulation in Rat Cerebellar Granule Neurons

Lead (Pb²⁺) is a common pollutant and potent central neurotoxin. We have studied its pathways of permeation by two-photon fluorescence microscopy in rat cerebellar granule neurons loaded with the fluorescent dye indo-1. Pb²⁺ binds indo-1 with high affinity acting as a quencher. Its permeation through the neuronal membrane was indicated by a decrease of the fluorescence emission, which occurred even in resting condition. In the presence of 20 μM Pb²⁺, uptake reached a plateau level (≈45% of initial fluorescence) in 4 min and was partially antagonized by 25 μM lanthanum. Subsequent addition of a membrane permeant ionophore caused a further (>70%) quenching of the dye, suggesting that previous saturation was due to inactivation of the transport system. Intracellular Pb²⁺ concentrations were evaluated from the fluorescence intensity and this estimate indicated that the concentration of free Pb²⁺ sufficient to inactivate the transport system is close to 50 pM.     

Immunolesioning of Glutamate Receptor GluR1-Containing Neurons in the Rat Neostriatum Using a Novel Immunotoxin

1. To investigate the potency of a novel immunotoxin that is specific for glutamate receptor GluR1, a subunit of the -amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type receptor channel, immunolesioning was performed.2. A ribosome-inactivating protein, trichosanthin (TCS), was isolated and conjugated to the goat anti-rabbit IgG antibody molecule. The anti-rabbit antibody–TCS complex was preincubated with GluR1-specific rabbit antibody to produce a GluR1-specific immunotoxin. The immunotoxin was unilaterally administered into either the neostriatum or the lateral ventricle of rats.3. Immunoreactivity for GluR1 or GluR4 was revealed in perfuse-fixed sections of the neostriatum obtained from the lesioned and control animals by immunocytochemistry. After ventricular or striatal injections of the immunotoxin, depletions of GluR1-immunoreactive neurons, the presumed GABAergic interneurons in the neostriatum, were found. Depletions of GluR4-immunoreactive perikarya, the presumed same subpopulation of striatal interneurons, were also found. In addition, no change in the pattern of distribution of immunoreactivity for GluR2 or glial fibrillary acidic protein was found in the lesioned neostriatum. These results indicate that the novel GluR1 immunotoxin is potent and specific.4. In addition, striatal application of the immunotoxin caused a greater depletion in the number of GluR1-immunoreactive neurons. The present results also indicate that the route of immunotoxin application may be important in producing specific lesions.     

New horizons in drug discovery of lymphocyte-specific protein tyrosine kinase (Lck) inhibitors: a decade review (2011–2021) focussing on structure–activity relationship (SAR) and docking insights

Lymphocyte-specific protein tyrosine kinase (Lck), a non-receptor Src family kinase, has a vital role in various cellular processes such as cell cycle control, cell adhesion, motility, proliferation, and differentiation. Lck is reported as a key factor regulating the functions of T-cell including the initiation of TCR signalling, T-cell development, in addition to T-cell homeostasis. Alteration in expression and activity of Lck results in numerous disorders such as cancer, asthma, diabetes, rheumatoid arthritis, atherosclerosis, and neuronal diseases. Accordingly, Lck has emerged as a novel target against different diseases. Herein, we amass the research efforts in literature and pharmaceutical patents during the last decade to develop new Lck inhibitors. Additionally, structure-activity relationship studies (SAR) and docking models of these new inhibitors within the active site of Lck were demonstrated offering deep insights into their different binding modes in a step towards the identification of more potent, selective, and safe Lck inhibitors.     

Neuroglobin Involvement in the Course of Arsenic Toxicity in Rat Cerebellar Granule Neurons

Exposure to arsenic in drinking water results in a widespread environmental problem in the world, and the brain is a major target. Neuroglobin is a vertebrate heme protein regarded as playing neuroprotective role in hypoxia or oxidative stress. In this study, we investigated the toxic effects of sodium arsenite (NaAsO₂) on primary cultured rat cerebellar granule neurons (CGNs) and detected neuroglobin (Ngb) expression in rat CGNs exposed to NaAsO₂. Our results show that apoptosis was obviously induced by NaAsO₂ treatment in rat CGNs by annexin V-fluorescein isothiocyanate assay. Intracellular reactive oxygen species generation increased significantly in the cells exposed to NaAsO₂, and the apoptotic effects could be partially reversed by antioxidant N-acetyl-l-cysteine. Ngb protein and mRNA expression were significantly downregulated in rat CGNs shortly after NaAsO₂ exposure and then upregulated after a longer time of exposure. Furthermore, mRNA expression changed more than protein expression and the toxic effect of NaAsO₂ on Ngb expression is dose dependent. Higher Ngb expression was also detected in rat cerebellum, but not in other parts (cerebrum, hippocampus, and midbrain) of the brain exposed to NaAsO₂ for 16 weeks. Taken together, cytotoxic effects of NaAsO₂ on rat CGNs is induced at least partly by oxidative stress and Ngb may influence the course of arsenic toxicity in rat CGNs and rat cerebellum.     

Tyrosine Phosphorylation in a Model of Ischemia Using the Rat Hippocampal Slice: Specific, Long-Term Decrease in the Tyrosine Phosphorylation of the Postsynaptic Glycoprotein PSD-GP180

Abstract: The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M_r 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M_r 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro “ischemia” was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N₂ instead of O₂ and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca²⁺ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca²⁺ was omitted from the incubation media. Reoxygenation of ischemic slices also resulted in the Ca²⁺-dependent, transient tyrosine phosphorylation of p42. The major PSD-associated, tyrosine-phosphorylated glycoprotein of molecular mass 180 kDa has recently been identified as the NR2B subunit of the NMDA receptor. The results suggest that changes in tyrosine phosphorylation after an ischemic insult may modulate the NMDA receptor or signal transduction pathways in the postsynaptic cell and are consistent with a role for tyrosine phosphorylation in the sequence of events leading to neuronal cell damage and death.     

Activation and Tyrosine Phosphorylation of 44-kDa Mitogen-Activated Protein Kinase (MAPK) Induced by Electroconvulsive Shock in Rat Hippocampus

Abstract: Electroconvulsive shock (ECS) has been reported to induce the phosphorylation and activation of 42-kDa, but not 44-kDa, mitogen-activated protein kinase (MAPK) in rat hippocampus. We studied the activation and tyrosine phosphorylation of MAPKs in rat brain after ECS. We observed the increase of the activities of both 42- and 44-kDa MAPKs in rat hippocampus after ECS. The activities reached peak at 2 min and returned to basal levels by 15 min after ECS. We also observed the increased phsophorylation on the tyrosine residue of 42-kDa MAPK in rat hippocampus after ECS, but not on that of 44-kDa MAPK. However, when we examined the immunoprecipitated 44-kDa MAPK, we could demonstrate that the tyrosine phosphorylation of 44-kDa MAPK at 2 min after ECS was markedly increased, in accordance with the increase of kinase activity. These results indicate that ECS induces the transient activation and tyrosine phosphorylation of 44-kDa MAPK, as well as 42-kDa MAPK, in rat hippocampus, although the amount of tyrosine phosphorylation is far less and the kinase activity is lower in 44-kDa MAPK than in 42-kDa MAPK.     

Long-Term Induction of Tyrosine Hydroxylase Expression: Compensatory Response to Partial Degeneration of the Dopaminergic Nigrostriatal System in the Rat Brain

Abstract: The present study was undertaken to examine the adaptive changes occurring 1 and 6 months after moderate or severe unilateral 6-hydroxydopamine-induced lesions confined to the lateral part of the rat substantia nigra pars compacta (SNC). The expression of tyrosine hydroxylase (TH) enzyme was analyzed in the remaining dopaminergic nigral cell bodies and in the corresponding striatal nerve endings. In the cell bodies of the lesioned SNC, TH mRNA content was increased (+20 to +30%) 6 months after the lesion without changes in cellular TH protein amounts. The depletion of TH protein in the nerve terminal area was less severe than the percentage of cell loss observed in the SNC at 1- and 6-month postlesion intervals. Moreover, the decrease in TH protein in the ipsilateral striatum was less pronounced 6 months after lesion than 1 month after. That no corresponding change in TH protein content was observed in the cell bodies at a time when TH increased in nerve terminals suggests that the newly synthesized protein is probably rapidly transported to the striatal fibers. These results suggest the existence of a sequence of changes in TH expression between cell bodies and fibers, occurring spontaneously after partial denervation of the nigrostriatal pathway.     

Activation of Mitogen-Activated Protein Kinase in Cultured Rat Hippocampal Neurons by Stimulation of Glutamate Receptors

Abstract: Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca²⁺ ionophore, remained intact. Lavendustin A, a tyrosine kinase inhibitor, was without effect. In experiments with ³²P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c- raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca²⁺-dependent pathways and that the activation of Raf-1 is not involved.     

Changes in the Tyrosine Phosphorylation of Mitogen-Activated Protein Kinase in the Rat Hippocampus During and Following Severe Hypoglycemia

Abstract: The changes in the levels of tyrosine-phosphorylated proteins in the cytosolic fraction of the rat hippocampus subjected to severe hypoglycemia were analyzed. A marked increase in tyrosine phosphorylation of a 43-kDa protein was observed at 30 min of isoelectric EEG and 30 min and 1 h of recovery. Immunostaining of the same blot with antibody against mitogen-activated protein (MAP) kinase demonstrated a double band of ∼42 and 43 kDa. The increased tyrosine phosphorylation of MAP kinase during hypoglycemic coma and the early recovery period suggests that MAP kinase may be involved in neuronal degeneration and repair.     

Expression and Electrophysiological Function of Actin in Chick Cerebellar Neurons

Among several monoclonal antibodies obtained by immunizing Balb/c mice with cerebellar synaptic membrane fractions from E20 chick embryos, the antibody, named M35, suppressed Ca-spikes in immature cultured chick cerebellar neurons. M35 immunoprecipitated 43kDa protein from a ¹²⁵I-labeled embryonic crude cerebellar membrane fraction. Immunohistochemically, the M35 antigen was expressed most intensively in Purkinje cells, but its expression was limited to highly motile structures at developmental neuronal remodeling. Electrophysiologically, M35 facilitated current responses to AMPA and inhibited the responses to GABA in cultured cerebellar Purkinje neurons. The several peptides derived from the affinity-purified 43kDa protein were found to have homologous amino acid sequences to non-muscle actins. These results suggest that the antigen recognized by M35 may play an essential role probably as membrane ion channels modulating synaptic functions in not only the development and growth but also the neuronal activity of chick cerebellar Purkinje cells.     

p56lck SH2 domain binding motifs from bead binding screening of peptide libraries containing phosphotyrosine surrogates

Phosphorylation reactions are key mediators in a variety of biochemical signal processes. Research into the selective inhibition of protein tyrosine kinases to generate anticancer agents has made O-phosphotyrosyl analogues important pharmacological tools. The simple procedures reported here involving the formation of iterative peptide libraries together with the development of a selective and sensitive bead-binding assay have made it possible to rapidly screen peptides incorporating O-phosphotyrosyl surrogates (including O-phospho-2,3,5,6-tetrafluorotyrosine, 4-(phosphono)hydroxymethyl-phenylalanine and 4-(phosphono)fluoromethyl-phenylalanine) for their potential to inhibit the protein tyrosine kinase p56^lck. These procedures can be easily adapted to combinatorial peptide libraries.     

p56lck SH2 domain binding motifs from bead binding screening of peptide libraries containing phosphotyrosine surrogates

Summary Phosphorylation reactions are key meditors in a variety of biochemical signal processes. Research into the selective inhibition of protein tyrosine kinases to generate anticancer agents has madeO-phosphotyrosyl analogues important pharmacological tools. The simple procedures reported here involving the formation of interative peptide libraries together with the development of a selective and sensitive bead-binding assay have made it possible to rapidly screen peptides incorporatingO-phosphotyrosyl surrogates (includingO-phospho-2,3,5,6-tetrafluorotyrosine, 4-(phosphono)hydroxymethyl-phenylalanine and 4-(phosphono)fluoromethyl-phenylalanine) for their potential to inhibit the protein tyrosine kinase p56^lck. These procedures can be easily adapted to combinatorical peptide libraries.