Vmax. activation of pp60c-src tyrosine kinase from neuroblastoma neuro-2A.
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ABSTRACT:
SUBMITTER: Matten WT
PROVIDER: S-EPMC1148604 | biostudies-other | 1987 Dec
REPOSITORIES: biostudies-other
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Project description:c-src is a nonreceptor tyrosine protein kinase that is highly concentrated in synaptic regions, including synaptic vesicles and growth cones. Here, we report that the mRNA signal of pp60c-src is widely distributed in the rat brain with particularly high concentrations in the hippocampus. After spatial maze learning, up-regulation of c-src mRNA was observed in the CA3 region of the hippocampus, which was accompanied by increases in pp60c-src protein in hippocampal synaptosomal preparations. Training also triggered an increase in c-src protein tyrosine kinase activity that was correlated with its tyrosine dephosphorylation in the synaptic membrane fraction. After training, pp60c-src from hippocampus showed enhanced interactions with synaptic proteins such as synapsin I, synaptophysin, and the type 2 N-methyl-d-aspartate receptor, as well as the cytoskeletal protein actin. The association of pp60c-src with insulin receptor in the synaptic membrane fraction, however, was temporally decreased after training. Furthermore, in vitro results showed that Ca(2+) and protein kinase C might be involved in the regulation of protein-protein interactions of pp60c-src. These results suggest, therefore, that pp60c-src participates in the regulation of hippocampal synaptic activity during learning and memory.
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Project description:Protein kinases are enzymes that catalyze the covalent transfer of the γ-phosphate of an adenosine triphosphate (ATP) molecule onto a tyrosine, serine, threonine, or histidine residue in the substrate and thus send a chemical signal to networks of downstream proteins. They are important cellular signaling enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Unregulated protein kinase activity is often associated with a wide range of diseases, therefore making protein kinases major therapeutic targets. A prototypical system of central interest to understand the regulation of kinase activity is provided by tyrosine kinase c-Src, which belongs to the family of Src-related non-receptor tyrosine kinases (SFKs). Although the broad picture of autoinhibition via the regulatory domains and via the phosphorylation of the C-terminal tail is well characterized from a structural point of view, a detailed mechanistic understanding at the atomic-level is lacking. Advanced computational methods based on all-atom molecular dynamics (MD) simulations are employed to advance our understanding of tyrosine kinase activation. The computational studies suggest that the isolated kinase domain (KD) is energetically most favorable in the inactive conformation when the activation loop (A-loop) of the KD is not phosphorylated. The KD makes transient visits to a catalytically competent active-like conformation. The process of bimolecular trans-autophosphorylation of the A-loop eventually locks the KD in the active state. Activating point mutations may act by slightly increasing the population of the active-like conformation, enhancing the availability of the A-loop to be phosphorylated. The Src-homology 2 (SH2) and Src-homology 3 (SH3) regulatory domains, depending upon their configuration, either promote the inactive or the active state of the kinase domain. In addition to the roles played by the SH3, SH2, and KD, the Src-homology 4-Unique domain (SH4-U) region also serves as a key moderator of substrate specificity and kinase function. Thus, a fundamental understanding of the conformational propensity of the SH4-U region and how this affects the association to the membrane surface are likely to lead to the discovery of new intermediate states and alternate strategies for inhibition of kinase activity for drug discovery. The existence of a multitude of KD conformations poses a great challenge aimed at the design of specific inhibitors. One promising computational strategy to explore the conformational flexibility of the KD is to construct Markov state models from aggregated MD data.
| S-EPMC6602532 | biostudies-literature
Project description:To identify high-confidence NMD targets in mouse N2A neuroblastoma cells, we used our established transcriptome-wide RNA sequencing (RNA-seq) methodologies. Through parallel analyses of RNA-seq upon UPF1-knockdown (KD) and RNA immunoprecipitation (RIP-seq) footprinting of p-UPF1-bound RNAs, we identified 1027 high-confidence neuronal NMD targets.
2021-10-25 | GSE180136 | GEO
Project description:Elevated level of palmitic acid (PA), a long-chain saturated fatty acid (SFA), is lipotoxic to many different types of cells including Neuro-2a (N2a) neuroblastoma cells. CD36 is a multifunctional membrane glycoprotein that acts as a fatty acid translocase (FAT) facilitating the transport of long-chain free fatty acids (FFAs) into cells, serves a fatty acid (FA) sensing function in areas including taste buds and the proximal gut, and acts as a scavenger receptor that binds to many ligands, including FAs, collagen, oxidized low-density lipoproteins, and anionic phospholipids. However, the involvement of CD36 in FA uptake and PA lipotoxicity in N2a cells remains unclear. In this study, we examined FA uptake in BSA- and PA-treated N2a cells and investigated the involvement of CD36 in FA uptake and PA lipotoxicity in N2a cells. Our data showed that PA treatment promoted FA uptake in N2a cells, and that treatment with sulfo-N-succinimidyl oleate (SSO), a CD36 inhibitor, significantly decreased FA uptake in BSA- and PA-treated N2a cells, and ameliorated PA-induced decrease of cell viability, decrease of diploid cells, and increase of tetraploid cells. We also found that CD36 knockdown significantly decreased FA uptake in both BSA- and PA-treated cells as compared to their corresponding wild-type controls, and dramatically attenuated PA-induced cell cycle defects in N2a cells. Our data suggest that CD36 may play a critical role in FA uptake and PA lipotoxicity in N2a cells. CD36 may therefore represent a regulatory target against pathologies caused by excess FAs.
| S-EPMC8615720 | biostudies-literature
Project description:Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.
| S-EPMC1134817 | biostudies-other
Project description:Protein phosphatase 2A (PP2A) is a major phospho-Ser/Thr phosphatase and a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer's disease (AD), PP2A regulation remains relatively poorly understood. It has been reported that the PP2A catalytic subunit (PP2Ac) is inactivated by a single phosphorylation at the Tyr307 residue by tyrosine kinases such as v-Src. However, multiple mass spectrometry studies have revealed the existence of other putative PP2Ac phosphorylation sites in response to activation of Src and Fyn, two major Src family kinases (SFKs). Here, using PP2Ac phosphomutants and novel phosphosite-specific PP2Ac antibodies, we show that cellular pools of PP2Ac are instead phosphorylated on both Tyr127 and Tyr284 upon Src activation, and on Tyr284 following Fyn activation. We found these phosphorylation events enhanced the interaction of PP2Ac with SFKs. In addition, we reveal SFK-mediated phosphorylation of PP2Ac at Y284 promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity; the phospho-deficient Y127/284F and Y284F PP2Ac mutants prevented SFK-mediated phosphorylation of Tau at the CP13 (pSer202) epitope, a pathological hallmark of AD, and SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings demonstrate a novel PP2A regulatory mechanism that challenges the existing dogma on the inhibition of PP2A catalytic activity by Tyr307 phosphorylation. We propose dysregulation of SFK signaling in cancer and AD can lead to alterations in PP2A phosphorylation and subsequent deregulation of key PP2A substrates, including ERK and Tau.
| S-EPMC9396060 | biostudies-literature
Project description:Tyrosine kinases are important cellular signaling allosteric enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Their activity must be tightly controlled, and malfunction can lead to a variety of diseases, particularly cancer. The nonreceptor tyrosine kinase c-Src, a prototypical model system and a representative member of the Src-family, functions as complex multidomain allosteric molecular switches comprising SH2 and SH3 domains modulating the activity of the catalytic domain. The broad picture of self-inhibition of c-Src via the SH2 and SH3 regulatory domains is well characterized from a structural point of view, but a detailed molecular mechanism understanding is nonetheless still lacking. Here, we use advanced computational methods based on all-atom molecular dynamics simulations with explicit solvent to advance our understanding of kinase activation. To elucidate the mechanism of regulation and self-inhibition, we have computed the pathway and the free energy landscapes for the "inactive-to-active" conformational transition of c-Src for different configurations of the SH2 and SH3 domains. Using the isolated c-Src catalytic domain as a baseline for comparison, it is observed that the SH2 and SH3 domains, depending upon their bound orientation, promote either the inactive or active state of the catalytic domain. The regulatory structural information from the SH2-SH3 tandem is allosterically transmitted via the N-terminal linker of the catalytic domain. Analysis of the conformational transition pathways also illustrates the importance of the conserved tryptophan 260 in activating c-Src, and reveals a series of concerted events during the activation process.
| S-EPMC5398919 | biostudies-literature
Project description:Src is the representative member of the Src-family kinases (SFKs), a group of tyrosine kinases involved in several cellular processes. Its main function has been for long confined to the plasma membrane/cytoplasm compartment, being a myristoylated protein anchored to the cell membrane and functioning downstream to receptors, most of them lacking intrinsic kinase activity. In the last decades, new roles for some SFKs have been described in the nuclear compartment, suggesting that these proteins can also be involved in directly regulating gene transcription or nucleoskeleton architecture. In this review, we focused on those nuclear functions specifically attributable to Src, by considering its function as both tyrosine kinase and adapting molecule. In particular, we addressed the Src involvement in physiological as well as in pathological conditions, especially in tumors.
| S-EPMC7215861 | biostudies-literature
Project description:BackgroundIntermolecular autophosphorylation at Tyr416 is a conserved mechanism of activation among the members of the Src family of nonreceptor tyrosine kinases. Like several other tyrosine kinases, Src can catalyze the thiophosphorylation of peptide and protein substrates using ATPγS as a thiophosphodonor, although the efficiency of the reaction is low.ResultsHere, we have characterized the ability of Src to auto-thiophosphorylate. Auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni(2+), resulting in kinase activation. Other tyrosine kinases (Ack1, Hck, and IGF1 receptor) also auto-thiophosphorylate in the presence of Ni(2+). Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase.ConclusionsSrc and other tyrosine kinases catalyze auto-thiophosphorylation in the presence of Ni(2+). Thiophosphorylation of Src occurs at Tyr416 in the activation loop, and results in enhanced kinase activity. Tyr416-thiophosphorylated Src could serve as a stable, persistently-activated mimic of Src.
| S-EPMC4936181 | biostudies-literature
Project description:To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2. On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src. The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents. Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor. One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c. Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme. Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax. of 620 nmol/min per mg. The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared. Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers. This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.
| S-EPMC1133104 | biostudies-other