Dataset Information

Vmax. activation of pp60c-src tyrosine kinase from neuroblastoma neuro-2A.

ABSTRACT: A kinetic analysis of the tyrosine-specific protein kinase of pp60c-src from the C1300 mouse neuroblastoma cell line Neuro-2A and pp60c-src expressed in fibroblasts was carried out to determine the nature of the increased specific activity of the neuroblastoma enzyme. In immune-complex kinase assays with ATP-Mn2+ and the tyrosine-containing peptide angiotensin I as phosphoacceptor substrate, pp60c-src from the neuroblastoma cell line was characterized by a maximum velocity (Vmax.) that was 7-15-fold greater than the Vmax. of pp60c-src from fibroblasts. The neuroblastoma enzyme exhibited Km values for ATP (16 +/- 3 microM) and angiotensin I (6.8 +/- 2.6 mM) that were similar to Km values for ATP (25 +/- 3 microM) and angiotensin I (6.5 +/- 1.7 mM) of pp60c-src from fibroblasts. pp60v-src expressed in Rous-sarcoma-virus-transformed cells exhibited an ATP Km value (25 +/- 4 microM) and an angiotensin I Km value (6.6 +/- 0.5 mM) that approximated the values determined for pp60c-src in neuroblastoma cells and fibroblasts. These results indicate that the pp60c-src kinase from neuroblastoma cells has a higher turnover number than pp60c-src kinase from fibroblasts, and that the neural form of the enzyme would be expected to exhibit increased catalytic activity at the saturating concentrations of ATP that are found intracellularly.

SUBMITTER: Matten WT

PROVIDER: S-EPMC1148604 | biostudies-other | 1987 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

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.

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.

Dataset Information

Vmax. activation of pp60c-src tyrosine kinase from neuroblastoma neuro-2A.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure