Study of cross talk between phosphatases and OGA on a ZO-3-derived peptide

Amino Acids - O-GlcNAcylation, like phosphorylation, is a dynamic and rapid posttranslational modification which regulates many cellular processes. Phosphorylation on tyrosine and O-GlcNAcylation...     

Endogenous phosphotyrosine signaling in zebrafish embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.     

Kinetic analysis of the mass transport limited interaction between the tyrosine kinase lck SH2 domain and a phosphorylated peptide studied by a new cuvette-based surface plasmon resonance instrument

We explored the use of a newly developed cuvette-based surface plasmon resonance (SPR) instrument (IBIS) to study peptide-protein interactions. We studied the interaction between the SH2 domain of lck and a phosphotyrosine peptide EPQY*EEIPIYL which was immobilized on a sensor chip. No indications for mass transport limitation (MTL) were observed when standard kinetic approaches were used. However, addition of competing peptide during dissociation revealed a high extent of rebinding. A dissociation rate constant (k(d)) of 0.6+/-0.1 s(-1) was obtained in the presence of large amounts of peptide. A simple bimolecular binding model, applying second-order kinetics for the cuvette system, could not adequately describe the data. Fits were improved upon including a step in the model which describes diffusion of the SH2 domain from the bulk to the sensor, especially for a surface with high binding capacity. From experiments in glycerol-containing buffers, it appeared that the diffusion rate decreased with higher viscosity. It is demonstrated that MTL during association and dissociation can be described by the same diffusion rate. A binding constant (K(D)) of 5.9+/-0.8 nM was obtained from the SPR equilibrium signals by fitting to a Langmuir binding isotherm, with correction for loss of free analyte due to binding. An association rate constant k(a) of 1.1(+/-0.2)x10(8) M(-1) x s(-1) was obtained from k(d)/K(D). The values for k(a) and k(d) obtained in this way were 2-3 orders larger than that from standard kinetic analysis, ignoring MTL. We conclude that in a cuvette the extent of MTL is comparable to that in a flow system.     

Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the K(m) for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or K(m) for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches.     

Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.     

The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi–Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2–JH2 linker region participates in controlling activity by reducing the affinity for ATP.     

Direct effects of acute hypoxia on the reactivity of peripheral arteries of the chicken embryo

In the chicken embryo, acute hypoxemia results in cardiovascular responses, including an increased peripheral resistance. We investigated whether local direct effects of decreased oxygen tension might participate in the arterial response to hypoxemia in the chicken embryo. Femoral arteries of chicken embryos were isolated at 0.9 of incubation time, and the effects of acute hypoxia on contraction and relaxation were determined in vitro. While hypoxia reduced contraction induced by high K(+) to a small extent (-21.8 +/- 5.7%), contractile responses to exogenous norepinephrine (NE) were markedly reduced (-51.1 +/- 3.2%) in 80% of the arterial segments. This effect of hypoxia was not altered by removal of the endothelium, inhibition of NO synthase or cyclooxygenase, or by depolarization plus Ca(2+) channel blockade. When arteries were simultaneously exposed to NE and ACh, hypoxia resulted in contraction (+49.8 +/- 9.3%). Also, relaxing responses to ACh were abolished during acute hypoxia, while the vessels became more sensitive to the relaxing effect of the NO donor sodium nitroprusside (pD(2): 5.81 +/- 0.21 vs. 5.31 +/- 0.27). Thus, in chicken embryo femoral arteries, acute hypoxia blunts agonist-induced contraction of the smooth muscle and inhibits stimulated endothelium-derived relaxation factor release. The consequences of this for in vivo fetal hemodynamics during acute hypoxemia depend on the balance between vasomotor influences of circulating catecholamines and those of the endothelium.     

A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates

A versatile assay for protein tyrosine phosphatases (PTP) employing 3-nitrophosphotyrosine containing peptidic substrates is described. These therapeutically important phosphatases feature in signal transduction pathways. The assay involves spectrophotometric detection of 3-nitrotyrosine production from 3-nitrophosphotyrosine containing peptidic substrates, which are accepted by many PTPs. Compared to conventional chromogenic phosphate derivatives, the more realistic peptidic substrates allow evaluating substrate specificity. The assay’s applicability is demonstrated by determining kinetic parameters for several PTP-substrate combinations and inhibitor evaluation, as well as detection of PTP activity in lysates. The convenient new assay may assist further adoption of PTPs in drug development.     

Maturation-dependent changes of angiotensin receptor expression in fowl

An angiotensin (ANG) receptor homologous to the type 1 receptor (AT1) has been cloned in chickens (cAT1). We investigated whether cAT1 expression in various tissues shows maturation/age-dependent changes. cAT1 mRNA levels detected in renal glomeruli [in situ hybridization (ISH)] and kidney extract (RT-PCR) are significantly (P < 0.01) higher in 19-day embryos (EB) than in chicks (CH, 2-3 wk) and pullets/cockerels (PL/CK, 14-16 wk). The levels in adrenal glands (concentrated in subcapsular regions) are high in EB and further increased in CH and PL/CK. cAT1 mRNA is also detectable in smooth muscle (SM)/adventitia of EB and CH aorta and in the adventitia, but not SM, from PL/CK aortas. The endothelia from small arteries and arterioles, but not from aorta, express cAT1 mRNA (ISH). In all age groups, ANG II induces profound endothelium-dependent relaxation of abdominal aorta, partly (37-47%) inhibitable (P < 0.01) by Nomega-nitro-l-arginine methyl ester (l-NAME, 10(-4) M), suggesting the presence of ANG receptor in endothelium. l-NAME-resistant ANG II relaxation, examined in a limited number of EB or CH aortas, was reduced by 125 mM K+ or apamin plus charybdotoxin. The results suggest that 1) cAT1 is present in kidney, adrenal gland, and vascular endothelium (heterogeneity exists among arteries) of EB, CH, and PL/CK, and in aortic SM/adventitia of EB/CH but only in adventitia of PL/CK; 2) levels of cAT1 gene expression change during maturation in a tissue-specific manner; and 3) ANG II-induced relaxation may be partly attributable to nitric oxide and potassium channel activation.