Antibody-free detection of phosphoserine/threonine containing peptides by homogeneous time-resolved fluorescence.

Protein phosphorylation is a critical signaling mechanism in cellular regulation and stress response, and more than 95% of the phosphorylations are targeted toward Ser or Thr amino-acid residues. The classical techniques for analyzing phospho-amino acid residues use radioisotopes or sequence-specific antibodies.
However, both practical and economical limitations have prevented their development, and we here propose an original approach for the detection of phospho-Ser/Thr residues.
It requires no antibody and exploits the patented homogeneous time-resolved fluorescence (HTRF) technology, in association with a 3-step chemical transformation of phospho-amino acids into fluorescent derivatives.
The process involves: (i) alkaline β-elimination of the phosphorylated group, (ii) Michael addition of a bifunctional group, and then (iii) introduction of cyanin-5 as fluorescent acceptor for HTRF. The donor fluorescent moiety at the N-terminus of the phosphorylated peptide is a streptavidin europium cryptate conjugate.
After its development, the detection system has been validated on synthetic peptide substrates of Chk2, a key protein kinase activated in response to DNA damage and involved in cell cycle arrest.
The results showed a good correlation with known specificity profiles. Interestingly, the detection system is versatile, easy to implement, and suitable for multiple parallel analyses.

Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the beta2-adrenergic receptor using phosphoserine-specific antibodies.

Agonist-stimulated desensitization of the beta2-adrenergic receptor (beta2AR) is caused by both a potent cAMP-dependent protein kinase (PKA)-mediated phosphorylation and a less potent, occupancy-dependent, G protein-coupled receptor kinase (GRK)-mediated phosphorylation that leads to beta-arrestin binding and internalization.
In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355, Ser356 of the human beta2AR overexpressed in human embryonic kidney (HEK) 293 cells were characterized using phosphoserine-specific antibodies.
Specificity of the antibodies was shown by their lack of reactivity with mutant beta2ARs lacking the respective sites. In addition, overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. Epinephrine, prostaglandin E1, and forskolin at maximum concentrations stimulated phosphorylation of the beta2AR PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. Epinephrine stimulated PKA site phosphorylation with an EC50 of 20 to 40 pM. In contrast, epinephrine stimulated GRK site phosphorylation (Ser355,Ser356) with an EC50 of 200 nM (1-min treatments), which is more than 4000-fold higher relative to PKA site phosphorylation, consistent with an occupancy-driven process. After 10 to 30 min, the EC50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still approximately 200-fold greater than for the PKA site.
The EC50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation. The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the beta2AR lacking the PKA consensus sites.
The initial levels (2 min) of a range of agonist-stimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase, namely epinephrine>> or = formoterol = fenoterol>> terbutaline = zinterol = albuterol>> salmeterol>> dobutamine>> or = ephedrine. However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as albuterol and salmeterol, stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine, and the correlation breaks down.
The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous beta2AR expressed in A431 human epidermoid carcinoma cells.
To summarize, our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation, heterologous stimulation of the PKA site phosphorylation, no dependence of GRK site phosphorylation on PKA sites, and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists.

Changes in anti-phosphoserine and anti-phosphothreonine antibody binding during the sleep-waking cycle and after lesions of the locus coeruleus.

Cellular responses to many extracellular signals occur through phosphorylation or dephosphorylation of intracellular proteins.
To determine whether changes in protein phosphorylation accompany the electrophysiological changes occurring during the sleep-waking cycle, immunocytochemical mapping of cells labeled with anti-phosphoserine and anti-phosphothreonine antibodies was performed on brain sections of sleeping and waking rats.
Animals implanted for chronic polysomnographic recordings were sacrificed after either 3h of sleep or 3h of sleep deprivation by gentle handling.
Anti-phosphoserine and anti-phosphothreonine staining was mainly localized in neurons and was high in some brain regions, such as cerebral cortex and hypothalamus, and low in others, such as the thalamus. In all cases, the number of cells labeled with either antibody in the cerebral cortex was markedly higher in rats sacrificed after 3h of waking than in rats sacrificed after 3h of sleep.
Unilateral lesions of the locus coeruleus by local injection of 6-hydroxydopamine were performed in other animals to determine whether the increase in protein phosphorylation during waking was influenced by the activity of the noradrenergic system, which is higher in waking than in sleep.
In animals sacrificed after 3h of spontaneous or forced waking, the number of labeled neurons in the cerebral cortex was decreased on the side in which noradrenergic fibers had been lesioned.
These results suggest that 1) neurons exist physiologically in different states of phosphorylation, ranging from a state of very high phosphorylation (e.g., in the cerebral cortex) to a state of very low phosphorylation (e.g., in many thalamic nuclei); 2) the fraction of highly phosphorylated neurons in cerebral cortex is higher in waking than in sleep and 3) part of the immunoreactive phosphorylation present in highly labeled cortical neurons is controlled by the locus coeruleus.

Identification of a high-affinity anti-phosphoserineantibody for the development of a homogeneous fluorescence polarization assay of protein kinase C.

In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions.
Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available.
Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 +/- 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO(4))LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO(4))LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates.
Phosphatidylserine, which enhances the kinase activity of PKC in a Ca(2+)-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay.
The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.

Differential hormone-dependent phosphorylation of progesterone receptor A and B forms revealed by a phosphoserine site-specific monoclonal antibody.

Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven sites) in a manner that involves distinct groups of sites coordinately regulated by hormone and different kinases. Progress on defining a functional role for PR phosphorylation has been hampered both by the complexity of phosphorylation and the lack of simple, nonradioactive methods to detect the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo.
Toward this end, we have produced monoclonal antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a basal site at Ser 190 (MAb P190) and a hormone-induced site at Ser 294 (MAb P294).
Biochemical experiments showed the differential reactivity of the P190 and P294 MAbs for phosphorylated and unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under different experimental conditions including denatured PR protein by Western blots and PR in its native conformation in solution or complexed to specific target DNA.
As detected by Western blot of T47D cells treated with hormone for different times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This difference in kinetics suggests that the Ser 294 site is more labile than basal sites and is acted upon by distinct phosphatases.
A strong preferential hormone-dependent phosphorylation of Ser 294 was observed on PR-B as compared with the amino-terminal truncated A form of PR.
This was unexpected because Ser 294 and flanking sequences are identical on both proteins, suggesting that a distinct conformation of the N-terminal domain of PR-A inhibits phosphorylation of this site.
That Ser 294 lies within an inhibitory domain that mediates the unique repressive functions of PR-A raises the possibility that differential phosphorylation of Ser 294 is involved in the distinct functional properties of PR-A and PR-B.

Anti-phosphoserine and anti-phosphothreonine antibodies modulate autophosphorylation of the insulin receptor but not EGF receptor.

We examined the effect of anti-phosphothreonine and anti-phosphoserine antibodies on insulin receptor autophosphorylation. These antibodies did not affect insulin binding activity of the receptor. These antibodies, however, inhibited insulin-stimulated autophosphorylation of insulin receptor, while did not affect EGF-stimulated autophosphorylation of EGF receptor.

Phosphoserine antibody

70R-PR015 Fitzgerald 50 ug 241 EUR

Phosphoserine antibody

20C-CR1332RP Fitzgerald 100 ug 133 EUR

Phosphoserine antibody

10R-P125a Fitzgerald 250 ul 673 EUR

Phosphoserine antibody

10R-7895 Fitzgerald 100 ug 322 EUR

Phosphoserine Antibody

20-abx134715 Abbexa
  • 356.00 EUR
  • 537.00 EUR
  • 217.00 EUR
  • 100 ul
  • 200 ul
  • 30 ul

Phosphoserine Antibody

abx020673-100ug Abbexa 100 ug 1066 EUR

Phosphoserine Antibody

abx448438-400ul Abbexa 400 ul 537 EUR

Phosphoserine Antibody

20-abx159643 Abbexa
  • 272.00 EUR
  • 230.00 EUR
  • 100 ug
  • 50 ug

Phosphoserine antibody (biotin)

60R-PR001bt Fitzgerald 100 ug 840 EUR

Phosphoserine Antibody (APC)

abx448359-400ul Abbexa 400 ul 592 EUR

Phosphoserine Antibody (Biotin)

abx448367-400ul Abbexa 400 ul 592 EUR

Phosphoserine Antibody (FITC)

abx448373-400ul Abbexa 400 ul 578 EUR

Phosphoserine Antibody (PerCP)

abx448393-400ul Abbexa 400 ul 592 EUR

Phosphoserine Antibody (RPE)

abx448401-400ul Abbexa 400 ul 592 EUR

Phosphoserine Antibody (Streptavidin)

abx448409-400ul Abbexa 400 ul 592 EUR

Phosphoserine Antibody (HRP)

abx448439-400ul Abbexa 400 ul 606 EUR

Phosphoserine (pSer) Antibody

abx415747-50ug Abbexa 50 ug 787 EUR

Phosphoserine Monoclonal Antibody

ABM40198-003ml Abbkine 0.03ml 158 EUR

Phosphoserine Monoclonal Antibody

ABM40198-01ml Abbkine 0.1ml 289 EUR

Phosphoserine Monoclonal Antibody

ABM40198-02ml Abbkine 0.2ml 414 EUR

Phosphoserine Monoclonal Antibody

EM1189-100ul ELK Biotech 100ul 279 EUR

Phosphoserine Monoclonal Antibody

EM1189-50ul ELK Biotech 50ul 207 EUR

Antibody for Phosphoserine

SPC-149F Stressmarq 0.4ml 362 EUR

Antibody for Phosphoserine

SPC-149F-A390 Stressmarq 0.4ml 409 EUR

Antibody for Phosphoserine

SPC-149F-A488 Stressmarq 0.4ml 408 EUR

Antibody for Phosphoserine

SPC-149F-A565 Stressmarq 0.4ml 408 EUR

Antibody for Phosphoserine

SPC-149F-A594 Stressmarq 0.4ml 408 EUR
The inhibition was reversed by adding large amounts of phosphoserine or phosphothreonine. These data suggest that phosphoserine and phosphothreonine on insulin receptor play an important role in insulin-induced conformational change of the receptor.