Humanization of a phosphothreonine peptide-specific chicken antibody by combinatorial library optimization of the phosphoepitope-binding motif.

Detection of protein phosphorylation at a specific residue has been achieved by using antibodies, which have usually been raised by animal immunization. However, there have been no reports of the humanization of phosphospecific non-human antibodies. Here, we report the humanization of a chicken pT231 antibody specific to a tau protein-derived peptide carrying the phosphorylated threonine at residue 231 (pT231 peptide) as a model for better understanding the phosphoepitope recognition mechanism. In the chicken antibody, the phosphate group of the pT231-peptide antigen is exclusively recognized by complementarity determining region 2 of the heavy chain variable domain (VH-CDR2). Simple grafting of six CDRs of the chicken antibody into a homologous human framework (FR) template resulted in the complete loss of pT231-peptide binding. Using a yeast surface-displayed combinatorial library with permutations of 11 FR residues potentially affecting CDR loop conformations, we identified 5 critical FR residues. The back mutation of these residues to the corresponding chicken residues completely recovered the pT231-peptide binding affinity and specificity of the humanized antibody. Importantly, the back mutation of the FR 76 residue of VH (H76) (Asn to Ser) was critical in preserving the pT231-binding motif conformation via allosteric regulation of ArgH71, which closely interacts with ThrH52 and SerH52a residues on VH-CDR2 to induce the unique phosphate-binding bowl-like conformation. Our humanization approach of CDR grafting plus permutations of FR residues by combinatorial library screening can be applied to other animal antibodies containing unique binding motifs on CDRs specific to posttranslationally modified epitopes.

Dual regulation of a Dictyostelium STAT by cGMP and Ca2+ signalling.

When cells are exposed to hyperosmotic stress, the Dictyostelium STAT orthologue STATc is rapidly tyrosine phosphorylated. Previous observations suggest a non-paradigmatic mode of STAT activation, whereby stress-induced serine phosphorylation of the PTP3 protein tyrosine phosphatase inhibits its activity towards STATc. We show that two serine residues in PTP3, S448 and S747, are rapidly phosphorylated after osmotic stress. cGMP is a second messenger for hyperosmotic stress response and 8-bromo-cGMP, a membrane-permeable form of cGMP, is a known activator of STATc. GbpC, a cGMP-binding Ras guanine nucleotide exchange factor protein, is a founder member of a protein family that includes LRRK2, the gene commonly mutated in familial Parkinson's disease. Genetic ablation of gbpC prevents STATc activation by 8-bromo-cGMP. However, osmotic-stress-induced activation of STATc occurs normally in the gbpC null mutant. Moreover, 8-bromo-cGMP does not stimulate phosphorylation of S448 and S747 of PTP3 in a wild-type strain. These facts imply the occurrence of redundant activation pathways. We present evidence that intracellular Ca(2+) is a parallel second messenger, by showing that agents that elevate intracellular Ca(2+) levels are potent STATc activators that stimulate phosphorylation of S448 and S747. We propose that stress-induced cGMP signalling exerts its stimulatory effect by potentiating the activity of a semi-constitutive tyrosine kinase that phosphorylates STATc, whereas parallel, stress-induced Ca(2+) signalling represses STATc dephosphorylation through its inhibitory effect on PTP3.

Identification of the autophosphorylation sites of LRRK2.

Parkinson's disease (PD) is a major adult-onset neurodegenerative disorder affecting the extrapyramidal motor system. A subset of patients develop PD as an autosomal dominant trait, of which PARK8 caused by mutations in the leucine-rich repeat kinase 2 (LRRK2) gene is highlighted because of its high frequency and clinicopathological similarity to sporadic PD. Previous studies have suggested that overactivation of LRRK2 caused by missense mutations leads to neuronal toxicity in PARK8, although the regulatory mechanism that governs the kinase activity of LRRK2 remains unknown. In this study, we expressed the carboxyl-half fragments of LRRK2 (DeltaN-LRRK2) that harbors the kinase as well as the ras-like (ROC) domains in Sf9 cells, subjected them to in vitro phosphorylation reaction, and analyzed the autophosphorylation by matrix assisted laser desorption/ionization- time of flight (MALDI-TOF) mass spectrometer. We identified Ser1403, Thr1404, Thr1410, Thr1491 located within the ROC domain, as well as Thr1967 and Thr1969 in the kinase domain, as the autophosphorylation sites. Substitution of Thr1967, an autophosphorylation site located within the kinase domain, to Ala caused a significant decrease in the kinase activity, implicating Thr1967 in the kinase activity of LRRK2. Phosphospecific antibodies to the autophosphorylation sites specifically recognized full-length LRRK2 subjected to in vitro phosphorylation reaction, indicating that the autophosphorylation takes place in holoproteins. Further analysis of autophosphorylation will clarify the mechanism of activation of LRRK2, as well as the pathomechanism of PD in relation to overactivation of LRRK2.

Imaging protein kinase Calpha activation in cells.

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.

The absence of endogenous beta-endorphin selectively blocks phosphorylation and desensitization of mu opioid receptors following partial sciatic nerve ligation.

Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the beta-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid beta-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.

Autophosphorylation of type II CaM kinase in hippocampal neurons: localization of phospho- and dephosphokinase with complementary phosphorylation site-specific antibodies.

We have visualized the distribution of autophosphorylated type II CaM kinase in neural tissue with the use of two complementary antibodies: a monoclonal antibody that binds to the alpha and beta subunits of the kinase only when they are autophosphorylated at threonine-286 (287 in beta) and affinity-purified rabbit antibodies that bind to both subunits only when they are not phosphorylated at these residues. We used these antibodies to double-label organotypic hippocampal cultures, detecting the mouse monoclonal antibody with rhodamine and the rabbit polyclonal antibodies with fluorescein. In double-exposed photographs, the ratios of intensities of the two fluorophores revealed the relative proportion of autophosphorylated and nonphosphorylated kinase in individual neurons throughout the cultures. We found that autophosphorylated and nonphosphorylated kinase are colocalized throughout most neurons rather than segregated within distinct cells or subcellular domains. However, the variations in intensity of the two fluorophores indicated that the proportion of autophosphorylated kinase is consistently higher in neuronal somas than in the neuropil. Incubation of the cultures in Ca2+ free medium dramatically reduced both the level of autophosphorylated kinase detected biochemically and the relative intensity of fluorescent staining with the phosphokinase specific monoclonal antibody. These results support the hypothesis that regulation of Ca(2+)-independent CaM kinase activity in vivo occurs by a dynamic equilibrium between autophosphorylation and dephosphorylation and that this equilibrium is maintained, at varying steady-state levels, in all parts of neurons.

Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21.

Replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in various processes, including nucleotide excision repair and DNA replication. The 32 kDa subunit of RPA (RPA32) is phosphorylated in response to various DNA-damaging agents, and two protein kinases, ataxia-telangiectasia mutated (ATM) and the DNA-dependent protein kinase (DNA-PK) have been implicated in DNA damage-induced phosphorylation of RPA32. However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported. Here we generated a phosphospecific antibody that recognizes Thr21-phosphorylated RPA32. We show that both DNA-PK and ATM phosphorylate RPA32 on Thr21 in vitro. Ionizing radiation (IR)-induced phosphorylation of RPA32 on Thr21 was defective in ATM-deficient cells, while camptothecin (CPT)-induced phosphorylation of RPA32 on Thr21 was defective in cells lacking functional DNA-PK. Neither ATM nor DNA-PK was required for etoposide (ETOP)-induced RPA32 Thr21 phosphorylation. However, two inhibitors of the ATM- and Rad3-related (ATR) protein kinase activity prevented ETOP-induced Thr21 phosphorylation. Inhibition of DNA replication prevented both the IR- and CPT-induced phosphorylation of Thr21, whereas ETOP-induced Thr21 phosphorylation did not require active DNA replication. Thus, the regulation of RPA32 Thr21 phosphorylation by multiple DNA damage response protein kinases suggests that Thr21 phosphorylation of RPA32 is a crucial step within the DNA damage response.

Oxytocin induces dephosphorylation of eukaryotic elongation factor 2 in human myometrial cells.

The oxytocin (OT) receptor (OTR) mediates a wide spectrum of biological actions and is expressed in a large number of different tissues, including uterine, breast, and lung tumors. To define more completely the intracellular signaling mechanisms linked to OTR activation, we have used a phosphoproteomics approach and have characterized changes in the phosphorylation states of intracellular proteins in response to OTR activation in OTR-expressing cell lines. Using a specific antiphosphothreonine antibody, we observed several distinct changes in the threonine phosphorylation patterns. The most prominent change involved dephosphorylation of a 95-kDa moiety. Purification by ion exchange chromatography combined with one- and two-dimensional polyacrylamide gel electrophoresis followed by N-terminal micro-sequence analysis revealed that the 95-kDa moiety corresponded to eukaryotic elongation factor 2. This protein is a key regulator of cellular protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. Dose-response curves in myometrial cells expressing the endogenous OTR indicated a significant effect of OT on eukaryotic elongation factor 2 dephosphorylation at 1 nM, a concentration close to the dissociation constant (K(d)) of OT. Time course analysis indicates that the effect is rapid with a significant effect occurring at 5 min. To determine directly the effect of OT on protein synthesis, the incorporation of [35S]Met into total protein was assessed. In myometrial cells, OTR activation led to significant 29% increase in total protein synthesis over a 2-h period. These findings establish a novel link between OTR activation and cellular protein synthesis and thus define a mechanism by which OT assumes a so far unrecognized, physiologically relevant trophic function.

WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt substrate.

Recent evidence indicates that mutations in the gene encoding the WNK1 [with no K (lysine) protein kinase-1] results in an inherited hypertension syndrome called pseudohypoaldosteronism type II. The mechanisms by which WNK1 is regulated or the substrates it phosphorylates are currently unknown. We noticed that Thr-60 of WNK1, which lies N-terminal to the catalytic domain, is located within a PKB (protein kinase B) phosphorylation consensus sequence. We found that PKB phosphorylated WNK1 efficiently compared with known substrates, and both peptide map and mutational analysis revealed that the major PKB site of phosphorylation was Thr-60. Employing a phosphospecific Thr-60 WNK1 antibody, we demonstrated that IGF1 (insulin-like growth factor) stimulation of HEK-293 cells induced phosphorylation of endogenously expressed WNK1 at Thr-60. Consistent with PKB mediating this phosphorylation, inhibitors of PI 3-kinase (phosphoinositide 3-kinase; wortmannin and LY294002) but not inhibitors of mammalian target of rapamycin (rapamycin) or MEK1 (mitogen-activated protein kinase kinase-1) activation (PD184352), inhibited IGF1-induced phosphorylation of endogenous WNK1 at Thr-60. Moreover, IGF1-induced phosphorylation of endogenous WNK1 did not occur in PDK1-/- ES (embryonic stem) cells, in which PKB is not activated. In contrast, IGF1 still induced normal phosphorylation of WNK1 in PDK1(L155E/L155E) knock-in ES cells in which PKB, but not S6K (p70 ribosomal S6 kinase) or SGK1 (serum- and glucocorticoid-induced protein kinase 1), is activated. Our study provides strong pharmacological and genetic evidence that PKB mediates the phosphorylation of WNK1 at Thr-60 in vivo. We also performed experiments which suggest that the phosphorylation of WNK1 by PKB is not regulating its kinase activity or cellular localization directly. These results provide the first connection between the PI 3-kinase/PKB pathway and WNK1, suggesting a mechanism by which this pathway may influence blood pressure.

Thylakoid protein phosphorylation in evolutionally divergent species with oxygenic photosynthesis.

Phosphothreonine antibody was used to explore reversible thylakoid protein phosphorylation in vivo in evolutionally divergent organisms with oxygenic photosynthesis. Three distinct groups of organisms were found. Cyanobacteria and red algae, both with phycobilisome antenna system, did not show phosphorylation of any of the photosystem II (PSII) proteins and belong to group 1. Group 2 species, consisting of a moss, a liverwort and a fern, phosphorylated both the light-harvesting chlorophyll alb proteins (LHCII) and the PSII core proteins D2 and CP43, but not the D1 protein. Reversible phosphorylation of the D1 protein seems to be the latest event in the evolution of PSII protein phosphorylation and was found only in seed plants, in group 3 species. Light-intensity-dependent regulation of LHCII protein phosphorylation was similar in group 2 and 3 species, with maximal phosphorylation of LHCII at low light and nearly complete dephosphorylation at high light.

A generic time-resolved fluorescence assay for serine/threonine kinase activity: application to Cdc7/Dbf4.

The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phospho-threonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the K(m) for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.

Natural variation in phosphorylation of photosystem II proteins in Arabidopsis thaliana: is it caused by genetic variation in the STN kinases?

Reversible phosphorylation of photosystem II (PSII) proteins is an important regulatory mechanism that can protect plants from changes in ambient light intensity and quality. We hypothesized that there is natural variation in this process in Arabidopsis (Arabidopsis thaliana), and that this results from genetic variation in the STN7 and STN8 kinase genes. To test this, Arabidopsis accessions of diverse geographical origins were exposed to two light regimes, and the levels of phospho-D1 and phospho-light harvesting complex II (LHCII) proteins were quantified by western blotting with anti-phosphothreonine antibodies. Accessions were classified as having high, moderate or low phosphorylation relative to Col-0. This variation could not be explained by the abundance of the substrates in thylakoid membranes. In genotypes with atrazine-resistant forms of the D1 protein, low D1 and LHCII protein phosphorylation was observed, which may be due to low PSII efficiency, resulting in reduced activation of the STN kinases. In the remaining genotypes, phospho-D1 levels correlated with STN8 protein abundance in high-light conditions. In growth light, D1 and LHCII phosphorylation correlated with longitude and in the case of LHCII phosphorylation also with temperature variability. This suggests a possible role of natural variation in PSII protein phosphorylation in the adaptation of Arabidopsis to diverse environments.

Antibody specific for the Thr-286-autophosphorylated alpha subunit of Ca2+/calmodulin-dependent protein kinase II.

We report the production of an antibody specific for Ca2+/calmodulin-dependent protein kinase II (CaM-KII) autophosphorylated only at Thr-286 of the alpha subunit. Peptide Y-66 [sequence MHRQETVDC (Met-281 to Cys-289 of alpha subunit of CaM-KII)] was synthesized and phosphorylated by the CaM-KII endogenous to synaptic cytoskeleton (postsynaptic density-enriched fraction); the phosphorylated amino acid residue threonine corresponds to Thr-286 in the kinase alpha subunit. The phosphorylated Y-66 peptide was separated from the unphosphorylated peptide by HPLC and used as an immunogen after being coupled to hemocyanin. The antibodies that reacted with hemocyanin and unphosphorylated Y-66 peptide were adsorbed, and then IgG was purified. ELISA proved that the IgG obtained reacted specifically with phosphorylated Y-66 peptide. Immunoblot analysis showed that the antibody reacted specifically to the autophosphorylated CaM-KII both in purified and synaptic cytoskeleton-associated form. Appearance of CaM-KII subunits immunoreactive to anti-phosphorylated Y-66 antibody paralleled the generation of Ca(2+)-independent kinase activity. Immunocytochemical experiments clearly showed expression of the Thr-286- or Thr-287-autophosphorylated form of CaM-KII in cultured hippocampal cells treated with N-methyl-D-aspartate. Thus, this antibody could be extremely useful for studying the biological functions of CaM-KII.

Antibodies directed against phosphothreonine residues as potent tools for studying protein phosphorylation.

Here we report the development of novel antibodies which specifically react with phosphothreonine residues [anti-(P-Thr)antibodies]. The specificity of the antibodies was assessed in radioimmunoassays where we could demonstrate that half-maximal and maximal binding of the antibodies to plates coated with BSA - P-Thr occurred at serum dilutions of 1:4000 and 1:1000, respectively. P-Thr inhibited antibody binding with a half-maximal effect at 40 microM. P-Ser was 200-fold less potent while P-Tyr was essentially ineffective. Anti-(P-Thr) antibodies could specifically bind to phosphothreonine-containing proteins on Western blots. Using such a procedure we could demonstrate enhanced threonine phosphorylation of the EGF receptor upon treatment of intact unlabeled A431 cells with EGF. We could further demonstrate antibodies binding to proteins present in extracts of rat hepatoma cells (Fao). P-Thr at 10 microM completely inhibited antibody binding while P-Ser, P-Tyr, Thr or Ser, each present at tenfold higher concentrations, had no such inhibitory effect. Anti-(P-Thr) antibodies were also capable of specifically immunoprecipitating 32P-labeled phosphoproteins present in Triton extracts of Fao cells. Immunoprecipitation of proteins of 38 kDa, 55 kDa, 85 kDa, 100 kDa and 155 kDa was inhibited by 1 mM P-Thr but not by P-Tyr. These findings suggest that anti-(P-Thr) antibodies could be powerful tools in studies aimed at monitoring alterations in threonine phosphorylation of specific proteins as they occur under physiological conditions in response to various extracellular stimuli. Identification of such proteins can be conveniently monitored by immunoblotting.

Preparation of anti-phosphoserine and anti-phosphothreonine antibodies and their application in the study of insulin- and EGF-induced phosphorylation.

We prepared antibodies against phosphoserine (P-Ser) and phosphothreonine (P-Thr) by immunizing rabbits with P-Ser or P-Thr conjugated to bovine serum albumin. The antibodies (anti-P-Ser and anti-P-Thr) were purified using P-Ser or P-Thr affinity columns. Anti-P-Thr was highly specific for P-Thr, while anti-P-Ser showed weak cross-reactivity with P-Thr. We showed that these antibodies can immunodetect serine/threonine phosphorylated insulin and epidermal growth factor (EGF) receptors and several proteins which are phosphorylated on serine/threonine residues in response to insulin or EGF stimulation. The antibodies will certainly provide a good tool for discovering novel kinases and substrates involved in signal transduction.

Phosphorylation of light-harvesting complex II and photosystem II core proteins shows different irradiance-dependent regulation in vivo. Application of phosphothreonine antibodies to analysis of thylakoid phosphoproteins.

An immunological approach using a polyclonal phosphothreonine antibody is introduced for the analysis of thylakoid protein phosphorylation in vivo. Virtually the same photosystem II (PSII) core phosphoproteins (D1, D2, CP43, and the psbH gene product) and the light-harvesting chlorophyll a/b complex II (LHCII) phosphopolypeptides (LHCB1 and LHCB2), as earlier identified by radiolabeling experiments, were recognized in both pumpkin and spinach leaves. Notably, the PSII core proteins and LHCII polypeptides were found to have a different phosphorylation pattern in vivo with respect to increasing irradiance. Phosphorylation of the PSII core proteins in leaf discs attained the saturation level at the growth light intensity, and this level was also maintained at high irradiances. Maximal phosphorylation of LHCII polypeptides only occurred at low light intensities, far below the growth irradiance, and then drastically decreased at higher irradiances. These observations are at variance with traditional studies in vitro, where LHCII shows a light-dependent increase in phosphorylation, which is maintained even at high irradiances. Only a slow restoration of the phosphorylation capacity for LHCII polypeptides at the low light conditions occurred in vivo after the high light-induced inactivation. Furthermore, if thylakoid membranes were isolated from the high light-inactivated leaves, no restoration of LHCII phosphorylation took place in vitro. However, both the high light-induced inactivation and low light-induced restoration of LHCII phosphorylation seen in vivo could be mimicked in isolated thylakoid membranes by incubating with reduced and oxidized dithiothreitol, respectively. We propose that stromal components are involved in the regulation of LHCII phosphorylation in vivo, and inhibition of LHCII phosphorylation under increasing irradiance results from reduction of the thiol groups in the LHCII kinase.

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. 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.

Identification of calponin as a novel substrate of Rho-kinase.

Calponin, an F-actin-associated protein implicated in the regulation of smooth muscle contraction, is known to be phosphorylated in vitro by protein kinase C (PKC) and Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II). Unphosphorylated calponin binds to F-actin and inhibits the actin-activated myosin ATPase activity; these properties are lost on phosphorylation. In the present study, we found that Rho-kinase phosphorylated basic calponin stoichiometrically in vitro. We identified the sites of phosphorylation of calponin by Rho-kinase as Thr-170, Ser-175, Thr-180, Thr-184, and Thr-259, and prepared antibodies that specifically recognized calponin phosphorylated at Thr-170 and Thr-184. We showed that the phosphorylation of calponin by Rho-kinase inhibited the binding of calponin to F-actin. Taken together, these results suggest that calponin is a substrate of Rho-kinase and that Rho-kinase regulates the interaction of calponin with F-actin.