Functional endothelin/sarafotoxin receptors in rat heart myocytes: structure-activity relationships and receptor subtypes

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) family were characterized in newborn rat heart myocytes using human and rat endothelins (ET-1 and ET-3, respectively), SRTX-b and SRTX-c. Binding studies in intact cells and homogenates revealed significantly higher affinities of ET-1 and SRTX-b than of ET-3 and SRTX-c towards these receptors. This binding profile of ET/SRTX peptides points to their interaction with the receptor subtype designated E-S alpha. All four peptides induced time- and dose-dependent phosphoinositide hydrolysis with the following rank order of potency: ET-1 greater than SRTX-b greater than SRTX-c greater than ET-3. Thus, ET-3 which possesses an intermediate affinity toward the receptor was the least effective with regard to this response. These results confirm and extend our earlier report that the ET/SRTX peptides interact with a newly characterized receptor(s) associated with phosphoinositide metabolism and Ca2+ mobilization. The initiation of inositol phosphate formation is largely independent of extracellular Ca2+, verapamil and nifedipine, indicating that the ET/SRTX peptides are not agonists for the voltage-dependent Ca2+-channels.     

Sarafotoxin expression in the bronchopulmonary tract: immunohistochemical occurrence and colocalization with endothelins

The immunohistochemical occurrence of sarafotoxin (SRTX), a snake venom peptide under strong evolutionary control, was investigated in the pulmonary diffuse neuroendocrine system (PDNES) of newborn cats and rats. By applying the avidin-biotin-peroxidase complex method on serial lung sections, we have demonstrated its distribution and colocalization with different endothelin (ET) isoforms. A light microscopic study revealed apparent immunostaining for SRTX in neuronal components and smooth muscle tissue and in neuroepithelial bodies (NEB), while isolated neuroendocrine cells (NEC) remain unlabelled. Comparison of the SRTX reactivity pattern with that of different ET peptides on adjacent lung sections showed colocalization of SRTX-b with ET-3 in NEB, intrapulmonary ganglion cells and nerve fibres, on the one hand, and with ET-1 in airway and vascular smooth muscle cells, on the other. These findings, in addition to the remarkable functional and structural similarities between SRTX and ET peptides, suggest a common evolutionary origin and biological significance of sarafotoxin and endothelins. Moreover, this is the first time that a toxic peptide has been demonstrated in the PDNES.     

Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide.     

Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2.

MAP kinase-activated protein (MAPKAP) kinase-2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SB 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N-terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase-2, and this constitutively active form may be useful for studying its physiological roles.     

Characterization of PknC, a Ser/Thr kinase with broad substrate specificity from the cyanobacterium Anabaena sp. strain PCC 7120.

Eukaryotic-like protein Ser/Thr and Tyr kinases have only recently been discovered in prokaryotes. In most cases, their biochemical properties have been poorly characterized. The nitrogen-fixing and heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 houses a family of eukaryotic-like Ser/Thr kinases. Some of these enzymes are required for cell growth or development under certain conditions. None of them, however, has been shown experimentally to possess Ser/Thr kinase activity. A gene, pknC, encoding a novel putative Ser/Thr kinase was isolated from Anabaena sp. PCC 7120. The recombinant PknC was shown to be phosphorylated on a Thr residue. This phosphorylation was probably due to the autophosphorylation activity of PknC itself because mutation of two amino acid residues within the subdomain II of its catalytic domain eliminated the phosphorylation of PknC. PknC displayed also a Ser kinase activity towards several nonspecific substrates, and the two residues needed for PknC autophosphorylation was equally required for the phosphorylation of other substrates. PknC is thus a Ser/Thr kinase with broad substrate specificity. The activity of PknC is likely to be regulated in vivo in order to limit the spectrum of its substrate specificity.     

Assembly protein precursor (pUL80.5 homolog) of simian cytomegalovirus is phosphorylated at a glycogen synthase kinase 3 site and its downstream "priming" site: phosphorylation affects interactions of protein with itself and with major capsid protein

Capsid assembly among the herpes-group viruses is coordinated by two related scaffolding proteins. In cytomegalovirus (CMV), the main scaffolding constituent is called the assembly protein precursor (pAP). Like its homologs in other herpesviruses, pAP is modified by proteolytic cleavage and phosphorylation. Cleavage is essential for capsid maturation and production of infectious virus, but the role of phosphorylation is undetermined. As a first step in evaluating the significance of this modification, we have identified the specific sites of phosphorylation in the simian CMV pAP. Two were established previously to be adjacent serines (Ser156 and Ser157) in a casein kinase II consensus sequence. The remaining two, identified here as Thr231 and Ser235, are within consensus sequences for glycogen synthase kinase 3 (GSK-3) and mitogen-activated protein kinase, respectively. Consistent with Thr231 being a GSK-3 substrate, its phosphorylation required a downstream "priming" phosphate (i.e., Ser235) and was reduced by a GSK-3-specific inhibitor. Phosphorylation of Ser235 converts pAP to an electrophoretically slower-mobility isoform, pAP*; subsequent phosphorylation of pAP* at Thr231 converts pAP* to a still-slower isoform, pAP**. The mobility shift to pAP* was mimicked by substituting an acidic amino acid for either Thr231 or Ser235, but the shift to pAP** required that both positions be phosphorylated. Glu did not substitute for pSer235 in promoting phosphorylation of Thr231. We suggest that phosphorylation of Thr231 and Ser235 causes charge-driven conformational changes in pAP, and we demonstrate that preventing these modifications alters interactions of pAP with itself and with major capsid protein, suggesting a functional significance.     

Threonine 41 in beta-catenin serves as a key phosphorylation relay residue in beta-catenin degradation

Beta-catenin phosphorylation at serine 45 (Ser45), threonine 41 (Thr41), Ser37, and Ser33 is critical for beta-catenin degradation, and regulation of beta-catenin phosphorylation is a central part of the canonical Wnt signaling pathway. Beta-catenin mutations at Ser45, Thr41, Ser37, and Ser33 perturb beta-catenin degradation and are frequently found in cancers. It is established that Ser45 phosphorylation by casein kinase I (CKI) initiates phosphorylation at Thr41, Ser37, and Ser33 by glycogen synthase kinase 3 (GSK3) and that phosphorylated Ser37 and Ser33 are recognized by the F-box protein beta-TrCP, a component of a ubiquitin ligase complex that mediates beta-catenin degradation. While the roles of Ser45, Ser37, and Ser33 are well documented, the function of Thr41 remains less defined. Here we show that Thr41 strictly acts as a phosphorylation relay residue and that the Ser-X-X-X-Ser (X is any amino acid) motif is obligatory for beta-catenin phosphorylation by GSK3. Beta-catenin phosphorylation/degradation and its regulation by Wnt can occur normally in the absence of Thr41 as long as the Ser-X-X-X-Ser motif/spacing is preserved. These results suggest that Thr41 functions to bridge sequential phosphorylation from Ser45 to Ser37 and provide further insights into the discrete steps and logic in beta-catenin phosphorylation-degradation.     

PKA modulates GSK-3beta- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3beta (GSK-3beta), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3beta, but the site-specific modulation of GSK-3beta-catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3beta-catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases.     

A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.     

Regulation of phosphorylation of tau by cyclin-dependent kinase 5 and glycogen synthase kinase-3 at substrate level

Microtubule associated protein tau, which is expressed in six alternatively spliced molecular isoforms in human brain, is abnormally hyperphosphorylated in Alzheimer disease and related tauopathies. Here, we show (i) that GSK-3alpha and neither GSK-3beta nor cdk5 can phosphorylate tau at Ser262 and phosphorylation at Ser235 by cdk5 primes phosphorylation at Thr231 by GSK-3alpha/beta; (ii) that tau isoforms with two N-terminal inserts (tau4L, tau3L) are phosphorylated by cdk5 plus GSK-3 at Thr231 markedly more than isoforms lacking these inserts (tau4, tau3); and (iii) that Thr231 is phosphorylated approximately 50% more in free tau than in microtubule-bound tau, and the phosphorylation at this site results in the dissociation of tau from microtubules. These findings suggest that the phosphorylation of tau at Thr231 and Ser262 by cdk5 plus GSK-3, which inhibits its normal biological activity, is regulated both by its amino terminal inserts and its physical state.     

Structure-receptor binding relationships of sarafotoxin and endothelin in porcine cardiovascular tissues

The specific binding of 125I-sarafotoxin S6b was observed in the microsomal fractions from porcine thoracic aorta, and two vasoconstrictive peptides with strikingly homologous structures, sarafotoxin (SRT) and endothelin (ET), interact with a common receptor of the vasculature. The order of the potency of an each endothelin or sarafotoxin analogue as a competitor against 125I-sarafotoxin S6b binding was ET-1 greater than ET-2 greater than SRT S6b greater than ET-3 much greater than SRT S6c. The hydrophobic carboxyl-terminal tail and intramolecular disulfide bridges are essential for the binding activity. In addition, Ser4, Ser5 and Lys9 seem to be important for the activity while the 6th residue does not affect the activity.     

Phosphorylation of phosphatase inhibitor-2 at centrosomes during mitosis

Inhibitor-2 (I-2) is a regulator of protein phosphatase type-1 (PP1), known to be phosphorylated in vitro by multiple kinases. In particular Thr72 is a Thr-Pro phosphorylation site conserved from yeast to human, but there is no evidence that this phosphorylation responds to any physiological signals. Here, we used electrophoretic mobility shift and immunoblotting with a site-specific phospho-Thr72 antibody to establish Thr72 phosphorylation in HeLa cells and show a 25-fold increase in phosphorylation during mitosis. Mass spectrometry demonstrated I-2 in actively growing HeLa cells was also phosphorylated at three other sites, Ser120, Ser121, and an additional Ser located between residues 70 and 90. In vitro kinase assays using recombinant I-2 as a substrate showed that the Thr72 kinase(s) was activated during mitosis, and sensitivity to kinase inhibitors indicated that the principal I-2 Thr72 kinase was not GSK3 but instead a member of the cyclin-dependent protein kinase family. Immunocytochemistry confirmed Thr72 phosphorylation of I-2 during mitosis, with peak intensity at prophase, and revealed subcellular concentration of the phospho-Thr72 I-2 at centrosomes. Together, the data show dynamic changes in I-2 phosphorylation during mitosis and localization of phosphorylated I-2 at centrosomes, suggesting involvement in mammalian cell division.     

Two threonine residues and two serine residues in the second and third intracellular loops are both involved in histamine H1 receptor downregulation

Human histamine H1 receptor (H1R) contains five possible phosphorylation residues (Thr140, Thr142, Ser396, Ser398 and Thr478) and the substitution of all these five residues to alanine completely impairs agonist-induced receptor downregulation. In the present study, to determine which residue(s) are responsible for receptor downregulation, we used mutant H1Rs in which single or multiple residues were substituted with alanine. The results suggested that two groups, i.e., residues Thr140 and Thr142, and residues Ser396 and Ser398, independently contributed to H1R downregulation. Thr140 and Ser398 mainly contributed to downregulation, and Thr142 or Ser396 had a slight inhibitory effect on Thr140- or Ser398-mediated process, respectively.     

Characterization of a Hank’s Type Serine/Threonine Kinase and Serine/Threonine Phosphoprotein Phosphatase in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn(2+)-, but not a Ca(2+)- or a Mg(2+)-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.     

Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells

MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2/cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle.     

Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes

Akt, also known as protein kinase B, is implicated in many cellular processes. Akt is phosphorylated at two residues, Thr308 and Ser473. Thr308-phosphorylated Akt is present in pericentriolar materials, while localization of Ser473-phosphorylated Akt was similar to that of microtubules in metaphase oocytes. Spindles were shorter and aberrant in oocytes injected with Thr308- or Ser473-phosphorylated Akt antibodies. Specifically, Thr308- and Ser473-phosphorylated Akts function individually and are both necessary to assemble the metaphase II (MII) spindle. Moreover, the functions of Thr308- and Ser473-phosphorylated Akts differ in MII oocytes. Although oocytes exhibited second polar body (PB2) emission after the injection of a peptide for Thr308, the chromosomal alignment and microtubular organization were aberrant. In contrast, the injection of a peptide for Ser473 caused a failure of PB2 emission. These results suggest that Thr308- and Ser473-phosphorylated Akts are individually involved in fertilization to complete meiosis, including different roles (i.e., Ser473-phosphorylated Akts are involved in PB2 emission, whereas Thr308-phosphorylated Akts regulate the organization of microtubules).     

Role of conformational sampling of Ser16 and Thr17-phosphorylated phospholamban in interactions with SERCA

Phosphorylation of phospholamban (PLB) at Ser16 and/ or Thr17 is believed to release its inhibitory effect on sarcoplasmic reticulum calcium ATPase. Ser16 phosphorylation of PLB has been suggested to cause a conformational change that alters the interaction between the enzyme and protein. Using computer simulations, the conformational sampling of Ser16 phosphorylated PLB in implicit membrane environment is compared here with the unphosphorylated PLB system to investigate these conformational changes. The results suggest that conformational changes in the cytoplasmic domain of PLB upon phosphorylation at Ser16 increase the likelihood of unfavorable interactions with SERCA in the E2 state prompting a conformational switch of SERCA from E2 to E1. Phosphorylation of PLB at Thr17 on the other hand does not appear to affect interactions with SERCA significantly suggesting that the mechanism of releasing the inhibitory effect is different between Thr17 phosphorylated and Ser16 phosphorylated PLB.     

Mechanism of Activation of PKB/Akt by the Protein Phosphatase Inhibitor Calyculin A

The protein phosphatase inhibitor calyculin A activates PKB/Akt to ~50% of the activity induced by insulin-like growth factor 1 (IGF1) in HeLa cells promoting an evident increased phosphorylation of Ser473 despite the apparent lack of Thr308 phosphorylation of PKB. Nevertheless, calyculin A-induced activation of PKB seems to be dependent on basal levels of Thr308 phosphorylation, since a PDK1-dependent mechanism is required for calyculin A-dependent PKB activation by using embryonic stem cells derived from PDK1 wild-type and knockout mice. Data shown suggest that calyculin A-induced phosphorylation of Ser473 was largely blocked by LY294002 and SB-203580 inhibitors, indicating that both PI3-kinase/TORC2-dependent and SAPK2/p38-dependent protein kinases contributed to phosphorylation of Ser473 in calyculin A-treated cells. Additionally, our results suggest that calyculin A blocks the IGF1-dependent Thr308 phosphorylation and activation of PKB, likely due to an enhanced Ser612 phosphorylation of insulin receptor substrate 1 (IRS1), which can be inhibitory to its activation of PI3-kinase, a requirement for PDK1-induced Thr308 phosphorylation and IGF1-dependent activation of PKB. Our data suggest that PKB activity is most dependent on the level of Ser473 phosphorylation rather than Thr308, but basal levels of Thr308 phosphorylation are a requirement. Additionally, we suggest here that calyculin A regulates the IGF1-dependent PKB activation by controlling the PI3-kinase-associated IRS1 Ser/Thr phosphorylation levels.     

A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun

We have used phospho-specific antibodies to re-examine the multisite phosphorylation of c-Jun in murine RAW macrophages and embryonic fibroblasts. Our results indicate that JNK isoforms are required and sufficient for the phosphorylation of Thr91 and Thr93, as well as the phosphorylation of Ser63 and Ser73, in response to LPS or anisomycin in macrophages and TNFalpha or anisomycin in fibroblasts. However, the phorbol ester (TPA) and EGF-induced phosphorylation of Ser63 and Ser73 is mediated by ERK1/ERK2, as well as JNK1/JNK2, in fibroblasts from wild-type mice and by ERK1/ERK2 alone in fibroblasts from JNK-deficient mice. The phosphorylation of Thr239 is catalysed by GSK3 and the phosphorylation of Ser243 by an as yet unidentified protein kinase. The inhibition of GSK3 is not required for the dephosphorylation of Thr239 in response to LPS, and nor is the phosphorylation of Thr91 and Thr93 required for the TPA- or EGF-induced dephosphorylation of Thr239 in fibroblasts. The agonist-induced dephosphorylation of Thr239 may involve a conformational change that exposes Thr239 to dephosphorylation and/or the activation of a Thr239 phosphatase.