Carboxypeptidase digestion of human chorionic gonadotropin

Native human chorionic gonadotropin (hCG) was resistant to carboxypeptidase digestion even in the presence of urea. Isolated alpha subunit of the hormone (hCG-alpha), though unreactive to enzyme treatment in the absence of denaturant, released up to four amino acid residues from the C-terminus on incubation with a mixture of carboxypeptidases A and B in urea. While an hCG-alpha product which lacked Ser-92 recombined completely with intact hCG-beta, hCG-alpha from which Ser-92 recombined completely with intact hCG-beta, hCG-alpha from which Ser-92 and Lys-91 were removed showed only partial recombination. The two recombinants were devoid of any in vivo biologic activity, but retained some of the immunologic activity of the native recombinant. These findings indicate that the integrity of the C-terminal residue of serine in hCG-alpha is essential for the expression of in vivo biologic activity of the native hormone.     

Dephosphorylation of Ser-226 facilitates plasma membrane retention of Ntcp

Ntcp is a phosphoprotein, and its translocation by cAMP to the plasma membrane is associated with dephosphorylation. However, the phosphorylation site(s) of Ntcp is not known. Thus, the aim of the present study was to determine the potential Ntcp phosphorylation sites and whether any of these phosphorylation sites is involved in Ntcp translocation. To determine the potential phosphorylation sites, metabolically labeled [32P]Ntcp isolated from hepatocytes was digested with clostripain and then subjected to SDS-PAGE followed by autoradiography. Clostripain digestion resulted in two phosphorylated peptides, and cAMP decreased phosphorylation of one of the peptides (7.8 K(d)), which contains the putative third cytoplasmic loop with three serine (Ser-213, Ser-226, and Ser-227) and two threonine (Thr-219 and Thr-225) residues. To determine whether any one of these serine/threonine residues is phosphorylated and/or is involved in Ntcp translocation, each of these serine/threonine residues were mutated to alanine. HuH-7 cells were transiently transfected with the wild-type and the mutated Ntcps followed by determination of taurocholate uptake and Ntcp expression, translocation and phosphorylation. Mutation of only Ser-226 resulted in 30% decrease in Ntcp phosphorylation and in 2.5 and 3.2-fold increases in taurocholate uptake and Ntcp retention in the plasma membrane, respectively. Cyclic AMP failed to further decrease phosphorylation and increase translocation of S226A-Ntcp. Taken together, these results suggest that the Ser-226 in the third cytoplasmic loop of Ntcp is phosphorylated and cAMP may increase Ntcp translocation to the plasma membrane by dephosphorylating Ntcp at this site.     

Thermodynamics,cooperativity and stability of the tetracycline repressor (TetR) upon tetracycline binding

Allosteric regulation of the Tet repressor (TetR) homodimer relies on tetracycline binding that abolishes the affinity for the DNA operator. Previously, interpretation of circular dichroism data called for unfolding of the α-helical DNA-binding domains in absence of binding to DNA or tetracycline. Our small angle X-ray scattering of TetR(D) in solution contradicts this unfolding as a physiological process. Instead, in the core domain crystal structures analyses show increased immobilisation of helix α9 and two C-terminal turns of helix α8 upon tetracycline binding. Tetracycline complexes of TetR(D) and four single-site alanine variants were characterised by isothermal titration calorimetry, fluorescence titration, X-ray crystal structures, and melting curves. Five crystal structures confirm that Thr103 is a key residue for the allosteric events of induction, with the T103A variant lacking induction by any tetracycline. The T103A variant shows anti-cooperative inducer binding, and a melting curve of the tetracycline complex different to TetR(D) and other variants. For the N82A variant inducer binding is clearly anti-cooperative but triggers the induced conformation.     

The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli

The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning alpha helices of domain II of the beta subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the alpha helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved alpha helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.     

Structural studies on transmembrane proteins. 1. Model study using bacteriorhodopsin mutants containing single cysteine residues

In developing new approaches to structural studies of polytopic transmembrane proteins, we have prepared bacteriorhodopsin mutants containing single cysteine residues at selected sites in different topological domains. Four such mutants were prepared: Gly-72----Cys and Ser-169----Cys in the presumed looped-out regions on the opposite sides of the membrane bilayer and Thr-90----Cys and Leu-92----Cys in the membrane-embedded helix C. The four mutants folded and regenerated the characteristic chromophore in detergent/phospholipid micelles and pumped protons like the wild-type bacteriorhodopsin. After reconstitution in asolectin vesicles, the sulfhydryl groups in the mutants Gly-72----Cys and Ser-169----Cys reacted with iodo[2-3H]acetic acid, while the sulfhydryl groups in the membrane-embedded mutants, Thr-90----Cys and Leu-92----Cys, did not. The sulfhydryl groups in all four mutants could be derivatized in the denatured state by reaction with iodoacetic acid or 6-acryloyl-2-(dimethylamino)naphthalene. Of these derivatives, the two from the mutants Gly-72----Cys and Ser-169----Cys folded like the wild-type bacterioopsin, whereas of the two from the helix C mutants, Thr-90----Cys and Leu-92----Cys, only the latter folded normally. However, the folding of Leu-92----Cys was also impaired when treated with the bulky 5-(iodoacetamido)fluorescein. The reactivity and the folding behavior of the cysteine mutants can thus report on the topographic domain as well as on the orientation of the helices within the membrane.     

Glycine-rich transmembrane helix 10 in the staphylococcal tetracycline transporter TetA(K) lines a solvent-accessible channel

The staphylococcal TetA(K) tetracycline exporter is classified within the major facilitator superfamily of transport proteins and contains 14 alpha-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K) transporter were individually replaced with cysteine. The level of solvent accessibility to each of the targeted amino acid positions was determined as a measure of fluorescein maleimide reactivity and demonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend from at least V298 on the periplasmic side. TMS 10 was found to be amphiphilic containing at least partially solvent accessible amino acid residues along the length of one helical face, suggesting that this helix may line a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significant role for a number of amino acid residues, including a predominance of glycine residues which were further analyzed by alanine substitution. These residues are postulated to allow interhelical interactions between TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transport mechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters for activity.     

Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.     

Site-specific phosphorylation differentiates active from inactive forms of the human T-cell leukemia virus type 1 Tax oncoprotein

The human T-cell leukemia virus type 1 oncoprotein Tax is a phosphoprotein with a predominately nuclear subcellular localization that accomplishes multiple functions via protein-protein interactions. It has been proposed that regulation of this protein's pleiotropic functions may be accomplished through phosphorylation of specific amino acid residues. We have conducted a phosphoryl mapping of mammalian-expressed Tax protein using a combination of affinity purification, liquid chromatography tandem mass spectrometry, and site-directed substitution mutational analysis. We achieved physical coverage of 77% of the Tax sequence and identified four novel sites of phosphorylation at Thr-48, Thr-184, Thr-215, and Ser-336. Previously identified potential serine phosphorylation sites at Ser-10, Ser-77, and Ser-274 could not be confirmed by mass spectrometry. The functional significance of these novel phosphorylation events was evaluated by mutational analysis and subsequent evaluation for activity via both CREB and NF-kappaB-responsive promoters. Our results demonstrate that phosphorylation at Thr-215 is associated with loss of both Tax functions, phosphorylation at Thr-48 was specifically deficient for activation via NF-kappaB, and phosphorylation at Thr-184 and Ser-336 had no effect on these Tax functions. Semiquantitation of phosphopeptides revealed that the majority of Tax was phosphorylated at Thr-48, Thr-184, Thr-215, and Ser-336, whereas only a minor population of Tax was phosphorylated at either Ser-300 or Ser-301. These results suggest that both positive and negative phosphorylation signals result in the maintenance of a subfraction of Tax as "active" protein.     

Identification of mPer1 phosphorylation sites responsible for the nuclear entry

Casein kinase 1 epsilon (CK1 epsilon) is an essential component of the circadian clock in mammals and Drosophila. The phosphorylation of Period (Per) proteins by CK1 epsilon is believed to be implicated in their subcellular localization and degradation, but the precise mechanism by which CK1 epsilon affects Per proteins has not been determined. In this study, three putative CK1 epsilon phosphorylation motif clusters in mouse Per1 (mPer1) were identified, and the phosphorylation status of serine and threonine residues in these clusters was examined. Phosphorylation of residues within a region defined by amino acids 653-663 and in particular of Ser-661 and Ser-663, was identified as responsible for the nuclear translocation of mPer1. Furthermore, phosphorylation of these residues may influence the nuclear translocation of a clock protein complex containing mPer1. These findings indicate that mPer1 phosphorylation is a critical aspect of the circadian clock mechanism.     

Spatiotemporal Regulation of Early Lipolytic Signaling in Adipocytes

Hormone-sensitive lipase (HSL) is a key enzyme regulating the acute activation of lipolysis. HSL functionality is controlled by multiple phosphorylation events, which regulate its association with the surface of lipid droplets (LDs). We determined the progression and stability of HSL phosphorylation on individual serine residues both spatially and temporally in adipocytes using phospho-specific antibodies. Within seconds of β-adrenergic receptor activation, HSL was phosphorylated on Ser-660, the phosphorylated form appearing in the peripheral cytosol prior to rapid translocation to, and stable association with, LDs. In contrast, phosphorylation of HSL on Ser-563 was delayed, the phosphorylated protein was predominantly detected on LDs, and mutation of the Ser-659/Ser-660 site to Ala significantly reduced subsequent phosphorylation on Ser-563. Phosphorylation of HSL on Ser-565 was observed in control cells; the phosphorylated protein was translocated to LDs with similar kinetics to total HSL, and the degree of phosphorylation was inversely related to phospho-HSL^Ser-563. These results describe the remarkably rapid, sequential phosphorylation of specific serine residues in HSL at spatially distinct intracellular locales, providing new insight into the complex regulation of lipolysis.     

Contributions of Conserved Serine Residues to the Interactions of Ligands with Dopamine D2 Receptors

Four dopamine D2 receptor mutants were constructed, in each of which an alanine residue was substituted for one of four conserved serine residues, i.e., Ser-193, Ser-194, Ser-197, and Ser-391. Wild-type and mutant receptors were expressed transiently in COS-7 cells and stably in C6 glioma cells for analysis of ligand-receptor interactions. In radioligand binding assays, the affinity of D2 receptors for dopamine was decreased 50-fold by substitution of alanine for Ser-193, implicating this residue in the binding of dopamine. Each mutant had smaller decreases in affinity for one or more of the ligands tested, with no apparent relationship between the class of ligand and the pattern of mutation-induced changes in affinity, except that the potency of agonists was decreased by substitution for Ser-193. The potency of dopamine for inhibition of adenylyl cyclase was reduced substantially by substitution of alanine for Ser-193 or Ser-197. Mutation of Ser-194 led to a complete loss of efficacy for dopamine and p-tyramine, which would be consistent with an interaction between Ser-194 and the p-hydroxyl substituent of dopamine that is necessary for activation of the receptors to occur. Because mutation of the corresponding residues of beta 2-adrenergic receptors has very different consequences, we conclude that although the position of these serine residues is highly conserved among catecholamine receptors, and the residues as a group are important in ligand binding and activation of receptors by agonists, the function of each of the residues considered separately varies among catecholamine receptors.     

Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.

To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.     

Cysteine-scanning mutagenesis around transmembrane segment VI of Tn10-encoded metal-tetracycline/H(+) antiporter

Each amino acid in putative transmembrane helix VI and its flanking regions, from Ser-156 to Thr-185, of a Cys-free mutant of the Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) was individually replaced by Cys. All of the cysteine-scanning mutants showed a normal level of tetracycline resistance except for the S156C mutant, which showed moderate resistance, indicating that there is no essential residue located in this region. All 20 mutants from S159C to W178C showed no reactivity with N-ethylmaleimide (NEM), whereas the mutants of the flanking regions from S156C to H158C and F179C to T185C were highly or moderately reactive with NEM. These results indicate that like transmembrane helices III and IX, the transmembrane helix VI comprising residues Ser-159-Trp-178 is totally embedded in the hydrophobic environment.     

Salivary cystatin SA-III, a potential precursor of the acquired enamel pellicle, is phosphorylated at both its amino- and carboxyl-terminal regions

Cystatin SA-III was purified from human submandibular/sublingual glandular secretions by adsorption to hydroxyapatite, gel filtration chromatography, and reversed-phase HPLC. The amino acid sequence of its amino-terminus was deduced by sequential Edman degradation and found to be identical to the first 10 residues of cystatin HSP-12. The purified protein was digested with endoproteinase Asp-N and the digestion products were subjected to fast atom bombardment mass spectroscopy. m/z values corresponding to 12 peptides were aligned to the sequence of cystatin S preceded by the eight-residue amino-terminal peptide detected in HSP-12. This process resulted in the assignment of peptides corresponding with 118 out of the 121 amino acid residues predicted from the nucleotide sequence for cystatin SA-III. In order to align several peptides, it was necessary to substitute four residues of phosphoserine for four residues of serine. Fast atom bombardment mass spectrometry and additional Edman degradation procedures localized the phosphate moieties to Ser-3, Ser-99, Ser-112, and Ser-116. This is the first report of the structure of cystatin SA-III deduced by amino acid sequencing techniques and indicates the sites of phosphoserine within the molecule. Based on these assignments, cystatin SA-III is unique among salivary proteins in that it possesses phosphate groups at its amino-terminus as well as its carboxyl-terminus.     

Dual specificity protein kinase activity of testis-specific protein kinase 1 and its regulation by autophosphorylation of serine-215 within the activation loop

TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitro kinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the "activation loop" of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.     

Retaining of the assembly capability of vimentin phosphorylated by mitogen-activated protein kinase-activated protein kinase-2

Intermediate filament (IF) networks can be regulated by phosphorylation of unit proteins, such as vimentin, by specific kinases leading to reorganization of the IF filamentous structure. Recently, we identified mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) as a vimentin kinase (Cheng and Lai [1998] J. Cell. Biochem. 71:169-181). Herein we describe the results of further in vitro studies investigating the effects of MAPKAP kinase-2 phosphorylation on vimentin and the effects of the phosphorylation on the filamentous structure. We show that MAPKAP kinase-2 mainly phosphorylates vimentin at Ser-38, Ser-50, Ser-55, and Ser-82, residues all located in the head domain of the protein. Surprisingly, and in stark contrast to phosphorylation by most other kinases, phosphorylation of vimentin by MAPKAP kinase-2 has no discernable effect on its assembly. It suggested that structure disassembly is not the only obligated consequence of phosphorylated vimentin as regulated by other kinases. Finally, a mutational analysis of each of the phosphorylated serine residues in vimentin suggested that no single serine site was primarily responsible for structure maintenance, implying that the retention of filamentous structure may be the result of the coordinated action of several phosphorylated serine sites. This also shed new lights on the functional task(s) of vimentin that is intermediate filament proteins might provide a phosphate reservoir to accommodate the phosphate surge without any structural changes.     

Role of human immunodeficiency virus type 1 matrix phosphorylation in an early postentry step of virus replication

The matrix domain (MA) is important for targeting of human immunodeficiency virus type 1 Gag assembly to the plasma membrane, envelope incorporation into virions, preintegration complex import into the nucleus, and nuclear export of viral RNA. Myristylation and phosphorylation are key regulatory events for MA function. Previous studies have indicated that MA phosphorylation at serine (Ser) residues is important for viral replication. This study defines the molecular mechanisms of virus particle assembly and infectivity through a detailed study of the role of MA serine phosphorylation. We show that the combined mutation of Ser residues at positions 9, 67, 72, and 77 impairs viral infectivity in dividing and nondividing cells, although the assembly of these Ser mutant viruses is comparable to that of wild-type virus. This defect can be rescued by pseudotyping these mutant viruses with vesicular stomatitis virus G protein, suggesting that these serine residues are critical in an early postentry step of viral infection. The phosphorylation level of MA in defective mutant viruses was severely reduced compared to that of the wild type, suggesting that phosphorylation of Ser-9, -67, -72, and -77 is important for an early postentry step during virus infection.     

Regulation of Microtubule Dynamics through Phosphorylation on Stathmin by Epstein-Barr Virus Kinase BGLF4

Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes.