Serine 167 is the major estradiol-induced phosphorylation site on the human estrogen receptor

Serine 167 has been identified by radiolabel and amino acid sequencing as the major estrogen-induced phosphorylation site on the human estrogen receptor (hER) from human MCF-7 mammary carcinoma cells. The phosphorylation of the hER on serine 167 was estrogen-dependent, increasing 4-fold upon estradiol treatment of MCF-7 cells and accounted for almost half of the total [32P]phosphate incorporated into the recombinant hER from Sf9 insect cells and the native hER from MCF-7 cells. Casein kinase II was found to phosphorylate the purified recombinant hER on serine 167 in vitro. In addition, estradiol binding enhanced by 2-fold the phosphorylation of the purified recombinant hER by casein kinase II in vitro. Western blot analysis and [32P]phosphate incorporation confirmed the presence of casein kinase II in Sf9 cells. These results demonstrate that the hER is phosphorylated on serine 167 by casein kinase II in a hormone-dependent manner.     

Hormone- and DNA-binding mechanisms of the recombinant human estrogen receptor

We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix. The recombinant hER bound estradiol with a positively cooperative mechanism. At hER concentrations in excess of 13 nM the Hill coefficient reached a maximal value of 1.6, whereas, at lower hER concentrations, the Hill coefficient approached 1.0, suggesting that the hER was dissociated to the monomeric species and site-site interactions were diminished. The hER specifically bound an estrogen responsive element (ERE) from chicken vitellogenin II gene as measured by the gel mobility assay, ethylation, and thymine interference footprinting. Specific interference patterns suggest a two-fold symmetry of the hER binding to the ERE with each monomer of the hER bound in the major groove of the DNA. These data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor.     

Ecto-protein tyrosine phosphatase activity in Trypanosoma cruzi infective stages

Live T. cruzi trypomastigotes and amastigotes possess ecto-protein tyrosine phosphatase activity as indicated by the ability of intact cells to catalyze dephosphorylation of tyrosine phosphorylated myelin basic protein, [32P]TyrRaytide, phosphotyrosine, or the phosphotyrosine analog p-nitrophenylphosphate (p-NPP). The dephosphorylation of myelin basic protein (MBP) and p-NPP was inhibited by sodium o-vanadate, zinc chloride and NaF, while dephosphorylation of [32P]TyrRaytide was insensitive to zinc chloride but sensitive to o-vanadate and NaF. In contrast, live cells were not able to dephosphorylate serine or threonine phosphorylated peptides ([32P]Kemptide) or proteins ([32P]RCM-lysozyme and [32P]MBP).     

The interaction of human estrogen receptor with DNA is modulated by receptor-associated proteins

To better define the role of accessory protein as mediators of estrogen receptor (ER) function, we have used immuno-, steroid-, and site-specific DNA-affinity chromatography to identify and characterize proteins that associate with ER in extracts from ER-expressing Chinese hamster ovary (CHO-ER) cells. Two associated proteins [70 and 55 kilodaltons (kDa)] were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis silver stain analysis of all three column eluates. Two additional proteins (45 and 48 kDa) were preferentially retained by the ER-specific DNA affinity column. The 70-kDa protein was subsequently identified by Western blot analysis as a heat shock protein (hsp70). The 55-kDa protein was identified by N-terminal microsequencing as a member of the protein disulfide isomerase family. The 48- and 45-kDa proteins remain unidentified. To determine the possible differential effects of estrogen agonists and antagonists on human (h) ER interaction with these proteins, CHO-ER cells were labeled with [35S]methionine in the presence of estradiol, 4-hydroxytamoxifen (OH-Tam; partial agonist/antagonist), or ICI 182,780 (complete antagonist). None of these ligands altered the pattern of associated proteins when hER complexes were isolated by adsorption to the vitellogenin A2 estrogen response element (ERE). However, when hER was isolated by immunoadsorption, a reduction in the level of associated hsp70 was observed following treatment with estradiol or OH-Tam, compared to no treatment or treatment with ICI 182,780. By gel retardation analysis, maximal interaction of affinity-purified hER with ERE occurred in the presence of all four associated proteins. Removal of the 48- and 45-kDa proteins and/or hsp70 resulted in a decrease in hER/ERE association, which could be restored by the addition of purified hsp70 and/or a mixture of the 48- and 45-kDa proteins. The increased stability of the restored complex was due primarily to an increase in the association rate of hER with ERE. These results suggest that accessory proteins may be required for maximal ER interaction with EREs and that estrogens and estrogen antagonists may promote differential retention of hsp70 in the presence or absence of a specific ERE.     

Cyclic adenosine 3',5'-monophosphate-dependent regulation of purified bovine aortic calcium/calmodulin-dependent myosin light chain kinase

Myosin light chain kinase was extracted from bovine aortic muscularis by a low ionic strength buffer containing 50% glycerol. It was purified 130-fold with a 10% yield by anion-exchange chromatography followed by affinity chromatography on calmodulin-Sepharose. The enzyme was 95% calcium/calmodulin-dependent and exhibited a specific activity of 2-6 mumol/min per mg. It phosphorylated the myosin regulatory light chain exclusively. The apparent Kd for calmodulin was 6.3 nM. Upon phosphorylation of the enzyme by the catalytic subunit of cyclic AMP-dependent protein kinase, its affinity for calmodulin decreased 4-fold, without alteration of the V. When examined by SDS-polyacrylamide gel electrophoresis, the purified enzyme was made up of two major peptides (Mr 142 000 and 131 000, respectively), with a minor 80 000 dalton peptide. All these peptides were 32P-labeled after incubation with [gamma-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. Also, after non-denaturing polyacrylamide gel electrophoresis, they all exhibited myosin light chain kinase activity, suggesting that the 131 000 and 80 000 dalton species are proteolytic products of the native enzyme of Mr 142 000. Vascular smooth muscle myosin light chain kinase is therefore soluble, calcium/calmodulin dependent and phosphorylatable by cyclic AMP-dependent protein kinase with concomitant decrease in its affinity for calmodulin. These features account for the beta-adrenergic relaxation of vascular smooth muscle.     

Expression, purification, and reconstitution of receptor for pituitary adenylate cyclase-activating polypeptide. large-scale purification of a functionally active G protein-coupled receptor produced in Sf9 insect cells

Human pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was expressed in Sf9 insect cells and Chinese hamster ovary (CHO) cells. The recombinant receptor in Sf9 cell membranes had low affinity for 125I-PACAP27 (Kd = 155.3 pM) and was insensitive to guanosine 5'-O-3-thiotriphosphate (GTPgammaS), whereas the receptor in CHO membranes had a high affinity (Kd = 44.4 pM) and was GTPgammaS sensitive. The receptor in Sf9 membranes was converted to a high affinity state (Kd = 20-40 pM) following solubilization with digitonin. A large quantity (2 mg from 8 liters of insect cells) of the purified PACAP receptors (Bmax = 23.9 nmol/mg of protein) were obtained in a digitonin-induced high affinity state (Kd = 17.3 pM) using biotinylated ligand affinity chromatography. The apparent molecular weight of the purified receptor (Mr = 48,000) was smaller than that of the receptor from CHO cells (Mr = 58,000) due to differences in asparagine-linked sugar chains. The purified receptor reverted to a low affinity state (Kd = 182.6 pM) upon reconstitution into lipid vesicles, however, the receptor reconstituted with Gs protein had a high affinity (Kd = 40.2 pM) and was GTPgammaS sensitive. [35S]GTPgammaS binding to the reconstituted Gs protein was enhanced by PACAP27 and PACAP38 (EC50 = 42.5 and 9.4 pM, respectively) but not by antagonist PACAP(6-38), indicating that the purified receptor was functionally active.     

Sodium butyrate stimulates PKC activation and induces differential expression of certain PKC isoforms during erythroid differentiation

Inside-out submitochondrial particles from potato tuber mitochondria were incubated with [gamma-32P]ATP. More than 16 phosphorylated polypeptides were detected by autoradiography on an SDS-gel. Two phosphoproteins, migrating at 22 and 28 kDa, were excised from the SDS-gel, electroeluted, and purified further by anion chromatography. The phosphoproteins were N-terminally sequenced. Over the regions sequenced, the 22 and 28 kDa phosphoproteins had 100% sequence identity with potato proteins identified as the delta'-subunit of the F1-ATPase and the b-subunit of the F0-ATPase, respectively. We suggest that phosphorylation of these proteins may control the interaction between F1 and F0 and regulate energy coupling in oxidative phosphorylation.     

Properties and phosphorylation sites of baculovirus-expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1)

NIPP-1 is the RNA-binding subunit of a major species of protein phosphatase-1 in the nucleus. We have expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1) in Sf9 cells, using the baculovirus-expression system. The purified recombinant protein was a potent (Ki = 9.9 +/- 0.3 pM) and specific inhibitor of protein phosphatase-1 and was stoichiometrically phosphorylated by protein kinases A and CK2. At physiological ionic strength, phosphorylation by these protein kinases drastically decreased the inhibitory potency of free NIPP-1. Phosphorylation of NIPP-1 in a heterodimeric complex with the catalytic subunit of protein phosphatase-1 resulted in an activation of the holoenzyme without a release of NIPP-1. Sequencing and phosphoamino acid analysis of tryptic phosphopeptides enabled us to identify Ser178 and Ser199 as the phosphorylation sites of protein kinase A, whereas Thr161 and Ser204 were phosphorylated by protein kinase CK2. These residues all conform to consensus recognition sites for phosphorylation by protein kinases A or CK2 and are clustered near a RVXF sequence that has been identified as a motif that interacts with the catalytic subunit of protein phosphatase-1.     

The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase

The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues. We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.     

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.     

Proteoglycan alterations in skin fibroblast cultures from patients affected with pseudoxanthoma elasticum

The heat-stable enterotoxin STa of E. coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion. We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro. Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with [32P]-phosphate using polyclonal anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and 32P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC. Phosphorylation was inhibited 40% by 5 microM of a synthetic peptide corresponding to the sequence around Ser1029 of the STa receptor, a site previously proposed as a potential PKC phosphorylation site. Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold. We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser1029 of the STaR/GC remains a candidate phosphorylation site by PKC.     

Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.     

Homozygous deletions of the CDKN2 gene and loss of heterozygosity of 9p in primary hepatocellular carcinoma

To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, 32P-labeled 3T3-L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin for 15 min. eEF-1 was purified by affinity chromatography on tRNA-Sepharose and shown to be phosphorylated on the alpha, beta and delta subunits. Phosphorylation of eEF-1alpha was stimulated sixfold in response to insulin, beta was stimulated fourfold and delta was threefold. The rate of elongation assayed with eEF-1 from insulin-stimulated cells was over twofold greater than with eEF-1 from serum-deprived cells. When eEF-1 from insulin-treated cells was subjected to two-dimensional tryptic phosphopeptide mapping, nine phosphopeptides were obtained with the alpha subunit, one with the beta subunit and three with the delta subunit. When compared with phosphopeptide maps of alpha, beta and delta subunits of eEF-1 phosphorylated in vitro by the insulin-stimulated multipotential protein kinase, the maps of the beta and delta subunits were identical. Five phosphopeptides obtained with the alpha subunit in vivo were identical to those obtained with S6 kinase in vitro; the remainder were unique. To examine whether protein kinase C had a role in phosphorylation of eEF-1 in response to insulin, protein kinase C was down-regulated by prolonged exposure of 3T3-L1 cells to 4beta-phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the alpha, beta and delta subunits was stimulated 2.5-fold in response to insulin, with elongation activity stimulated to a similar extent, suggesting that protein kinase C had no effect on stimulation of elongation in response to insulin. Thus, stimulation of eEF-1 activity in response to insulin appears to be mediated primarily by multipotential S6 kinase. This data is consistent with previous studies on stimulation of initiation via phosphorylation of initiation factors by multipotential S6 kinase [Morley, S. J. & Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989].     

Overexpression, one-step purification and characterization of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase

HASPP28 (heat- and acid-stable phosphoprotein of 28 kDa) has been purified to near homogeneity from the acid-stable protein fraction of rat brain extract. Based on the N-terminal 40 amino acid sequence, a pair of highly degenerate primers was used to generate a 107-bp probe from rat brain RNA by RT-PCR. From the rat brain lambda gt11 library, this probe identified two positive clones that together provided a cDNA of 837 bp with an open reading frame of 546 bp. This cDNA was extended by 3'RACE to 1.2 kb that included a polyadenylation signal and a poly(A) tail. The 180-amino-acid sequence derived from the open reading frame, which did not correspond to any known protein, was predicted to have phosphorylation sites for protein kinase C, casein kinase II (CKII), and protein kinase A. Indeed, both the purified rat brain HASPP28 and the recombinant HASPP28 (rHASPP28) can be phosphorylated by these kinases. Northern blot analysis indicated that HASPP28 was present in all rat tissues tested, including those from the brain, lung, spleen, kidney, liver, heart, and muscle, in decreasing order of abundance. Phosphopeptide analysis of rHASPP28 phosphorylated in vitro by various kinases showed different tryptic patterns on two-dimensional mapping and isoelectric focusing gels. From [32P]PO4-labeled N1E115 neuroblastoma cells, HASPP28 can be immunoprecipitated with a polyclonal antiserum raised against rHASPP28. The immunoprecipitated protein showed a phosphopeptide pattern similar to that of rHASPP28 phosphorylated by CK II in vitro. Furthermore, the immunoprecipitates from cells treated with phorbol 12-myristate 13-acetate or 8-bromo-cAMP did not show any increased phosphorylation over those of untreated ones, and the phosphopeptide patterns of the immunoprecipitates again were similar to that of CK II phosphorylated protein. These results suggest that HASPP28 is a novel phosphoprotein that can be phosphorylated by several kinases in vitro. In intact cells, CK II seems to be solely responsible for the phosphorylation of HASPP28.     

Phosphorylation of the cytoplasmic domain of E-selectin is regulated during leukocyte-endothelial adhesion

E-selectin, a selectin expressed on activated vascular endothelium, supports rolling and stable adhesion of leukocytes at sites of inflammation. Previously, we have reported that leukocyte adhesion to cultured endothelial cells induces association of the cytoplasmic domain of E-selectin with cytoskeletal elements, suggesting that outside-in signaling may occur during E-selectin-mediated adhesion. To investigate this potential signaling function of E-selectin, HUVEC activated with recombinant human IL-1beta (10 U/ml, 4 h) were labeled with [32P]orthophosphate, and E-selectin was immunoprecipitated using mAb H18/7. Autoradiography revealed constitutive phosphorylation of E-selectin in these cells and time-dependent dephosphorylation following adhesion of HL-60 cells. Cross-linking of cell surface E-selectin using H18/7 and a polyclonal secondary Ab induced E-selectin dephosphorylation, as did adhesion of beads coated with recombinant P-selectin glycoprotein ligand-1 (PSGL-1), an E-selectin ligand. Using adenoviral vector-mediated transfection in HUVEC of a tail-less E-selectin and phosphoamino acid analysis, we documented phosphorylation occurring exclusively within the cytoplasmic domain and involving serine residues. Additional experiments using a series of cytoplasmic domain mutants of E-selectin expressed in COS-7 cells localized the regions that were constitutively phosphorylated. Preincubation with okadaic acid and sodium vanadate abrogated adhesion-induced dephosphorylation of E-selectin. Thus, E-selectin, which is constitutively phosphorylated in cytokine-activated human endothelial cells, undergoes an enzymatically regulated dephosphorylation following leukocyte adhesion. This process appears to be triggered by multivalent ligand binding and/or cross-linking of cell surface E-selectin. Ligand-dependent regulation of the phosphorylation of E-selectin's cytoplasmic domain provides additional evidence for a transmembrane signaling function of this molecule during leukocyte-endothelial interactions.     

Purification of a protein phosphatase from chloroplast stroma capable of dephosphorylating the light-harvesting complex-II

A protein phosphatase was purified from the stroma of Pea (Pisum sativum L.) chloroplasts that is capable of dephosphorylating synthetic phosphopeptides. Following chromatographic purification of greater than 400-fold, two-dimensional electrophoresis indicated that the stromal protein phosphatase is a 29-kD protein. A similar molecular size was determined for the protein-phosphatase activity using gel-permeation chromatography, indicating that the stromal protein phosphatase is probably a monomer. The purified enzyme was able to dephosphorylate synthetic phosphopeptides, which mimic the thylakoid light-harvesting complex II (LHC-II) N terminus, as well as LHC-II in thylakoid membranes, but did not dephosphorylate the major 64-kD phosphoprotein in the stroma. The stromal protein phosphatase did not discriminate between dephosphorylation of phosphothreonine and phosphoserine residues in synthetic peptide substrates, providing further evidence that this enzyme is distinct from the protein phosphatase localized in thylakoid membranes. The exact physiological role of the stromal protein phosphatase has yet to be determined, but it may function in the dephosphorylation of LHC-II.     

HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172

Calmodulin-dependent protein kinase II (Cam kinase II) is known to desensitise epidermal growth factor receptor (HER-1) tyrosine kinase activity by a process involving phosphorylation at serines 1046/47 in the cytoplasmic tail. We have developed an experimental system to investigate phosphorylation of the related HER-2/c-erbB2 proto-oncogene utilising purified Cam kinase II and recombinant glutathione-S-transferase (GST) fusion proteins. The cDNA for rat Cam kinase II-alpha was transfected into human embryonic kidney (HEK) 293 fibroblasts and the expressed protein purified to homogeneity by calmodulin-agarose affinity chromatography. A GST fusion protein comprising residues 1126-1255 of HER-2 was phosphorylated by purified Cam kinase II, in contrast to a GST protein comprising residues 1005-1125. Phosphoamino-acid analysis and site-directed mutagenesis indicated that HER-2 was phosphorylated on a single site at threonine-1172 which resides within a consensus Cam kinase II phosphorylation site (RAKT). HER-2 (threonine-1172-alanine), in the form of a ligand-inducible chimaera HER-1/2, was co-transfected into HEK-293 fibroblasts with a constitutively active form of Cam kinase II, followed by in vivo labelling of these cells with 32 P-orthophosphate. Immunoprecipitation of ligand-activated receptors followed by two-dimensional phosphopeptide mapping indicated that threonine-1172 in HER-2 is a newly identified in vivo site which can be hyper-phosphorylated by constitutively active Cam kinase II. In addition, when over-expressed in HEK-293 fibroblasts, HER-1/2 (threonine-1172-alanine) showed a defect in desensitisation and underwent a more sustained EGF-induced receptor autophosphorylation compared to wild-type HER-1/2.