Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase

As reported previously [Vulliet et al. (1985) FEBS Lett. 182 335-339], tyrosine hydroxylase purified from rat pheochromocytoma is phosphorylated at an identical site (site A) by cyclic AMP-dependent protein kinase, the calmodulin-dependent multiprotein kinase and protein kinase C, while the calmodulin-dependent multiprotein kinase also phosphorylates another unique site (site C). Preparations of tyrosine hydroxylase purified from this source are also contaminated with traces of a fourth protein kinase which phosphorylates another unique site (site E). We have isolated tryptic peptides containing each of these sites and determined their amino acid sequences. By comparison of these data with the known cDNA sequence for rat tyrosine hydroxylase, we have been able to identify these sites as Ser-8 (site E), Ser-19 (site C), and Ser-40 (site A). In some preparations of tyrosine hydroxlyase, cyclic AMP-dependent protein kinase also phosphorylated a secondary site which was identified as ser-153. All of these phosphorylation sites are in the amino-terminal region, where there is no significant homology with the closely related enzyme, phenylalanine hydroxylase. Our data also establish that the initiator methionine is removed by post-translational processing to leave pro-2 as the amino-terminus of the mature protein. The significance of these results for the mechanism of action of extracellular signals on catecholamine biosynthesis is discussed.     

Identification of the major sites of phosphorylation in IGF binding protein-3

Insulin-like growth factor binding protein-3 (IGFBP-3) is the major carrier of insulin-like growth dactor I and II in the circulation. IGFBP-3 is secreted by various tissues and cell lines as a glycosylated phosphoprotein. We have identified two major serine phosphorylation sites located at amino acids 111 and 113 of the human protein. These serine residues ngighboring amino acids potentially involved in defining a protrein kinase recognition sequence were mutated to alanine using PCR. Single and double point mutants were stably transfected into CHO-cells and analyzed for their level of phosphorylation. Mutation of both serines reduced phosphorylation by 80% in the full-length protein and completely abolished phosphorylation in a 17 kDa IGFBP-3 fragment, derived from digestion with EndoProteinase Lys-C. The 17kDa fragment containd serines 111 and 113. S111A/S113A, a double serine-to-alanine mutant at positions 111 and 113, showed a strongly reduced glycosylation pattern that appears to be the result of anino acid substitutions rather tha n lack of phosphrylation. Mutant S111A/S113A, despite being non-phosphorylated and non-glycosylated, is functionally similar to the wild-type IGFBP-3 in terms of IGF-1 binding. These results enhance our non-glycosylated, is functionally similar to the wild-type IGFBP-3 in terms of IGF-I binding. These results enhance our understanding on the functional role of glycosylation and phosphorylation of IGFBP-3.     

Identification of multiple in vivo phosphorylation sites in rabbit myelin basic protein

Myelin basic protein of rabbit brain (Mr = 18,200) was initially freed of the bulk of the nonphosphorylated species (mainly component 1) by Cm-cellulose chromatography at high pH. The remainder of the protein was subjected to peptic digestion at pH 6.00, which resulted in specific, essentially complete cleavage at several bonds (Phe-44--Phe-45, Phe-87--Phe-88, Leu-109--Ser-110, and Leu-151--Phe-152) and partial cleavage at the Tyr-14--Leu-15 bond. Gel filtration of the digest through Sephadex G-25 (fine) yielded three fractions, the first containing primarily peptides 1-44 and 45-87, the second peptides 15-44, 88-109, and 110-151, and the third peptides 1-14 and 152-168. Each fraction was chromatographed on Cm-cellulose at pH 8.2, and the resulting subfractions and partially purified peptides were analyzed for phosphoserine and phosphothreonine. Materials containing significant amounts of the phosphoamino acids were subsequently chromatographed on Cm-cellulose at pH 4.65, and the analyses for phosphoserine and phosphothreonine were repeated. The resulting purified peptic phosphopeptides were identified by amino acid analysis and tryptic peptide mapping. Comparison of the maps with those of the unphosphorylated counterparts located the tryptic phosphopeptides. These were recovered and their identities were established by amino acid analysis. In those cases where the phosphopeptide contained 2 Ser residues, the position of the phosphoserine was established by aminopeptidase M digestion. Five phosphorylation sites were found: Ser-7, Ser-56, Thr-96, Ser-113, and Ser-163. Only a small fraction of these sites was phosphorylated in the total basic protein, with values ranging from about 2 (ser-113) to 6% (Thr-96). With the possible exception of Ser-56, these sites are not the ones that have been reported to be phosphorylated in vitro by cyclic AMP-dependent protein kinase.     

Identification of protein binding sites in genomic DNA by two-dimensional gel electrophoresis.

We describe a simple two-dimensional electrophoresis procedure to identify the recognition sites of DNA-binding proteins within large DNA molecules. Using this approach, we have mapped E. coli IHF (Integration Host Factor) binding sites within phage Lambda (48 kb) and phage Mu (39 kb) DNA. We are also able to visualize IHF binding sites in E. coli chromosomal DNA (4,700 kb). We present an extension of this technique using direct amplification by PCR of the isolated restriction fragments, which should permit the cloning of a collection of recognition sequences for DNA binding proteins in complex genomes.     

DNA binding specificity and transactivation properties of SREBP-2 bound to multiple sites on the human apoA-II promoter.

DNase I footprinting of the apoA-II promoter using sterol regulatory element binding protein-2 [(SREBP-2 (1-458)] expressed in bacteria identified four protected regions, designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332) and AIIK (-760 to -743), which bind SREBP-2 and contain either palindromic or direct repeat motifs. Potassium permanganate and dimethyl sulfate interference experiments using the AIIAB region as probe showed that the nucleotides of a decameric palindromic repeat RTCAMVTGMY and two 5' T residues participate in DNA-protein interactions. SREBP-2 transactivated the intact (-911/+29) apoA-II promoter 1.7-fold and truncated apoA-II promoter segments which contain one, two or three SREBP-2 sites 11- to 17-fold in HepG2 cells. Transactivation of a promoter construct containing the binding site AIIAB and the apoA-II enhancer, which includes the binding site AIIK, was abolished by mutations in element AIIAB. An SREBP-2 mutant defective in DNA binding caused a dose-dependent repression of the apoA-II promoter activity. Repression was also caused by an SREBP-2 mutant which lacks the N-terminal activation domain (residues 1-93) but binds normally to its cognate sites. In contrast, a double SREBP-2 mutant which lacks both the DNA binding and the activation domains has no effect on the apoA-II promoter activity. Overall, the findings suggest that SREBP-2 can transactivate the apoA-II promoter by binding to multiple sites. Furthermore, the repression caused by the DNA binding deficient mutants results from squelching of positive activator(s) which appear to recognize the activation domain of SREBP-2.     

DNA binding properties of the nuclear matrix and individual nuclear matrix proteins. Evidence for salt-resistant DNA binding sites

The DNA binding characteristics of the rat nuclear matrix were investigated. A saturable and temperature-dependent, salt-resistant DNA binding to the nuclear matrix was discovered, with 70-80% of total bound DNA resistant to extraction with high concentrations of salt at 37 degrees C, compared to less than 5% at 0 degrees C. The initial binding of DNA to nuclear matrix is sensitive to salt concentration, indicating a transition to a salt-resistant binding state. The nuclear matrix shows a preference for single-stranded DNA, both in saturation and competition assays, with little binding of RNA or double-stranded DNA. Further competition studies show a preference for matrix-attached DNA probably involving predominantly AT-rich sequences, while a specific sequence defined previously as a matrix-attached region (MAR; Cockerill, P. N., and Garrard, W. T. (1986) Cell 46, 273-282) only showed preference for a limited number of the total matrix binding sites. These results and estimates from saturation data of approximately 150,000 single-stranded DNA binding sites per matrix lead us to propose that the nuclear matrix contains different classes of DNA binding sites, each with a separate sequence specificity. Binding of DNA to individual matrix polypeptides separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose blots was also temperature-dependent, salt-resistant, and showed a preference for binding DNA over RNA and nuclear matrix DNA over total genomic DNA. Subnuclear fractionation experiments further demonstrated that the nuclear matrix is enriched in the subset of higher molecular weight (greater than 50,000) DNA binding proteins of isolated nuclei and correspondingly depleted of the lower molecular weight ones. Of the approximately 12 major proteins separated on nonequilibrium two-dimensional gels, 7 were identified as specific DNA binding proteins including lamins A and C (but not B), and the internal nuclear matrix proteins, matrins D, E, F, G, and 4.     

DNA recognition properties of the N-terminal DNA binding domain within the large subunit of replication factor C.

Replication Factor C (RFC) is a five-subunit protein complex required for eukaryotic DNA replication and repair. The large subunit within this complex contains a C-terminal DNA binding domain which provides specificity for PCNA loading at a primer-template and a second, N-terminal DNA binding domain of unknown function. We isolated the N-terminal DNA binding domain from Drosophila melanogaster and defined the region within this polypeptide required for DNA binding. The DNA determinants most efficiently recognized by both the Drosophila minimal DNA binding domain and the N-terminal half of the human large subunit consist of a double-stranded DNA containing a recessed 5' phosphate. DNA containing a recessed 5' phosphate was preferred 5-fold over hairpined DNA containing a recessed 3' hydroxyl. Combined with existing data, these DNA binding properties suggest a role for the N-terminal DNA binding domain in the recognition of phosphorylated DNA ends.     

Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites

Cyclic AMP-dependent protein kinase catalyzes the incorporation of 3-4 mol of phosphate into the alpha subunit of rat brain sodium channels in vitro or in situ. Digestion of phosphorylated sodium channels with CNBr yielded three major phosphorylated fragments of 25, 31, and 33 kDa. These fragments were specifically immunoprecipitated with site-directed antisera establishing their location within an intracellular loop between the first and second homologous domains containing residues 448 to 630 of sodium channel RI or residues 450-639 of sodium channel RII. Five of the seven major tryptic phosphopeptides generated from intact sodium channel alpha subunits were contained in each of the 25-, 31-, and 33-kDa CNBr fragments, indicating that most cAMP-dependent phosphorylation sites are in this domain. Since CNBr digestion of sodium channels which had been metabolically labeled with 32P in intact neurons yielded the same phosphorylated fragments, the phosphorylated region we have identified is the major location of phosphorylation in situ. Only serine residues were phosphorylated by cAMP-dependent protein kinase in vitro, while approximately 16% of the phosphorylation in intact neurons was on threonine residues that must lie outside the domain we have identified. Since this domain is phosphorylated in intact neurons, our results show that it is located on the intracellular side of the plasma membrane. These results are considered with respect to models for the transmembrane orientation of the alpha subunit.     

Product inhibition studies of yeast phosphoglycerate kinase evaluating properties of multiple substrate binding sites.

Product inhibition studies on yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) have been performed with 1,3-P2-glycerate. The results indicate that: 1. The catalytic reaction can be affected via four substrate binding sites, two for MgATP2- and two for 3-P-glycerate. 2. There is one catalytic centre per enzyme molecule. 3. The catalytic reaction primarily occurs at the 'first' or 'high affinity' MgATP2- and 3-P-glycerate binding sites. The 'second' set of sub-sites for these substrates are located in a region for regulation of the catalytic reaction. 4. The products of the reaction, 1,3-P2-glycerate and ADP, are preferentially bound to the regulatory region. 5. MgATP2- and 1,3-P2-glycerate are able to bind simultaneously to this region. When liganded with MgATP2- the apparent Ki value for 1,3-P2-glycerate increases from 3 microM to 20 microM.     

DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage

Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.     

DNA footprint enhancement using tandem binding sites.

A concatenated DNA fragment containing a five-repeat binding site was used for DNase I footprinting. Under the same conditions, the tandem repeat assay greatly enhanced the DNA footprint as compared with a native DNA sequence with only one binding site. This technique provides an approach for improving poor DNA footprints.     

Identification of three oligosaccharide binding sites in ricin.

The galactoside-binding sites of ricin B chain can be blocked by affinity-directed chemical modification using a reactive ligand derived from asialoglycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is modified to contain a reactive dichlorotriazine moiety. Two separate galactoside-binding sites have been clearly established in the ricin B chain by X-ray crystallography [Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 260-269], and it is necessary to covalently attach two such reactive ligands to the B chain to block its binding to galactoside affinity matrixes. A method was developed using thiol-specific labeling of the ligand combined with subsequent immunoaffinity chromatography which allowed the isolation of ricin B chain peptides covalently linked to the ligand from proteolytic digests of purified blocked ricin. The sites of covalent attachment of the two ligands in blocked ricin were inferred from sequence analysis to be Lys 62 in domain 1 of the B chain and Tyr 148 in domain 2. A minor species of blocked ricin contains a third covalently attached ligand. From the analysis of peptides derived from blocked ricin enriched in this species, it is inferred that Tyr 67 in domain 1 is the specific site on the ricin B chain where a third reactive ligand becomes covalently linked to the protein. These results are interpreted as providing support for the notion that the ricin B chain has three oligosaccharide binding sites.     

Subunit structure and multiple phosphorylation sites of phospholamban

The phosphorylation-induced mobility shift of the high molecular weight form of phospholamban (24,500 daltons) in the cardiac sarcoplasmic reticulum produced on 3',5'-cyclic AMP (cAMP)-dependent phosphorylation with 5 mM ATP was resolved into five clear steps on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and on Ca2+-calmodulin-dependent phosphorylation into ten steps. The mobility shift of the low molecular weight form of phospholamban (less than 14,400 daltons) in these reactions occurred in one step and two steps, respectively. With the two protein kinase activities, the electrophoretic pattern of the mobility shifts of the high and low molecular weight forms of phospholamban was similar to that obtained with Ca2+-calmodulin-dependent protein kinase alone. The results of pulse-chase experiments involving the centrifuge column method suggested that the site(s) of phosphorylation by cAMP- and Ca2+-calmodulin-dependent protein kinase activities are on the same phospholamban molecule. Two-dimensional tryptic peptide maps of phosphorylated phospholamban indicated that cAMP-dependent protein kinase phosphorylates at a single site, A, and Ca2+-calmodulin-dependent protein kinase phosphorylates at sites C1 and C2 in the low molecular weight form, where A is different from C1 but may be the same as C2. The high molecular weight form of phospholamban is suggested to be a pentamer of identical monomers (low molecular weight form) having one phosphorylation site for cAMP-dependent protein kinase and two for Ca2+-calmodulin-dependent protein kinase.     

Identification of phosphorylation sites in native lamina-associated polypeptide 2 beta.

Lamina-associated polypeptide 2 beta (LAP 2 beta), an integral protein of the inner nuclear membrane, appears to be involved in the spatial organization of the interface between nucleoplasma, lamina, and nuclear envelope. Its ability to interact with other proteins and the structural integrity of the nuclear envelope is probably regulated by phosphorylation. Here, we report nonmitotic LAP 2 beta phosphorylation sites that are phosphorylated in the native protein when purified from nuclear envelopes of mouse neuroblastoma Neuro2a cells. Five phosphorylation sites were detected by nano-electrospray mass spectrometric analysis of tryptic LAP 2 beta peptides using parent ion scans specific for phosphopeptides. By mass spectrometric sequencing of these peptides, we identified as phosphorylated residues Thr 74, Thr 159, Ser 176, and Ser 179. Two of the phosphorylation sites, Thr 74 (within a region known to bind chromatin) and Thr 159, are part of consensus sequences of proline-directed kinases. Ser 179 is part of a consensus site for protein kinase C which is able to highly phosphorylate LAP 2 beta in vitro. Three phosphorylation sites, Thr 159, Ser 176, and Ser 179, are located within a stretch of 20 amino acids, thereby forming a highly phosphorylated protein domain which may integrate signaling by multiple protein kinases. Additionally, we identified for the first time at the protein level the LAP 2 splice variant LAP 2 epsilon in nuclear envelopes.     

DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization

Upon DNA damage, p53 has been shown to be modified at a number of N-terminal phosphorylation sites including Ser15 and -33. Here we show that phosphorylation is induced as well at a novel site, Ser20. Phosphorylation at Ser15, -20 and -33 can occur within minutes of DNA damage. Interestingly, while the DNA-binding activities of p53 appear to be dispensable, efficient phosphorylation at these three sites requires the tetramerization domain of p53. Substitution of an artificial tetramerization domain for this region also permits phosphorylation at the N-terminus, suggesting that oligomerization is important for DNA damage-induced signalling to p53.