Phosphorylation of nucleolin by a nucleolar type NII protein kinase

Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.     

Domain structure of the HSC70 cochaperone, HIP.

The domain structure of the HSC70-interacting protein (HIP), a 43-kDa cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor, has been probed by limited proteolysis and biophysical and biochemical approaches. HIP proteolysis by thrombin and chymotrypsin generates essentially two fragments, an NH2-terminal fragment of 25 kDa (N25) and a COOH-terminal fragment of 18 kDa (C18) that appear to be well folded and stable as indicated by circular dichroism and recombinant expression in Escherichia coli. NH2-terminal amino acid sequencing of the respective fragments indicates that both proteases cleave HIP within a predicted alpha-helix following the tetratricopeptide repeat (TPR) region, despite their different specificities and the presence of several potential cleavage sites scattered throughout the sequence, thus suggesting that this region is particularly accessible and may constitute a linker between two structural domains. After size exclusion chromatography, N25 and C18 elute as two distinct and homogeneous species having a Stokes radius of 49 and 24 A, respectively. Equilibrium sedimentation and sedimentation velocity indicate that N25 is a stable dimer, whereas C18 is monomeric in solution, with sedimentation coefficients of 3.2 and 2.3 S and f/f(o) values of 1.5 and 1.1 for N25 and C18, respectively, indicating that the N25 is elongated whereas C18 is globular in shape. Both domains are able to bind to the ATPase domain of HSC70 and inhibit rhodanese aggregation. Moreover, their effects appear to be additive when used in combination, suggesting a cooperation of these domains in the full-length protein not only for HSC70 binding but also for chaperone activity. Altogether, these results indicate that HIP is made of two structural and functional domains, an NH2-terminal 25-kDa domain, responsible for the dimerization and the overall asymmetry of the molecule, and a COOH-terminal 18-kDa globular domain, both involved in HSC70 and unfolded protein binding.     

Direct interaction between nucleolin and hepatitis C virus NS5B

Hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme in HCV replication. While studying the subcellular localization of a NS5B mutant lacking the C-terminal membrane-anchoring domain, NS5Bt, we found that expression of the green fluorescent protein (GFP)-fused form was exclusively nucleolar. Interestingly, the distribution of endogenous nucleolin changed greatly in the cells expressing GFP-NS5B, with nucleolin colocalized with GFP-NS5B in perinuclear regions in addition to the nucleolus, suggesting that NS5B retains the ability to bind nucleolin. The interaction between nucleolin and NS5B was demonstrated by GST pull-down assay. GST pull-down assay results indicated that C-terminal region of nucleolin was important for its binding to NS5B. Scanning clustered alanine substitution mutants library of NS5B revealed two sites on NS5B that binds nucleolin. NS5B amino acids 208-214 and 500-506 were both found to be indispensable for the nucleolin binding. We reported that the latter sequence is essential for oligomerization of NS5B, which is a prerequisite for the RdRP activity. C-terminal nucleolin inhibited the NS5B RdRP activity in a dose-dependent manner. Taken together, this indicates the binding ability of nucleolin may be involved in NS5B functions.     

Limited proteolysis as a probe of the conformation and nucleic acid binding regions of nucleolin.

Nucleolin, also called protein C23, is a RNA-associated protein implicated in the early stages of ribosome assembly. To study the general conformation and map the nucleic acid binding regions, rat nucleolin was subjected to limited proteolysis using trypsin and chymotrypsin in the presence or absence of poly(G). The cleavage sites were classified according to their locations in the three putative domains: the highly polar amino-terminal domain, the central nucleic acid binding domain, which contains four 90-residue repeats, and the carboxyl-terminal domain, which is rich is glycine, dimethylarginine, and phenylalanine. The most labile sites were found in basic segments of the amino-terminal domain. This region was stabilized by Mg2+. At low enzyme concentrations, cleavage by trypsin or chymotrypsin in the amino-terminal domain was enhanced by poly(G). Trypsin produced a relatively stable 48-kDa fragment containing the central and carboxyl-terminal domains. The enhanced cleavage suggests that binding of nucleic acid by the central domain alters the conformation of the amino-terminal domain, exposing sites to proteolytic cleavage. At moderate enzyme concentrations, the 48-kDa fragment was protected by poly(G) against tryptic digestion. At the highest enzyme concentrations, both enzymes cleaved near the boundaries between repeats 2, 3, and 4 with some sites protected by poly(G), suggesting that the repeats themselves form compact units. The carboxyl-terminal domain was resistant to trypsin but was cleaved by chymotrypsin either in the presence or in the absence of poly(G), indicating exposure of some phenylalanines in this region. These studies provide a general picture of the topology of nucleolin and suggest that the nucleic acid binding region communicates with the amino-terminal domain.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

Concerted activities of the RNA recognition and the glycine-rich C-terminal domains of nucleolin are required for efficient complex formation with pre-ribosomal RNA.

Nucleolin is an abundant nucleolar protein which is involved in the early stages of ribosome assembly. The central 40-kDa domain of nucleolin comprises four RNA recognition motifs (RRM) which are presumed to be involved in specific interactions with pre-rRNA. In order to examine in detail the role of this central domain and the contribution of the N-terminal and C-terminal domains of nucleolin to RNA binding, we have used an Escherichia coli expression system to synthezise polypeptides corresponding to various combinations of the three domains and their subdomains. By means of an in-vitro binding assay and a synthetic RNA corresponding to a specific recognition site in pre-rRNA we have been able to demonstrate conclusively that the central 40-kDa domain is indeed responsible for the specificity of RNA recognition and that the N-terminal domain can be removed without affecting RNA binding. Most interestingly, it appears that the C-terminal 10-kDa domain, which is rich in glycine and arginine residues, is essential for efficient binding of nucleolin to RNA, but does not itself contribute to the specificity of the interaction. Circular dichroic spectroscopic probing of the RNA component shows that the C-terminal domain significantly modifies the RNA-binding properties of the central RRM core. Finally, infrared spectroscopic studies reveal that the central 40-kDa domain is structured in alpha helices and beta sheets and that the interaction with the specific pre-rRNA site induces subtle changes in the beta sheet conformation.     

The Murine Nucleolin Protein Is an Inducible DNA and ATP Binding Protein Which Is Readily Detected in Nuclear Extracts of Lipopolysaccharide-Treated Splenocytes

A 100-kDa DNA binding protein was found to be dramatically up-regulated upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also found to exhibit moderate binding specificity for the immunoglobulin isotype switch DNA repeats. Furthermore, the induction of the 100-kDa protein by LPS was found to be mediated by both an increase in the protein's stability and an increase in the synthesis of the protein. In vitro phosphorylation experiments revealed that the 100-kDa DNA binding protein was one of the most heavily phosphorylated proteins in both lymphoid and nonlymphoid nuclear extracts. Although this in vitro phosphorylation initially appeared to be mediated by a potent nuclear kinase activity, it was later determined that a significant part of the detected labeling was due to the direct binding of ATP by the 100-kDa protein. Antibodies raised to the 100-kDa DNA binding protein were used to isolate cDNA clones from a lymphocyte cDNA λgt11 expression library. Nucleotide sequence analysis revealed that the cloned cDNAs were identical to the mouse nucleolin gene. The β-galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and a more severely truncated 45-kDa protein (encoded by exons 5-14 of nucleolin) were both found to bind strongly to DNA and ATP. Furthermore, the strength of DNA binding was found to be highly dependent on the overall dG content of the DNA probes. Our experiments also revealed that apart from binding ATP and G-rich DNA, nucleolin directly bound GTP, dATP, and dGTP, but not dCTP, dTTP, or dUTP. Computer analysis revealed that the putative ATP binding domains appear to fall within two of the phylogenetically conserved RNA binding domains of nucleolin.     

Structure of the mouse nucleolin gene. The complete sequence reveals that each RNA binding domain is encoded by two independent exons

Nucleolin is a multifunctional nucleolar protein involved in the synthesis, packaging and maturation of pre-rRNA in eukaryotic cells. We describe the molecular organization and complete sequence of the mouse nucleolin gene, the first higher eukaryotic gene encoding a protein that is both an RNA binding protein involved in rRNA processing and a specific nucleolar protein. The nucleolin gene extends over 9000 base-pairs and is split into 14 exons that encode the 706 amino acid residues of the protein. The promoter sequence is G + C-rich (67% G + C) with four G/C boxes, it lacks bona fide TATA and CAAT boxes and shows capping site heterogeneity. The existence of pyrimidine-rich motifs, similar to those found in the promoter of ribosomal protein genes, could be relevant to the co-regulation of genes whose products are involved in ribosome biogenesis. Nucleolin contains four RNA binding domains, each about 80 amino acid residues long, which include the 11-residue core ribonucleoprotein consensus motif. Each domain is encoded by two exons, with an intervening sequence interrupting the conserved core motif at roughly the same amino acid position. This latter result suggests that the RNA binding domains are composed of two independent subdomains, whose functions remain to be determined.     

Identification of a 110-kDa nonintegrin cell surface laminin-binding protein which recognizes an A chain neurite-promoting peptide.

Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identified a 110-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the 110-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact sequence identity with nucleolin, a major 110-kDa nucleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-peptide-binding 110-kDa protein. The 110-kDa protein appears to be a ligand for a specific site on laminin.     

A protein partially expressed on the surface of HepG2 cells that binds lipoproteins specifically is nucleolin

Nucleolin, a major nucleolar protein of rapidly growing eukaryotic cells, has been thought to be predominantly if not exclusively located in the nucleolus. Recent data however [Borer, R.A., Lehner, C.F., Eppenberger, H.M., & Nigg, N.A. (1989) Cell 56, 379-390] suggest that the protein shuttles constantly between the nucleus and cytoplasm. Ligand blotting studies of whole cell extracts of HepG2 cells identified, in addition to the LDL receptor, another LDL binding protein of Mr 109,000. The 109-kDa protein was partially purified by HPLC and, like the LDL receptor, bound apoB- and apoE-containing lipoproteins but not HDL. However, unlike the LDL receptor, the 109-kDa protein bound lipoproteins in the presence of EDTA and reducing agents, had a lower affinity for lipoproteins than the LDL receptor, and did not react with two antibodies raised against the LDL receptor. The protein sequences of three separate peptides derived from the partially purified 109-kDa species were determined and were identical except for one residue to three separate regions of the published sequence of nucleolin. On immunoblot analysis the 109-kDa protein reacted with a nucleolin-specific antibody, and purified nucleolin reacted both with anti-109-kDa antibody and with LDL. When intact HepG2 cells were treated with Pronase before harvest, there was a 46% decrease in 109-kDa protein while recovery of actin, an intracellular protein, was unaffected. When intact HepG2 cells were surface iodinated and the proteins subjected to HPLC fractionation, the 109-kDa protein was found to be iodinated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actin and tubulin binding domains of synapsins Ia and Ib

Synapsins Ia and Ib are neuronal phosphoproteins involved with the regulated clustering of small synaptic vesicles at the presynaptic terminus. In vitro they bind and bundle filaments of both actin and tubulin. Previously, we identified an actin binding domain in the NH2-terminal 25-kDa fragment (N25) generated by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage of synapsin I and found that a complementary COOH-terminal 52-kDa portion of the molecule (N52) contained either a second actin binding site or a site of self-association [Petrucci, T. P., & Morrow, J. S. (1987) J. Cell. Biol. 105, 1355]. Using direct binding assays between actin, tubulin, and specific synapsin NTCB-derived peptides, we confirm the ability of purified N25 to bind but not bundle actin and demonstrate that the complementary N52 (or N50) fragments from synapsins Ia and Ib and a 14-kDa fragment derived from the middle of the molecule also associate directly with actin. An antibody specific for N25 inhibits the actin binding activity of N25 and the actin bundling but not the actin binding activity of intact synapsin I. Similar studies conducted with purified tubulin and tubulin immobilized on Sepharose demonstrate that both tubulin and actin bind at approximately the same sites in the NH2-terminal half of synapsin I. Although the fragments derived from the COOH terminus of both synapsin Ia and synapsin Ib (N40b/N34) were devoid of measurable actin binding activity after NTCB cleavage, they were specifically labeled in the intact molecule by a photoactivated cross-linker bound to F-actin. Collectively, these results indicate that synapsins Ia and Ib possess two actin and tubulin binding domains located in the NH2-terminal half of the molecule and suggest that a third actin binding domain is located in the COOH-terminal region. The NH2-terminal sites are found in NTCB peptides N25 and N14, while the third site, apparently of lower affinity, resides in N40b/N34. It is hypothesized that, in the intact molecule, the two NH2-terminal domains contribute to a single high-affinity actin and/or tubulin binding site in the "globular" head region of synapsin I, while the third actin binding domain constitutes the topographically distinct site required for the actin bundling activity of the native molecule. The 45-residue COOH extension that distinguishes synapsin Ia from synapsin Ib appears not to be involved with actin binding, since no differences were found in the ability of N40b and N34 to be photo-cross-linked to actin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein and cDNA sequence of a glycine-rich, dimethylarginine-containing region located near the carboxyl-terminal end of nucleolin (C23 and 100 kDa)

By a combination of protein chemistry and recombinant DNA methods a glycine-rich region was found to be located near the carboxyl terminus of the nucleolar specific phosphoprotein, nucleolin, from Novikoff hepatoma (protein C23) and Chinese hamster ovary cells (100-kDa nucleolar protein). A sequence of 192 amino acid residues was derived from partial sequences of cyanogen bromide and N-bromosuccinimide fragments of protein C23 and deduced protein sequence from Chinese hamster ovary cell 100-kDa cDNA sequences. The 66 residues sequenced by protein methods were identical to the corresponding residues deduced by DNA sequencing. The multiple residues of NG,NG-dimethylarginine (DMA) contained in the nucleolin polypeptide were found to be limited to a segment of less than 10 kDa near the carboxyl-terminal end of the protein. This segment also contained internally repeated sequences (e.g. 7 copies of the sequence Gly-Gly-Arg-Gly-Gly were found) which were unrelated to sequences closer to the amino-terminal end. Most arginine residues in this region were surrounded by 2 or 3 glycine residues and were relatively close in sequence to phenylalanine residues.     

Isolation and molecular and functional properties of the amino-terminal domain of colicin A

A plasmid was constructed which allowed easy and efficient production and purification of the NH2-terminal domain of colicin A. In only three steps, an homogenous 18-kDa polypeptide was obtained. The NH2- and COOH-terminal sequences of the protein were determined and showed that it corresponded to the NH2-terminal 171 amino acid residues of the 63-kDa colicin A. Although colicin A is a highly asymmetric protein, hydrodynamic studies indicated that the NH2-terminal domain (designated AT) has a globular structure. This fragment is not the receptor-binding domain of colicin A but is required for the transfer of colicin A across the outer membrane of sensitive cells. However, it has a low affinity for phospholipid films and this affinity is not pH-dependent, in contrast to that of colicin A.     

Domain structure of human 72-kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions.

The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play a role in metastasis and in angiogenesis, forms a noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of enzyme activity. To define the regions of the 72-kDa gelatinase responsible for TIMP-2 binding, a series of NH2- and COOH-terminal deletions of the enzyme were constructed using the polymerase chain reaction technique. The full-length and the truncated enzymes were expressed in a recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two truncated enzymes ending at residues 425 (delta 426-631) and 454 (delta 455-631) were purified. Like the full-length recombinant 72-kDa gelatinase, both COOH-terminally truncated enzymes were activated with organomercurial and digested gelatin and native collagen type IV. In contrast to the full-length enzyme, delta 426-631 and delta 455-631 enzymes were less sensitive to TIMP-2 inhibition requiring 10 mol of TIMP-2/mol of enzyme to achieve maximal inhibition of enzymatic activity. The activated but not the latent forms of the delta 426-631 and delta 455-631 proteins formed a complex with TIMP-2 only when excess molar concentrations of inhibitor were used. We also expressed the 205-amino acid COOH-terminal fragment, delta 1-426, and found that it binds TIMP-2. In addition, a truncated version of the 72-kDa gelatinase lacking the NH2-terminal 78 amino acids (delta 1-78) of the proenzyme retained the ability to bind TIMP-2. These studies demonstrate that 72-kDa gelatinases lacking the COOH-terminal domain retain full enzymatic activity but acquire a reduced sensitivity to TIMP-2 inhibition. These data suggest that both the active site and the COOH-terminal tail of the 72-kDa gelatinase independently and cooperatively participate in TIMP-2 binding.