Insulin-like growth factor binding protein 2 is a growth inhibitory protein conserved in zebrafish

Fish serum contains several specific binding proteins for insulin-like growth factors (IGFBPs). The structure and physiological function of these fish IGFBPs are unknown. Here we report the complete primary sequence of a zebrafish IGFBP deduced from cDNA clones isolated by library screening and rapid amplification of cDNA ends. The full-length 1,757-bp cDNA encodes a protein of 276 aa, which contains a putative 22-residue signal peptide and a 254-residue mature protein. The mature zebrafish IGFBP has a predicted molecular size of 28,440 Da and shows high sequence identity with human IGFBP-2 (52%). The sequence identities with other human IGFBPs are <37%. Chinese hamster ovary cells stably transfected with the zebrafish IGFBP-2 cDNA secreted a 31-kDa protein, which bound to IGF-I and IGF-II with high affinity, but did not bind to Des(1–3)IGF-I or insulin. Northern blot analyses revealed that the zebrafish IGFBP-2 transcript is a 1.8-kb band expressed in many embryonic and adult tissues. In adult zebrafish, IGFBP-2 mRNA levels were greatly reduced by growth hormone treatment but increased by prolonged fasting. When overexpressed or added to cultured zebrafish and mammalian cells, the zebrafish IGFBP-2 significantly inhibited IGF-I-stimulated cell proliferation and DNA synthesis. These results indicate that zebrafish IGFBP-2 is a negative growth regulator acting downstream in the growth hormone-IGF-I axis.

Crystal structure of the ribosomal RNA domain essential for binding elongation factors

The structure of a 29-nucleotide RNA containing the sarcin/ricin loop (SRL) of rat 28 S rRNA has been determined at 2.1 Å resolution. Recognition of the SRL by elongation factors and by the ribotoxins, sarcin and ricin, requires a nearly universal dodecamer sequence that folds into a G-bulged cross-strand A stack and a GAGA tetraloop. The juxtaposition of these two motifs forms a distorted hairpin structure that allows direct recognition of bases in both grooves as well as recognition of nonhelical backbone geometry and two 5′-unstacked purines. Comparisons with other RNA crystal structures establish the cross-strand A stack and the GNRA tetraloop as defined and modular RNA structural elements. The conserved region at the top is connected to the base of the domain by a region presumed to be flexible because of the sparsity of stabilizing contacts. Although the conformation of the SRL RNA previously determined by NMR spectroscopy is similar to the structure determined by x-ray crystallography, significant differences are observed in the “flexible” region and to a lesser extent in the G-bulged cross-strand A stack.

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, ≈240 Å channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of ≈50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA.

The ribosome-in-pieces: Binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA

An oligoribonucleotide (a 27-mer) that mimics the sarcin/ricin (S/R) domain of Escherichia coli 23S rRNA binds elongation factor EF-G; the Kd is 6.9 μM, whereas for binding to ribosomes it is 0.7 μM. Binding saturates when EF-G and the S/R RNA are equimolar; at saturation 70% of the input RNA is in complexes with EF-G. Binding of EF-G to S/R RNA does not require GTP but is inhibited by GDP; the inhibition by GDP is overcome by GTP. The effects of mutations of the S/R domain nucleotides G2655, A2660, and G2661 suggest that EF-G recognizes the conformation of the RNA rather than the identity of the nucleotides. EF-G also binds to an oligoribonucleotide (an 84-mer) that has the thiostrepton region of 23S rRNA; however, EF-G binds independently to S/R and thiostrepton oligoribonucleotides.

Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: Regulation of HSP90-binding activity of FKBP52

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.

p53CP, a putative p53 competing protein that specifically binds to the consensus p53 DNA binding sites: A third member of the p53 family?

p53 tumor suppressor protein negatively regulates cell growth, mainly through the transactivation of its downstream target genes. As a sequence-specific DNA binding transcription factor, p53 specifically binds to a 20-bp consensus motif 5′-PuPuPuC(A/T) (T/A)GPyPyPyPuPuPuC(A/T)(T/A)GPyPyPy-3′. We have now identified, partially purified, and characterized an additional ≈40-kDa nuclear protein, p53CP (p53 competing protein), that specifically binds to the consensus p53 binding sites found in several p53 downstream target genes, including Waf-1, Gadd45, Mdm2, Bax, and RGC. The minimal sequence requirement for binding is a 14-bp motif, 5′-CTTGCTTGAACAGG-3′ [5′-C(A/T)(T/A)GPyPyPyPuPuPuC(A/T)(T/A)G-3′], which includes the central nucleotides of the typical p53 binding site with one mismatch. p53CP and p53 (complexed with antibody) showed a similar binding specificity to Waf-1 site but differences in Gadd45 and T3SF binding. Like p53, p53CP also binds both double- and single-stranded DNA oligonucleotides. Important to note, cell cycle blockers and DNA damaging reagents, which induce p53 binding activity, were found to inhibit p53CP binding in p53-positive, but not in p53-negative, cells. This finding suggested a p53-dependent coordinate regulation of p53 and p53CP in response to external stimuli. p53CP therefore could be a third member of the p53 family, in addition to p53 and p73, a newly identified p53 homolog. p53CP, if sequestering p53 from its DNA binding sites through competitive binding, may provide a novel mechanism of p53 inactivation. Alternatively, p53CP may have p53-like functions by binding and transactivating p53 downstream target genes. Cloning of the p53CP gene ultimately will resolve this issue.

Syndapin I, a Synaptic Dynamin-binding Protein that Associates with the Neural Wiskott-Aldrich Syndrome Protein

The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein–protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal.

Mutations at Phosphorylation Sites of Xenopus Microtubule-associated Protein 4 Affect Its Microtubule-binding Ability and Chromosome Movement during Mitosis

Microtubule-associated proteins (MAPs) bind to and stabilize microtubules (MTs) both in vitro and in vivo and are thought to regulate MT dynamics during the cell cycle. It is known that p220, a major MAP of Xenopus, is phosphorylated by p34cdc2 kinase as well as MAP kinase in mitotic cells, and that the phosphorylated p220 loses its MT-binding and -stabilizing abilities in vitro. We cloned a full-length cDNA encoding p220, which identified p220 as a Xenopus homologue of MAP4 (XMAP4). To examine the physiological relevance of XMAP4 phosphorylation in vivo, Xenopus A6 cells were transfected with cDNAs encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein. Mutations of serine and threonine residues at p34cdc2 kinase-specific phosphorylation sites to alanine interfered with mitosis-associated reduction in MT affinity of XMAP4, and their overexpression affected chromosome movement during anaphase A. These findings indicated that phosphorylation of XMAP4 (probably by p34cdc2 kinase) is responsible for the decrease in its MT-binding and -stabilizing abilities during mitosis, which are important for chromosome movement during anaphase A.

Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link between GLUT1 and the Cytoskeleton

Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin–Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, α-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.

Different Domains of the M-Band Protein Myomesin Are Involved in Myosin Binding and M-Band TargetingV⃞

Myomesin is a 185-kDa protein located in the M-band of striated muscle where it interacts with myosin and titin, possibly connecting thick filaments with the third filament system. By using expression of epitope-tagged myomesin fragments in cultured cardiomyocytes and biochemical binding assays, we could demonstrate that the M-band targeting activity and the myosin-binding site are located in different domains of the molecule. An N-terminal immunoglobulin-like domain is sufficient for targeting to the M-band, but solid-phase overlay assays between individual N-terminal domains and the thick filament protein myosin revealed that the unique head domain contains the myosin-binding site. When expressed in cardiomyocytes, the head domains of rat and chicken myomesin showed species-specific differences in their incorporation pattern. The head domain of rat myomesin localized to a central area within the A-band, whereas the head domain of chicken myomesin was diffusely distributed in the cytoplasm. We therefore conclude that the head domain of myomesin binds to myosin but that this affinity is not sufficient for the restriction of the domain to the M-band in vivo. Instead, the neighboring immunoglobulin-like domain is essential for the precise incorporation of myomesin into the M-band, possibly because of interaction with a yet unknown protein of the sarcomere.

Mapping of a Myosin-binding Domain and a Regulatory Phosphorylation Site in M-Protein, a Structural Protein of the Sarcomeric M Band

The myofibrils of cross-striated muscle fibers contain in their M bands cytoskeletal proteins whose main function seems to be the stabilization of the three-dimensional arrangement of thick filaments. We identified two immunoglobin domains (Mp2–Mp3) of M-protein as a site binding to the central region of light meromyosin. This binding is regulated in vitro by phosphorylation of a single serine residue (Ser76) in the immediately adjacent amino-terminal domain Mp1. M-protein phosphorylation by cAMP-dependent kinase A inhibits binding to myosin LMM. Transient transfection studies of cultured cells revealed that the myosin-binding site seems involved in the targeting of M-protein to its location in the myofibril. Using the same method, a second myofibril-binding site was uncovered in domains Mp9–Mp13. These results support the view that specific phosphorylation events could be also important for the control of sarcomeric M band formation and remodeling.

ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

Effects of Age on the Posttranscriptional Regulation of CCAAT/Enhancer Binding Protein α and CCAAT/Enhancer Binding Protein β Isoform Synthesis in Control and LPS-Treated Livers

The CCAAT/enhancer binding protein α (C/EBPα) and CCAAT/enhancer binding protein β (C/EBPβ) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPαs with molecular masses of 42, 38, 30, and 20 kDa and C/EBPβs of 35, 20, and ∼8.5 kDa. The DNA-binding activities and pool levels of p42C/EBPα and p30C/EBPα in control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPα and C/EBPβ isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42C/EBPα levels and binding activity, whereas those of p20C/EBPα and p20C/EBPβ are increased. However, translation of 42-kDa C/EBPα is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPα- and C/EBPβ-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPα and C/EBPβ isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.

The Small GTP-binding Protein R-Ras Can Influence Integrin Activation by Antagonizing a Ras/Raf-initiated Integrin Suppression Pathway

The rapid modulation of ligand-binding affinity (“activation”) is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.

The Phosphatidylinositol 3-Phosphate Binding Protein Vac1p Interacts with a Rab GTPase and a Sec1p Homologue to Facilitate Vesicle-mediated Vacuolar Protein Sorting

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein–protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.