Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA.

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.

Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase.

The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock. This mammalian enzyme is composed of a catalytic alpha subunit and noncatalytic beta and gamma subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of Saccharomyces cerevisiae. In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the alpha1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. C-terminal truncation of the 548-amino acid (aa) wild-type alpha1 protein to aa 312 or 392 abolishes the binding of the beta/gamma subunits and dramatically increases protein expression. The full-length wild-type alpha1 subunit is only minimally active in the absence of co-expressed beta/gamma, and alpha1(1-392) likewise has little activity. Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392. alpha-1(1-312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP. The increased levels of protein expression on transient transfection of either truncated alpha subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the alpha1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability. A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.

Overexpression, purification, and biophysical characterization of the heterodimerization domain of the core-binding factor beta subunit.

Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBF alpha subunit and a non-DNA binding CBF beta subunit that increases the affinity of CBF alpha for DNA. We have developed a procedure for overexpressing and purifying full-length CBF beta as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBF beta proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBF beta bind to a CBF alpha DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBF beta is a mixed alpha/beta protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy.

Inhibition and substrate competition kinetics in analysis of porcine thyroid alkaline ribonuclease's specificity toward synthetic RNA's and tRNA.

Inhibition and substrate competition kinetics demonstrated that tRNA is a highly preferred substrate of thyroid alkaline RNase. The pyrimidine-specific RNase cleaved poly(C) 2.8 x 10(5) faster than poly(U). kcat:K(M) ratios for tRNA and poly(C) based on molecular weights failed to predict preference when both were present. Competition experiments between poly(C) and tRNA revealed tRNA was a tight-binding competing substrate and the cytidylate residues in the 3'-CCA terminus to tRNA were preferred about 280:1 over those in poly(C). Poly(U) was competitive with tRNA. When poly(C) was the substrate, inhibition type by poly(G) depended on poly(G) concentration. Neither tRNA lacking its 3' terminal cytidylyl(3'-5')adenosine and terminating in a 2':3' cCMP residue, tRNA lacking its 3' terminal 5'AMP residue, guanosine, nor guanylyl(3'-5')guanylyl(3'-5')guanosine were inhibitors. Product inhibition by adenosine and 2':3' cCMP showed the kinetic mechanism for cleavage of tRNA was ordered uni bi.

Biochemical and biophysical properties of the core-binding factor alpha2 (AML1) DNA-binding domain.

The Runt domain is the DNA-binding domain defining a small family of transcription factors that are involved in important developmental processes. Developmental pathways controlled by Runt domain proteins include sex determination, neurogenesis, segmentation, and eye development in Drosophila and hematopoiesis in mammals. In addition to binding DNA, the Runt domain also mediates heterodimerization with another subunit called the core-binding factor beta (CBFbeta) subunit. In this study we overexpress the Runt domain from the mouse CBFalpha2 (AML1) protein in Escherichia coli, and purify it from the insoluble fraction. We determine the equilibrium constants for Runt domain binding to two different DNA sequences by surface plasmon resonance technology. Circular dichroism spectroscopy demonstrates that the Runt domain is a folded beta-domain with essentially no alpha-helical content. The single tryptophan residue in the CBFalpha2 Runt domain at amino acid 79 is shown by tryptophan fluorescence spectroscopy to reside in a polar environment. Finally, we demonstrate that ATP can be UV cross-linked to the Runt domain and that ATP binding is sensitive to an amino acid substitution in the putative Kinase-1a motif (P-loop).

Identification of a second human acetyl-CoA carboxylase gene.

Acetyl-CoA carboxylase (ACC), an important enzyme in fatty acid biosynthesis and a regulator of fatty acid oxidation, is present in at least two isoenzymic forms in rat and human tissues. Previous work has established the existence of a 265,000 Da enzyme in both the rat and human (RACC265; HACC265) and a higher-molecular-mass species (275,000-280,000 Da) in the same species (RACC280; HACC275). An HACC265 gene has previously been localized to chromosome 17. In the present study, we report cloning of a partial-length human cDNA sequence which appears to correspond to HACC275 and its rat homologue, RACC280, as judged by mRNA tissue distribution and cell-specific regulation of mRNA/protein expression. The gene encoding this isoenzymic form of ACC has been localized to the long arm of human chromosome 12. Thus, ACC is represented in a multigene family in both rodents and humans. The newly discovered human gene and its rat homologue appear to be under different regulatory control to the HACC265 gene, as judged by tissue-specific expression in vivo and by independent modulation in cultured cells in vitro.

Transcriptional activity of core binding factor-alpha (AML1) and beta subunits on murine leukemia virus enhancer cores.

Core binding factor (CBF), also known as polyomavirus enhancer-binding protein 2 and SL3 enhancer factor 1, is a mammalian transcription factor that binds to an element termed the core within the enhancers of the murine leukemia virus family of retroviruses. The core elements of the SL3 virus are important genetic determinants of the ability of this virus to induce T-cell lymphomas and the transcriptional activity of the viral long terminal repeat in T lymphocytes. CBF consists of two subunits, a DNA binding subunit, CBF alpha, and a second subunit, CBF beta, that stimulates the DNA binding activity of CBF alpha. One of the genes that encodes a CBF alpha subunit is AML1, also called Cbf alpha 2. This locus is rearranged by chromosomal translocations in human myeloproliferative disorders and leukemias. An exogenously expressed Cbf alpha 2-encoded subunit (CBF alpha 2-451) stimulated transcription from the SL3 enhancer in P19 and HeLa cells. Activity was mediated through the core elements. Three different isoforms of CBF beta were also tested for transcriptional activity on the SL3 enhancer. The longest form, CBF beta-187, increased the transcriptional stimulation by CBF alpha 2-451 twofold in HeLa cells, although it had no effect in P19 cells. Transcriptional activation by CBF beta required binding to the CBF alpha subunit, as a form of CBF beta that lacked binding ability, CBF beta-148, failed to increase activity. These results indicated that at least in certain cell types, the maximum activity of CBF required both subunits. They also provided support for the hypothesis that CBF is a factor in T lymphocytes that is responsible for recognition of the SL3 cores. We also examined whether CBF could distinguish a 1-bp difference between the enhancer core of SL3 and the core of the nonleukemogenic virus, Akv. This difference strongly affects transcription in T cells and leukemogenicity of SL3. However, no combination of CBF alpha and CBF beta subunits that we tested was able to distinguish the 1-bp difference in transcription assays. Thus, a complete understanding of how T cells recognize the SL3 core remains to be elucidated.

Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.

Sequence specificity of the core-binding factor.

The core-binding factor (CBF) binds the conserved core motif in mammalian type C retrovirus enhancers. We analyzed the phosphate contacts made by CBF on the Moloney murine leukemia virus enhancer by ethylation interference assay. The phosphate contacts span 9 bp centered around the consensus core site. To examine the sequence preferences for CBF binding, we employed the technique of selected and amplified binding sequence footprinting (T. K. Blackwell and H. Weintraub, Science 250:1104-1110, 1990). The consensus binding site for CBF defined by selected and amplified binding sequence footprinting is PyGPyG GTPy.

A casein kinase-like kinase phosphorylates beta-tubulin and may be a microtubule-associated protein.

The hypothesis that casein kinase II (CKII) is a microtubule-associated protein kinase was investigated using a neuronal cell line and bovine brain. Heparin, an inhibitor of CKII, inhibited the phosphorylation of a PC12 cytosolic protein whose molecular weight was similar to that of beta-tubulin. Partially purified PC12 CKII was immunoreactive to an antibody directed against bovine CKII and was able to phosphorylate purified beta-tubulin in a heparin-inhibitable manner when the concentration of tubulin was less than 50 micrograms/ml. To better determine if CKII is a microtubule-associated protein kinase, bovine brain tubulin was chromatographed on FPLC Mono Q and phosphocellulose columns. Several tubulin casein kinase (TCK) activities were apparent. All TCK activities phosphorylated tubulin and casein, but none was able to phosphorylate the CKII-specific synthetic peptide RRREEETEEE. One of these TCK fractions was immunoreactive to the antibody directed against CKII, and this antibody labeled a 50-kDa molecular mass band that had a molecular mass distinctly different from those of the subunits of CKII. Thus, we suggest that a CKII-like protein, but not CKII, might be a microtubule-associated protein.

Porcine thyroid cytosolic, latent alkaline ribonuclease: resistance to protein denaturants.

1. A ribonuclease isolated from porcine thyroid cytosol using phenol: sodium dodecylsulfate treatment was associated with RNA and identical to latent alkaline ribonuclease. 2. Distribution of activity between aqueous and phenolic phases depended on pH, RNA, and ribonuclease inhibitor. 3. The ribonuclease was totally resistant to urea, guanidinium: HCl, chloroform:isoamyl alcohol, ethanol, heating at 100 degrees C for 10 min or at 80 degrees C plus 100 mM NaCl. It was highly resistant to hydrolysis by proteinase K except in the presence of detergent. 4. The extreme stability and other properties of latent alkaline ribonuclease could be the result of its association with RNA.

Microporosity of the substratum regulates differentiation of MDCK cells in vitro.

We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [35S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.