A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.

In two previous studies we described the properties of a heat-stable DNA-binding protein present in rat liver nuclei. This protein, hereafter termed C/EBP, is capable of selective binding to the CCAAT homology of several viral promoters (Graves et al. 1986), as well as the core homology common to many viral enhancers (Johnson et al. 1987). We now report the isolation of a recombinant clone of the gene that encodes C/EBP. Expression of the clone in bacterial cells yields a protein that binds in vitro to both the CCAAT homology and the enhancer core homology, providing conclusive evidence that a single gene product accounts for both binding activities. By examining the properties of protease-derived fragments of C/EBP, we have localized its DNA-binding domain to a 14-kD fragment. A 60-amino-acid segment located within the DNA-binding domain of C/EBP bears sequence similarity to the products of the myc and fos oncogenes.

The receptor for the wilt-inducing phytotoxin fusicoccin was purified to homogeneity from plasma membranes of Commelina communis as a complex with the radioligand [3H]9'-nor-8'-hydroxyfusicoccin. The preparation consisted of two polypeptides with apparent molecular masses of 30.5 kDa and 31.5 kDa and with isoelectric points of around pH 5.2 and 5.3, respectively. The proteins were N-terminally blocked. Internal amino acid sequences were obtained for both polypeptides of the fusicoccin-binding complex. Sequence information, as well as subsequent immunological analysis, proved that both polypeptides are members of the eukaryotic 143-3 family, which comprises structurally conserved regulatory proteins of widespread occurrence and a wide range of functions. 143-3 isoform(s) constituting the fusicoccin receptor are distinguishable from other cellular 143-3 proteins by their tight association with the plasma membrane. Applying temperature-induced Triton X-l 14 phase separation experiments, they, as well as the target enzyme of fusicoccin action, the H'-ATPase, partitioned into the phospholipid-rich fraction which contains the most hydrophobic proteins. The results discussed herein provide a basis for the elucidation of the molecular mechanism of fusicoccin action.

A surge of luteinizing hormone (LH) from the pituitary gland triggers ovulation, oocyte maturation, and luteinization for successful reproduction in mammals. Because the signaling molecules RAS and ERK1/2 (extracellular signal-regulated kinases 1 and 2) are activated by an LH surge in granulosa cells of preovulatory follicles, we disrupted Erk1/2 in mouse granulosa cells and provide in vivo evidence that these kinases are necessary for LH-induced oocyte resumption of meiosis, ovulation, and luteinization. In addition, biochemical analyses and selected disruption of the Cebpb gene in granulosa cells demonstrate that C/EBPβ (CCAAT/Enhancerbinding protein-β) is a critical downstream mediator of ERK1/2 activation. Thus, ERK1/2 and C/ EBPβ constitute an in vivo LH-regulated signaling pathway that controls ovulation-and luteinization-related events.In the mammalian ovary, the female germ cells (oocytes) reside within the ovarian follicles and are surrounded by somatic cell-derived granulosa cells (GCs) and cumulus cells that have endocrine functions and control oocyte maturation. Female reproductive success depends on the growth of ovarian follicles and differentiation of GCs as well as oocyte maturation and ovulation (1,2). Although LH plays a critical role in the initiation of ovulation and in the terminal differentiation of GCs to luteal cells that compose the corpora lutea (CLs) and produce progesterone, the precise molecular targets in these processes remain ill-defined. Cyclic adenosine 3´,5´-monophosphate (cAMP) is a well-known mediator of LH action, but LH also induces expression of the epidermal growth factor

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C/EBP is a rat liver nuclear protein capable of sequence-specific interaction with DNA. The DNA sequences to which C/EBP binds in vitro have been implicated in the control of messenger RNA synthesis. It has therefore been predicted that C/EBP will play a role in regulating gene expression in mammalian cells. The region of the C/EBP polypeptide required for direct interaction with DNA has been identified and shown to bear amino acid sequence relatedness with the product of the myc, fos, and jun proto-oncogenes. The arrangement of these related amino acid sequences led to the prediction of a new structural motif, termed the "leucine zipper," that plays a role in facilitating sequence-specific interaction between protein and DNA. Experimental tests now provide support for the leucine zipper hypothesis.

This report describes the identification and purification of a nuclear protein from rat liver that binds selectively to DNA sequences associated with several animal virus enhancers. The binding activity was tracked by direct DNase I footprinting through four steps of biochemical fractionation. These procedures led to the identification of a polypeptide species exhibiting an apparent molecular weight of 20 kD that accounts for enhancer binding activity. DNase I and dimethyl sulfate footprinting assays were used to examine the manner in which the purified protein binds to enhancer elements associated with SV40, murine sarcoma virus, and polyoma virus. The results of these assays indicate that the initial interaction established between the 20-kD protein and each viral enhancer occurs via a common DNA sequence known as the enhancer core homology.[Key Words: Enhancers; DNA-binding proteins; DNA-protein interactions; transcriptional regulation; rat liver; Gillies et al. 1983;Queen and Baltimore 1983;Walker et al. 1983}. Although the mechanism by which long-range activation is achieved is not yet understood, several different lines of evidence have implicated the involvement of cellular factors that act in trans to potentiate enhancer utilization. For example, it has been observed that when a reporter gene linked to an enhancer is introduced into cultured animal cells, its expression can be reduced selectively by the inclusion of excess, unlinked copies of the enhancer DNA sequence {Scholer and Gruss 1984; Mercola et al. 1985}. The results of these experiments have been interpreted to reflect in vivo competition for cellular factors that bind to enhancer DNA sequences. More direct evidence for the interaction between cellular factors and enhancers has come from experiments that mapped the accessibility of guanine residues within enhancers to alkylation by dimethylsulfate (DMS) in isolated nuclei. The results of two such studies revealed l Present address: Department of Cellular, Viral and Molecular Biology, University of Utah Medical Center, Salt Lake City, Utah 84132 USA. DMS protection-enhancement pattems within the intragenic enhancer of the mouse heavy-chain immunoglobulin gene in myeloma cells {which express the immunoglobulin gene} but not in fibroblasts Ephrussi et al. 1985}. These altered patterns of DMS alkylation presumably reflect in situ contacts between cellular proteins and enhancer DNA sequences.The involvement of cellular factors in enhancer activation has been demonstrated most clearly in the case of the glucocorticoid receptor. In an elegant series of experiments Yamamoto and colleagues have shown that the long terminal repeat (LTRI of mouse mammary tumor virus {MMTV} behaves as an enhancer (Chandler et al. 1983), and that the hormone inducibility of MMTV transcription is mediated by direct binding of the glucocorticoid receptor protein to the MMTV enhancer {Payvar et al. 1983}.Various lines of evidence have raised the provocative notion that enhancers may consist of multiple domains that together c...

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