Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73.

Recent advances indicate a link between tumour promoters, transformation, and AP-1 activity. Protein kinase C activation increases AP-1 DNA-binding activity independently of new protein synthesis. AP-1 is also stimulated by transforming oncoproteins and growth factors. These proteins are thought to participate in a signalling cascade affecting the nuclear AP-1 complex composed of the Jun and Fos proteins. Because c-Jun is the most potent transactivator in the AP-1 complex and is elevated in Ha-ras-transformed cells, in which c-Fos is downregulated, we focused on it as a potential target. c-Jun could convert input from an oncogenic signalling cascade into changes in gene expression. Indeed, transformation of rat embryo fibroblasts by c-Jun requires an intact transcriptional activation domain and cooperation with oncogenic Ha-ras. Expression of oncogenic Ha-ras augments transactivation by c-Jun and stimulates its phosphorylation. Here we describe the mapping of the Ha-ras-responsive phosphorylation sites to serines 63 and 73 of c-Jun. Site-directed mutagenesis indicates that phosphorylation of these serines is essential for stimulation of c-Jun activity and for cooperation with Ha-ras in ocogenic transformation.

The transcriptional factor Egr-1 is synthesized by baculovirus-infected insect cells in an active, DNA-binding form.

The Egr-1 (zfp-6) gene encodes a zinc-finger-containing nuclear protein that is rapidly and transiently induced in quiescent cells treated with mitogens. We have constructed baculovirus vectors that synthesize mouse Egr-1 protein initiating at two putative ATG start sites. The ATG site producing the larger protein (Mr, 80,000) is similar, if not identical, to Egr-1 synthesized by serum-stimulated quiescent mouse fibroblasts, thus identifying the likely site for translation. The protein synthesized by the insect cells is active as assayed by its ability to bind to a specific DNA sequence that has been identified as an Egr-1 binding site. The insect cell system will allow further studies of the structure and function of the Egr-1 product, a protein that appears to be an important "master switch" for other genes.

Calcium-dependent distance changes in binary and ternary complexes of troponin.

Calcium-dependent distance changes have been determined by resonance energy transfer in binary and ternary troponin complexes in order to collect evidence for the structural rearrangements which are part of the hypothetical trigger mechanism of skeletal muscle contraction. Donor and acceptor fluorophores were either intrinsic tryptophans in subunits with a favourable sequence from different species, quasi-intrinsic Tb3+ ions bound to troponin C or extrinsic labels attached to specific cysteine or methionine residues. All chemically modified subunits proved fully active in conferring calcium sensitivity onto myosin ATPase. Nine distances were determined between five sites which allowed construction of a three-dimensional lattice representing the spatial distribution of four sites in the ternary complex of troponin C, I and T. Distances in binary complexes were nearly unaltered upon addition of the third subunit. Regulatory calcium binding caused distance changes of the order of 0.7-1.1 nm. In view of the large displacements of the hypothetical mechanism, they turned out to be smaller than anticipated. The fluorophoric sites selected may be localized in a zone of the troponin complex which happens to be relatively little affected by the mechanism. Alternatively, amplification of the moderate changes seen here would require the complete set of thin filament proteins.

A solution equivalent of the 2Zn----4Zn transformation of insulin in the crystal.

Circular dichroic spectroscopy clearly reveals a solvent-induced conformational change of insulin in the presence of zinc ions. The spectral change corresponds to an increase in helix content. The transition observed in solution is an equivalent of the 2Zn----4Zn insulin transformation in the crystal. This is inferred from a series of observations. (1) The spectral effects are compatible with the refolding of the B-chain N-terminus into a helix known from crystal studies. (2) The spectral effects are induced by the very same conditions which are known to induce the 2Zn----4Zn insulin transformation in the crystal (i.e. threshold concentrations of NaCl, KSCN, NaI, for example). (3) They fail to be induced by the same conditions that fail to induce the crystal transformation (e.g. Ni2+ instead of Zn2+). It is concluded that the potential to undergo the transition resides in the hexamer since neither insulin dimers nor monomeric des-pentapeptideB26-30-insulin respond detectably to high halide concentration. Secondly the ability of zinc ions to accommodate tetrahedral coordination allows the transition which is not permitted by other divalent metal ions. Thirdly the transition is independent of the off-axial tetrahedral zinc coordination sites since it occurs in [AlaB5]insulin which lacks the B5 histidine necessary for their formation. A symmetrically rearranged hexamer thus appears possible with two tetrahedrally coordinated zinc ions on the threefold axis; this is consistent with the observation that in native insulin two zinc ions per hexamer are sufficient to produce the full spectral effect. The amount of additional helix derived from the circular dichroic spectral change, however, cannot settle whether the transition comprises only three or all six of the subunits to yield a symmetrical hexamer. Finally the transformation in solution evidently still occurs in an intramolecularly A1-B29-cross-linked insulin in spite of the partially reduced flexibility.

The origin of the tyrosyl circular dichroism of tropomyosin.

1. The near ultraviolet circular dichroism of tropomyosin is due to tyrosine and to disulphide bonds. The optical activity of these chromophores can be distinguished by oxidising and reducing the protein. The circular dichroism due to tyrosine is exceptionally intense and is anomalous in that the shape of the spectrum and the wavelength of the maximum are different from those of the absorption spectrum. 2. The intense tyrosyl circular dichroism and the mismatch between circular dichroism and absorption spectra are likely to be due to tyrosine-tyrosine interactions at distances of less than 8 A. 3. The results are analysed in terms of the coiled coil model for tropomyosin and lead to the conclusion that the tyrosines of one helical subunit interact with those of the other. The in-register alignment of the helical chains, with five tyrosines of one chain opposite those of the other, accounts for the tyrosine-tyrosine interactions. 4. The disulphide circular dichroism of oxidized tropomyosin is intense and is consistent with intra-molecular disulphide bonds between helical subunits which can form in the non-staggered model for tropomyosin.