Phosphorylation of Ets1 regulates the complementation of a CSF-1 receptor impaired in mitogenesis.

Ets1, the founder member of the Ets transcription factor family, is involved in a variety of developmental and cellular processes. Previous studies have shown that serine phosphorylation of Ets1 inhibits its DNA binding activity, suggesting that phosphorylation is important in the regulation of Ets1 function. To further examine Ets1 phosphorylation, we ectopically expressed Ets1 in fibroblasts and stimulated these cells with serum. Using two-dimensional tryptic phosphopeptide analysis and site-directed mutagenesis, we found that Ets1 was phosphorylated on threonine 38, a residue conserved in several Ets proteins. Substitution of this residue with alanine enhanced CSF-1-dependent colony formation in semi-solid medium of NIH3T3 cells expressing a mitogenically defective CSF-1 receptor [Y809F]. Threonine 38 is part of a consensus amino-acid sequence frequently recognized and targeted by members of the MAP kinase family. Moreover, this residue is phosphorylated in vitro by recombinant ERK2, which suggests that the kinase which phosphorylates threonine 38 in vivo is a member of the MAP kinase family. In addition, phosphorylation on threonine 38 seems to negatively regulate Ets1 activity in response to growth-factor stimulation.

Calcium-induced phosphorylation of ETS1 inhibits its specific DNA binding activity.

Ets1, the founding member of the Ets gene family of transcriptional regulators, is a phosphoprotein which is highly expressed in cells of the T and B lymphoid lineages. Previous studies have shown that Ets1 becomes rapidly and transiently phosphorylated following antigen receptor (T cell (antigen) receptor (TCR) and membrane Ig) triggering a response which is absolutely dependent on ligand-induced calcium mobilization. By a combination of two-dimensional tryptic phosphopeptide and mutational analyses, the target residues of these calcium-dependent phosphorylation events are identified as 4 serine residues clustered in a domain of Ets1 adjacent to its DNA binding domain (Ets domain). From the comparison of the properties of wild type Ets1 with those of mutant proteins carrying serine-to-alanine substitution in target residues, calcium-dependent phosphorylation of Ets1 is shown to inhibit its binding to specific DNA sequences but does not affect its ability to accumulate in the nucleus, another property dependent on the Ets domain. Our data are consistent with a model in which the calcium-dependent phosphorylation of Ets1 represent the first step of a general clearance of Ets1 function during T and B cell activation.

Analysis of the DNA binding and transcriptional activation properties of the Ets1 oncoprotein.

The c-ets1 gene product (Ets1) is the prototype of a family of sequence-specific transcriptional activators which have been implicated in various developmental processes and in the response of cells to a variety of extracellular stimuli. We report here a structure-function analysis of the DNA binding and transcriptional activation properties of Ets1. The minimal region required for specific DNA binding is located at the carboxy-terminus of Ets1, a domain highly conserved in all known members of the Ets family. Transcriptional activation by Ets1 in mammalian cells requires an additional domain of 110 amino acids characterized by a high content of acidic residues and localized in the amino-terminal half of the protein. This domain also functions as a transcriptional activation domain in yeast cells when linked to the heterologous DNA binding domain of Gal4. In contrast to its conservation in Ets1 proteins across vertebrate species, this activation domain is not conserved in other members of the Ets family. These results indicate that an important level of specificity between different members of the Ets family may reside in the differential interactions of their respective activation domains with distinct general transcription factors or different associated coactivators.

Mode of action of the antitumor compound girodazole (RP 49532A, NSC 627434).

Girodazole (RP 49532A) or 3-amino-1-[4-(2 amino-1H-imidazolyl]-propanol, 2HCl is an experimental antitumor compound which inhibits protein synthesis in cell cultures and in cell free systems. The compound has been evaluated for its capacity to inhibit specific assays of initiation, elongation and termination of protein synthesis. Girodazole inhibited the release of nascent peptides from polyribosomes in rabbit reticulocyte lysates indicating that the major effect of the compound is on the protein synthesis termination step.

Antitumor activity and mechanism of action of the marine compound girodazole.

Girodazole, a new marine compound has been isolated from the sponge Pseudaxinyssa cantharella. Girodazole is active in vivo on several murine grafted tumors including leukemias (P388, L1210, i.p./i.p.) and solid tumors (MA 16/C mammary adenocarcinoma, M5076 histiocytosarcoma, s.c./i.v.). In addition, girodazole has identical cytotoxic properties in vitro on P388 and P388/DOX cells and retains antitumor activity in vivo on P388/DOX. Girodazole has a unique chemical structure different from those of known anticancer agents and of new compounds undergoing clinical trials. Biochemical studies indicate that girodazole inhibits protein synthesis during the elongation/termination steps. Toxicological studies have been done in mice and in dogs and did not reveal any major toxic effect which could preclude administration in patients. Girodazole is now undergoing phase I clinical studies.

Definition of an Ets1 protein domain required for nuclear localization in cells and DNA-binding activity in vitro.

Ets1 and Ets2 are nuclear phosphoproteins which bind to DNA in vitro and share two domains of strong identity. Deletion analyses of each of these conserved regions in Ets1 demonstrated that integrity of the carboxy-terminal domain, also conserved in the more distantly related elk and erg gene products, is essential for both nuclear targeting and DNA-binding activity in vitro.