The cytoplasmic zinc finger protein ZPR1 accumulates in the nucleolus of proliferating cells.

The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells.

The epidermal growth factor receptor is covalently linked to ubiquitin.

Incubation of cultured human fibroblasts with epidermal growth factor (EGF) causes a proliferative response that is mediated by the binding of the growth factor to specific cell surface receptors. One event that occurs rapidly following EGF binding is the covalent modification of the EGF receptor (EGF-R) by phosphorylation on Ser, Thr, and Tyr residues. Here we report the identification of ubiquitination as a second form of EGF-stimulated covalent modification of the receptor. The LGF receptor was not ubiquitinated in serum-starved cells. However, treatment with EGF caused a rapid increase in EGF-R ubiquitination. In contrast, no EGF-stimulated ubiquitination was found in experiments using cells that express a mutant tyrosine kinase-negative EGF-R. Similarly, ubiquitination of the EGF-R was not observed at 4 degrees C or if the cells are depleted of intracellular K+. Together, these data establish ubiquitination as a form of EGF-stimulated covalent modification of the EGF-R.

Mutational removal of the major site of serine phosphorylation of the epidermal growth factor receptor causes potentiation of signal transduction: role of receptor down-regulation.

The major site of epidermal growth factor receptor (EGF-R) serine phosphorylation is located within the COOH-terminal domain of the receptor at Ser1046/7. We have previously demonstrated that this phosphorylation site accounts for the acute desensitization of the EGF-R observed in EGF-treated cells. Here we show that the mutational removal of this negative regulatory phosphorylation site causes potentiation of signal transduction by the EGF-R. This potentiation can be accounted for in part by a block in the EGF-stimulated down-regulation of the EGF-R. These data indicate that the SER1046/7 phosphorylation site may have a regulatory role during long term incubation of cells with mitogenic concentrations of EGF.

Protein-tyrosine kinase activity of alternate protein products of the Drosophila Dsrc28C locus.

The Dsrc28C gene is a unique member of the extensive tyrosine kinase family. Two proteins, p66Dsrc28C and p55Dsrc28C, are encoded by the gene. Each contains a highly conserved tyrosine kinase domain and each lacks the usual amino-terminal myristylation signal. The protein-tyrosine kinase activity of the two proteins was investigated through a recombinant baculovirus expression system. p66Dsrc28C expressed from a recombinant baculovirus phosphorylated a large number of Sf9 cell proteins on tyrosine. A group of proteins of approximately 100 kDa were the preferred substrates. No evidence of p66Dsrc28C autophosphorylation was found. In contrast to p66Dsrc28C, p55Dsrc28C did not exhibit protein-tyrosine kinase activity when expressed from a recombinant baculovirus. A deletion derivative of p66Dsrc28C lacking the SH3 and SH2 domains also failed to phosphorylate Sf9 cell proteins. These results suggest that the protein-tyrosine kinase activity of Dsrc28C proteins is tightly regulated.

Signal transduction by the epidermal growth factor receptor is attenuated by a COOH-terminal domain serine phosphorylation site.

It has been proposed that the acute desensitization of epidermal growth factor receptor (EGF-R) function can be accounted for, in part, by the effect of EGF to increase phosphorylation of the receptor at Ser1046/7 (Countaway, J.L., Nairn, A.C., and Davis, R.J. (1992) J. Biol. Chem. 267, 1129-1140). Here, we show that the mutational removal of this phosphorylation site causes an activation of EGF-R function and a potentiation of signal transduction. The mechanism of potentiation results from 1) defective down-regulation of the EGF-R when cells are incubated with high concentrations of EGF; and 2) increased EGF-stimulated tyrosine phosphorylation. The increased EGF-stimulated phosphorylation is associated with an alteration of the apparent specificity of tyrosine phosphorylation and is independent of the down-regulation defect. Together, these data strongly support the hypothesis that Ser1046/7 is a biologically significant site of regulatory phosphorylation of the EGF-R.

Increased oncogenic potential of ErbB is associated with the loss of a COOH-terminal domain serine phosphorylation site.

The erbB oncogene encodes an altered form of the epidermal growth factor (EGF) receptor that lacks the extracellular ligand binding domain. This oncogene is exclusively leukemogenic. However, an increase in oncogenic potential and a broadening of the tissue specificity of tumor formation occurs after retroviral transduction of erbB. The increased oncogenic potential correlates with structural alterations within the erbB gene. One common event is the deletion of a serine phosphorylation site located within the COOH-terminal domain. This site of phosphorylation has been demonstrated to be required for EGF-induced desensitization of signaling by the EGF receptor (Countaway, J. L., Nairn, A. C., and Davis, R.J. (1992) J. Biol. Chem. 267, 1129-1140). Here we show that the mutation of erbB at this negative regulatory serine phosphorylation site causes fibroblast transformation in vitro and is associated with an increased oncogenic potential in vivo.

Gene encoding the 5.7-kilodalton chlorosome protein of Chloroflexus aurantiacus: regulated message levels and a predicted carboxy-terminal protein extension.

The major light-harvesting pigment of the green filamentous bacterium Chloroflexus aurantiacus is bacteriochlorophyll (Bchl) c, localized in chlorosomes attached to the inner surface of the cytoplasmic membrane. Chlorosomes consist of four polypeptides and associated pigments and lipids. Previous studies of the inducible assembly of the photosynthetic apparatus had indicated that the major chlorosomal polypeptides are present as high-molecular-weight aggregates before the appearance of mature chlorosomes, and a mechanism for posttranslational processing of a polyprotein had been proposed. We have isolated the gene (csmA) encoding the 5.7-kilodalton chlorosomal polypeptide from C. aurantiacus in order to determine whether this protein is synthesized as part of a polyprotein. Analysis of the nucleotide sequence of csmA indicates that the gene is not large enough to encode more than one known chlorosome polypeptide. Transcriptional analysis indicates that csmA is transcribed as a small message whose abundance is regulated in response to oxygen, so that no csmA message is detectable in cells grown aerobically in the dark. Comparison of the sequence predicted by csmA with the peptide sequence of the Bchl c binding protein purified from chlorosomes indicates that this protein is synthesized with a carboxy-terminal extension of 27 amino acids. We discuss possible roles for this carboxy-terminal extension in the assembly of chlorosomes.