Analysis of the autophosphorylation activity of transformation defective mutants of avian erythroblastosis virus

113

150

GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast Saccharomyces cerevisiae. GCN2, a translational activator of GCN4 expression, contains a domain homologous to the catalytic subunit of eucaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating GCN4 expression. Elevated GCN2 gene dosage led to derepression of GCN4 under nonstarvation conditions; however, we found that GCN2 mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated posttranslationally in amino acid-starved cells. Three dominant-constitutive GCN2 point mutations were isolated that led to derepressed GCN4 expression under nonstarvation conditions. Two of the GCN2(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which we suggested might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety. Deletions and substitutions in the HisRS-related sequences and in the carboxyl-terminal segment in which one of the GCN2(Con) mutation mapped abolished GCN2 positive regulatory function in vivo without lowering autophosphorylation activity in vitro. These results suggest that sequences flanking the GCN2 protein kinase moiety are positive-acting domains required to increase recognition of physiological substrates or lower the requirement for uncharged tRNA to activate kinase activity under conditions of amino acid starvation.In the yeast Saccharomyces cerevisiae, starvation for any one of several amino acids or a defective aminoacyl-tRNA synthetase leads to increased transcription of over 30 genes encoding amino acid-biosynthetic enzymes in several different pathways (reviewed in reference 19). The coordinate derepression of these biosynthetic pathways is known as general amino acid control. GCN4 is a positive regulatory protein that acts directly to stimulate transcription by binding to 5' noncoding sequences located upstream of each gene subject to the general control.Expression of GCN4 itself is regulated by amino acid availability at the level of translation initiation. This translational control involves four short upstream open reading frames (uORFs) present in the leader of GCN4 mRNA that inhibit translation initiation at the GCN4 start codon under nonstarvation conditions. The inhibitory effect of the four uORFs on GCN4 expression requires trans-acting negative regulators encoded by multiple GCD genes. This group of negative effectors was recently shown to include SUI2 and SUI3, the structural genes for the a and a subunits, respectively, of eucaryotic initiation factor 2 (42). Positive-acting factors encoded by the GCN2 and GCN3 genes are required to stimulate G...

Section: Resultsmentioning

confidence: 99%

Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression.

Wek¹,

Ramírez²,

Jackson³

et al. 1990

113

150

“…The monoclonal antibody 5E2 directed against phosphotyrosine was generously provided by Genentech, San Francisco, Calif. The rabbit anti-v-ErbB antibody knl4 was generated against a 588-bp BamHI fragment encoding the v-ErbB kinase domain as described previously (15). The Shc antiserum directed against the SH2 domain of the Shc protein has been described previously (38 (6).…”

Section: Methodsmentioning

confidence: 99%

Analysis of the role of the Shc and Grb2 proteins in signal transduction by the v-ErbB protein.

Meyer¹,

Labudda²,

McGlade³

et al. 1994

“…As ErbB lacks the EGF-binding domain, its kinase activity has been hypothesized to cause transformation by virtue of its activity being ligand independent. This study was undertaken to determine whether truncation of the ligand-binding domain affected the kinetics as well as the ligand dependence of kinase activity.Prior studies with ErbB proteins have demonstrated tyrosine kinase activity for autophosphorylation and the phosphorylation of synthetic peptides (3,8,9,10,15). Prior * Corresponding author.…”

mentioning

confidence: 99%

“…Prior studies with ErbB proteins have demonstrated tyrosine kinase activity for autophosphorylation and the phosphorylation of synthetic peptides (3,8,9,10,15). Prior * Corresponding author.…”

mentioning

confidence: 99%

Protein tyrosine kinase activities of the epidermal growth factor receptor and ErbB proteins: correlation of oncogenic activation with altered kinetics.

Nair¹,

Davis²,

Robinson³

1992

We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2', ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and -2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higherKm(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphorylation suggested that the ErbB proteins had lower catalytic efficiencies for the exogenous substrate than did the chEGFR. The three tested ErbB proteins had cytoplasmic domain mutations that conferred distinctive disease potentials. These mutations did not affect the kinetics for the phosphorylation of the exogenous substrate TK6. Two of the ErbB proteins contained all of the sites used for autophosphorylation. In these, a mutation that broadened oncogenic potential to endothelial cells caused an additional increase in Vmax(app) for autophosphorylation. Thus, mutations that change the EGFR into an ErbB oncogene cause multiple changes in the kinetics of protein tyrosine kinase activity.The v-erbB oncogenes of avian erythroblastosis viruses (AEV) arise by recombination of an avian leukosis virus with host sequences encoding the transmembrane and cytoplasmic domains of the chicken epidermal growth factor receptor (chEGFR). The EGFR consists of four functional domains: an extracellular epidermal growth factor (EGF)-binding N terminus, a short transmembrane domain, a tyrosine kinase domain, and a C terminal domain containing the major sites of EGF-stimulated autophosphorylation (for a review, see reference 28). Thirty to forty independent isolates of AEV have been identified (2,5,11,18,22). The EGF-binding domain is truncated in all of these isolates. Many also contain point mutations, truncations, or internal deletions in the cytoplasmic domain of the receptor. These mutations determine differences in the potential to induce erythroblastosis, angiosarcoma, hemangioma, or fibrosarcoma (7,21,22,24,27).The tyrosine kinase activity of the EGFR is required for its mitogenic activity (28). Analysis of the transformation potential of a series of linker insertion mutations in ErbB suggests that kinase activity is required for transformation (20). As ErbB lacks the EGF-binding domain, its kinase activity has been hypothesized to cause transformation by virtue of its activity being ligand independent. This study was undertaken to determine whether truncation of th...