Identification of autophosphorylation sites of HER2/neu.

HER2 or c-erbB-2 is a putative growth factor receptor with sequence homology to the epidermal growth factor receptor. It is the human homologue of the rat protooncogene neu and may have an important role in human malignancies such as breast and ovarian cancers. Like other growth factor receptors, HER2 has intrinsic protein tyrosine kinase activity and undergoes autophosphorylation. Recently, we have demonstrated that, similar to the epidermal growth factor receptor, all autophosphorylation sites of HER2 are localized in the carboxyl terminus of this protein. In the present study, immunopurified HER2 was allowed to autophosphorylate, and tryptic phosphopeptides were generated. After purification of these phosphopeptides by high performance liquid chromatography, microsequencing was performed. Utilizing this approach, two autophosphorylation sites were unequivocally identified at Y1023 and Y1248. The sequences of two other tyrosine phosphorylated tryptic peptides were determined, but the exact site of autophosphorylation could not be determined because multiple tyrosines were located on each peptide. However, each of these peptides contains tyrosines that correspond to major autophosphorylation sites of the epidermal growth factor receptor, suggesting that, in addition to Y1023 and Y1248, Y1139 and Y1222 also serve as autophosphorylation sites of HER2.

All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor.

Activation of the epidermal growth factor (EGF) receptor kinase leads to autophosphorylation and to the phosphorylation of various cellular substrates. The three known autophosphorylation sites of EGF receptor are located at the carboxyl-terminal tail where they probably act to compete with and thus modulate substrate phosphorylation. Mutational analysis and microsequencing techniques have been used to localize and identify new autophosphorylation site(s) of the EGF receptor. We have compared the phosphopeptide maps of human EGF receptor, and two deletion mutants lacking 63 and 126 amino acids from the carboxyl-terminal tail with the phosphopeptide maps of HER/neu and a chimeric EGF receptor containing the carboxyl-terminal tail of HER2/neu. HER2/neu is highly homologous to the EGF receptor, and it probably functions as a growth factor receptor for as yet unidentified growth factor. On the basis of this analysis, we have concluded that all autophosphorylation sites of EGF receptor and HER2/neu are located in their carboxyl-terminal tails. Utilizing the EGF receptors with carboxyl-terminal deletions, we were also able to identify tyr1086 as an additional autophosphorylation site of EGF receptor. Direct microsequencing of a phosphorylated tryptic peptide from the human EGF receptor confirmed this assignment.

Purification and characterization of tumor inhibitory factor-2: its identity to interleukin 1.

The tumor inhibitory factor-2 from the conditioned medium of the human rhabdomyosarcoma cell line A673 was purified and sequenced. The 19 N-terminal amino acid residues were identical to those of human interleukin 1 (IL-1), corresponding to the residues 119-137 of the IL-1 alpha precursor. The purified material had an apparent molecular weight similar to that of the mature secreted form of IL-1 alpha (Mr 17,400). In addition, similarly to IL-1, it induced the production of IL-2 by T-cells. The purified protein inhibited the growth of the A673 cells from which it was derived, suggesting that it may act as an autocrine growth inhibitor. It also inhibited the growth of a human adenocarcinoma of the lung and three human mammary carcinomas, but not of two human melanoma cell lines. In contrast, it stimulated the proliferation of normal human fibroblasts. These biological activities, previously assigned to a putative tumor inhibitory factor molecule, are apparently due to the production by the tumor cells of IL-1 alpha.

Molecular cloning of the human gene for von Willebrand factor and identification of the transcription initiation site.

A series of overlapping cosmid genomic clones have been isolated that contain the entire coding unit of the human gene for von Willebrand factor (vWf), a major component of the hemostatic system. The cloned segments span approximately 175 kilobases of human DNA sequence, and hybridization analysis suggests that the vWf coding unit is approximately 150 kilobases in length. Within one of these clones, the vWf transcription initiation site has been mapped and a portion of the vWf promoter region has been sequenced, revealing a typical "TATA box," a downstream "CCAAT box," and a perfect downstream repeat of the 8 base pairs containing the transcription start site. Sequencing of a segment of another genomic clone has revealed the vWf translation termination codon. Where tested, comparative restriction analysis of cloned and chromosomal DNA segments strongly suggests that no major alterations occurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-producing endothelial cells and nonexpressing leukocytes suggest that vWf gene expression is not accompanied by gross genomic rearrangements. In addition, there is significant homology of C-terminal coding sequences among the vWf genes of several vertebrate species.