Molecular cloning of HEK, the gene encoding a receptor tyrosine kinase expressed by human lymphoid tumor cell lines.

Abstract

We describe the molecular cloning of a receptor tyrosine kinase from a cell line (LK63) derived from a case of human pre-B-cell leukemia. We have previously shown that a monoclonal antibody (IIA4) raised against LK63 recognized a glycosylated, cell-surface 135-kDa molecule (HEK), which displayed tyrosine kinase activity in vitro. The (5), platelet-derived growth factor (6), colony-stimulating factor 1 (7), and stem cell factor (8). The recent isolation of lineagespecific (e.g., flk-2) (9) and developmentally regulated (e.g., CEK 5) (10) PTKs highlights an emerging role for this group of molecules in differentiation and development. As part of our study of human B-cell growth and differentiation, we have raised a number of monoclonal antibodies (mAbs) against early B-cell populations. One of these, designated IIIA4, has been shown to recognize a 135-kDa molecule (HEK) on the surface ofthe pre-B-cell leukemic cell line LK63 (11). Glycosylation studies demonstrated an estimated molecular mass of 95 kDa for the HEK core protein.By indirect immunofluorescence, the human T-cell line JM also expressed this antigen, but a wide range of other lymphoid and myeloid cell lines were negative. Fluorescence-activated cell sorter analysis of normal human lymphoid and bone marrow cells showed no detectable HEK expression. However, HEK expression was observed on a small number of fresh hemopoietic tumor specimens, raising the possibility that IIIA4 recognized the product of a sporadically overexpressed gene. Immunoprecipitated HEK protein was shown to have tyrosine kinase activity in vitro. These data suggested that IIIA4 recognized a membraneassociated PTK. We used the IIIA4 mAb to affinity purify HEK protein from LK63 cells. Amino acid sequencing demonstrated a unique N terminus and PTK-related internal sequences.In this report, we describe the isolation and sequence determination of a 4.5-kilobase (kb) cDNA encoding the HEK receptor tyrosine kinase (RTK)

EB-1, a tyrosine kinase signal transduction gene, is transcriptionally activated in the t(1;19) subset of pre-B ALL, which express oncoprotein E2a-Pbx1

“…Tyr-928 of ELK is a conserved SAM domain residue present in both SAM domains of EB-1 (Y497 and Y571). While the majority of Eph-related kinases are expressed in brain, certain members of the Eph family containing SAM domains are expressed predominantly in epithelial cells (ERK; Kiyokawa et al, 1994), in erythroid progenitors (HTK; Inada et al, 1997), and in pre-B and pre-T cells (HEK; Wicks et al, 1992). Expression of HEK in pre-B cells indicates that pre-B cells are likely to contain factors that bind SAM domains.…”

Eph receptors and ephrin ligands: embryogenesis to tumorigenesis

“…Interestingly, EphA2 expression levels were signi®cantly higher in cell lines from distant metastases than in primary melanomas. EphA3 was identi®ed as an antigen present on the surface of a pre-B cell leukemic cell line and was also found to be overexpressed in the absence of gene ampli®cation or rearrangements in some hemopoietic tumors and several lymphoid tumor cell lines (Wicks et al, 1992). EphB2 was found to be overexpressed in about one third of 31 human tumor cell lines examined, in 75% of the gastric tumors examined, and in some esophageal and colon cancers (Kiyokawa et al, 1994).…”

Dissecting the EphA3/Ephrin-A5 Interactions Using a Novel Functional Mutagenesis Screen

“…In addition to a C-terminal myc epitope (EEQKLISEEDL*), EcoRI and BglII cloning sites were introduced between the FLAG and Myc sequences to accommodate insertion of EphA3-ECD cDNAs. Accordingly, the 5Ј-EphA3 oligonucleotide ( 21 ELIPQPSNE 30 V (12)) was altered to introduce a unique (12). The symbols ␦ and indicate that this site is homologous to a residue in EphB2, which forms part of the dimerization and tetramerization interfaces, respectively.…”

“…The extracellular domain (ECD) consists of a unique N-terminal globular structure, necessary and sufficient for ephrin binding (10,11), followed by a cysteinerich linker, an EGF-like motif, and two type II fibronectin domains. For human EphA3 (12), these regions span amino acid sequence positions 29 -203, 204 -260, 271-324, 325-435, and 435-531, respectively. The minimal N-terminal globular domain has a ␤ jellyroll-like architecture (13), whereas structures of the cysteine-rich linker and adjoining EGF motif have not been solved to date.…”