Molecular profiling of tyrosine kinases in normal and cancer cells

Abstract

As the post-genome era is approaching, with vast amount of sequence information available and new technology developed, scientists are presented with opportunities to explore in simple analysis the structure and expression pattern of not just a single gene, but of an entire family of genes, if not the entire genome. The concept of 'molecular profiling' or 'expression array' has thus emerged. The need to simultaneously 'see' all genes in the same family is obvious under the precept of the combinatorial process being an underlying principle of complex biological systems: no gene exists in isolation, for virtually every molecule participates in intermolecular interactions. The activation of receptor tyrosine kinases through homo or hetero-dimerization is the prototypic example. In this review, a tyrosine kinase profile technique and its application to studying the expression of tyrosine kinases and the identification of novel kinases will be discussed. This serves as an introduction to the several interesting papers published in this special 'kinase' issue of the Journal of Biomedical Sciences, using this technique. A new simplified approach, kinase display, which is an extension of the profiling method and requires only restriction digestion and gel analysis will also be introduced.

Upregulation of Tyrosine Kinase TKT by the Epstein-Barr Virus Transactivator Zta

“…Because of the importance of tyrosine kinases in cellular control, we focused our investigation on the identification of Zta-regulated tyrosine kinases. Taking advantage of a two-step kinase display system, the tyrosine kinase profiles of Zta-expressing and control cells were displayed following amplification of tyrosine kinase transcripts using degenerate primers, which were based on the conserved DVW and DFG motifs found in most tyrosine kinases (27). The advantage of this newly developed assay is that each tyrosine kinase may be recognized by its unique restriction enzyme digestion pattern in the variable regions between the DVW and DFG motifs (27).…”

“…Taking advantage of a two-step kinase display system, the tyrosine kinase profiles of Zta-expressing and control cells were displayed following amplification of tyrosine kinase transcripts using degenerate primers, which were based on the conserved DVW and DFG motifs found in most tyrosine kinases (27). The advantage of this newly developed assay is that each tyrosine kinase may be recognized by its unique restriction enzyme digestion pattern in the variable regions between the DVW and DFG motifs (27). Analysis of the restriction profiles revealed that the most prominent, differentially regulated tyrosine kinase in the Zta-expressing clones is TKT (also known as DDR2) (23).…”

“…A tyrosine kinase display strategy was used to visualize the tyrosine kinase profile in Zta and vector transfectants (27). Total cellular RNA was extracted as specified by the manufacturer (REzol C&T; Protech, Taiwan, Republic of China).…”

“…The products were analyzed on a denaturing 7% polyacrylamide gel containing 6 M urea and 0.6ϫ TTE (glycerol tolerance buffer [USB, Cleveland, Ohio]). The patterns of tyrosine kinase expression may be determined from the pattern of restriction enzyme digestion products (27).…”

“…However, whether Zta directly or indirectly influences any cellular tyrosine kinase remains unknown. Utilizing a newly developed kinase display assay (27), we found that a cellular receptor tyrosine kinase, TKT (TRK-related tyrosine kinase, also called DDR2, Tyro 10, and CCK-2), could be upregulated by Zta at both the RNA and protein levels in a cell culture system (2,23,28). Importantly, the upregulation of phosphorylated TKT and the TKT downstream effector matrix metalloproteinase 1 (MMP-1) hinted that TKT might initiate a signaling cascade in Zta-expressing cells.…”