Molecular characterization of the murine syk protein tyrosine kinase cDNA, transcripts and protein

Abstract

Using a RT-PCR based cloning strategy and conventional cDNA library screening we have cloned the murine syk cDNA. Sequence analysis of the 5350bp full length cDNA revealed a 5' untranslated region (UTR) of 477bp, an open reading frame of 1884bp and an unusually long 3' UTR of 2989bp containing a polyadenylation signal. The cDNA encodes a putative protein of 628 amino acids with two SH2 domains located N-terminally of the protein tyrosine kinase domain. The highest overall homology, 98%, was observed to the rat syk. Northern blot analysis revealed that the murine syk protein is encoded by two transcripts of approximately 5.4 and 3.5kb, the difference in size being attributable to differences in the 3' UTR. Rabbit antisera raised against a pGEX-syk bacterial fusion protein recognized specifically a protein of approximately 67kd with intrinsic protein tyrosine kinase activity in lymphoid cell extracts. The size of the syk protein could be confirmed by in vitro transcription/translation of the full length clone. Expression of syk was found in a variety of mouse organs with the highest levels in spleen, heart, mammary gland and thymus and in several lymphoid cell lines. The majority of the expression observed in whole mammary glands originated from the lymph node. Upregulated expression of syk was observed in aggressive, metastasizing mammary gland tumours but not in well differentiated, non-metastasizing tumors.