The cloning and characterization of the TIK kinase.

Abstract

Protein phosphorylation is a mechanism of modulating protein activity which plays a central role in a wide variety of cellular functions. The ready reversibility of the phosphotransfer reaction makes it well-suited for rapid cellular responses, such as the responses to signals from extracellular stimuli. Members of the protein kinase family have been identified as components of cellular signal tranduction networks, and in fact many protein kinases mediate signals controlling cell growth, differentiation, and development. The work carried out in this thesis describes the cloning and characterization of the TIK kinase, a novel member of the protein kinase family. The TIK kinase was isolated and cloned from murine pre-B cells on the basis of its immunoreactivity with antibodies to phosphotyrosine, and was therefore originally thought to be a protein tyrosine kinase. Characterization of the in vitro and in vivo activity of this kinase, however, revealed that the TIK kinase possesses only serine and threonine phosphorylating ability. A kinase deficient TIK protein, constructed by site-directed mutagenesis, is not immunoreactive with the antiphosphotyrosine antibodies. These observations indicate the phosphoserine and/or phosphothreonine residues may be assuming a conformation within the context of this protein which mimicks the epitope(s) recognized by antiphosphotyrosine antibodies. The gene encoding the TIK kinase is transcribed to produce three distinct messenger RNA (mRNA) transcripts in all murine tissues examined, and in a number of murine leukemic cell lines. In the murine lymphocytic leukemia line L1210, however, three truncated mRNA molecules are produced in addition to the three normal transcripts detected in other tissues. Evidence is presented here that one allele of the TIK gene has undergone a genomic rearrangement in this cell line, and appears to giving rise to the abnormal pattern of mRNA expression observed. A cDNA corresponding to the smallest truncated mRNA transcript encoding the TIK kinase in the L1210 cell line has been obtained, and this cDNA encodes a mutant TIK protein lacking kinase activity. I postulate that the TIK kinase plays some role in the control of growth and differentiation during lymphocyte development, and that the mutation of the TIK gene has contributed to the initiation or maintenance of transformation in the L1210 cell line.