Characterization of a Phosphoprotein whose mRNA is Regulated by the Mitogenic Pathways in Dog Thyroid Cells

Authors


  • Note. The dog clone c5fw cDNA sequence has been deposited in the DDBJ, EMBL and Genbank databases under the accession number >emb|X99144|e996426. The human clone c5fw and clone c8fi cDNA sequences hove been deposited under the accession numbers [D87119] and [AJ0004HO], respectively.

J. E. Dumont, Institut de Recherche Intcrdisciplinaire, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Brussek, Belgium

Abstract

We have isolated cDNA clones encoding the dog and human forms of a novel protein whose function is still unknown. Sequence analysis indicates that dog clone c5fw protein contains 343 amino acid residues, several potential phosphorylation sites, and two of the 12 conserved subdomains (VIII and IX) that fold into a common catalytic core structure of the large family of protein kinases. Human clone c5fw shares 95% amino acid identity with its dog counterpart. We have also isolated another human-related clone c5fw sharing 70% amino acid identity with the dog sequence. We transiently expressed c-myc epitope-tagged clone c5fw protein in COS-7 cells and infected thyrocytes in primary culture with a recombinant adenovirus containing clone c5fw cDNA (adenovirus c5fw).In both experiments, a 46-kDa protein was detected and subsequently more extensively characterized. By two-dimensional gel electrophoresis and V8 protease digestion, we showed that this overexpressed protein is phosphorylated on different sites. Moreover, cells stimulated with thyrotropin or epiderinal growth factor, thyrotropin and fetal calf serum increased the level of clone c5fw protein produced after infection by adenovirus containing clone c5fw. The disappearance of this 46-kDa protein after 1 h of puromycin treatment indicates that it is a labile protein. Immunofluorescence and subcellular fractionation analysis have revealed that c-myc-tagged clone c5fw was insoluble and localized mainly in the cytoplasm, in the form of granules.

Abbreviations.
EGF

epidermal growth factor

TPA

12-O-tetradecanoylphorbol 13-acetate

HGF

hepatocyte growth factor

MBP

maltose binding protein

CMV

cytomegalovirus

SV40

simian virus 40

MOI

multiplicity of infection

FCS

fetal calf serum

PtdIns3-kinase

phosphatidylinositol 3-kinase

TSH

thyrotropin or thyroid stimulating hormone

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