Molecular Cloning of Human 14-3-3 cDNA with CD3 Zeta Peptide Containing Phosphosering Tag

Kang, Jae Seung

Abstract

14-3-3 proteins are highly conserved proteins of about 29 kDa and have a minimum of seven isoforms. 14- 3-3 proteins interact with many signalling proteins by the recognition of phosphoserine. For the identification of proteins which react with ITAM (immunoreceptor tyrosine-based activation motif) of CD3 zeta chain, labeled synthetic peptides representing the CD3 zeta chain structual motifs (ITAMs) with a tag of PKC substrate sequence were used for western blotting. One major protein band of approximately 29 kDa was identified in lysate of Jurkat T cell, B cells and HeLa cells. Screening of lamda gt 11 library derived from HeLa cell gave two clones of 14-3-3 protein cDNA. Inspection of their nucleotide sequences identified these two full length cDNA clones as the 29 kDa human homologue of rat 14-3-3 gamma and the human 14-3-3 zeta protein. The human 14-3-3 gamma isoform also showed high homology with other species in amino acid and nucleotide sequence. Although 14-3-3 proteins are phosphoserine-binding proteins, there may be another way of interaction between ITAMs of CD3 and 14-3-3 proteins.