Cloning and preliminary characterization of a 105 kDa protein with an N-terminal kinase-like domain

• Published in: Biochimica et biophysica acta Vol. 1517; no. 1; pp. 148 - 152
• Main Authors: Liu, Simon C.H., Lane, William S., Lienhard, Gustav E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.12.2000
• Subjects: 3T3 Cells; Adipocyte; Adipocytes - metabolism; Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Cytosol - enzymology; DNA, Complementary - chemistry; Expressed Sequence Tags; Mice; Microsomes - enzymology; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Fragments - genetics; Protein kinase; Protein-Serine-Threonine Kinases; Proteins - chemistry; Proteins - genetics; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-akt; Adipocyte; PKB, protein kinase B; SDS–PAGE, sodium dodecyl–polyacrylamide gel electrophoresis; Protein kinase; est, expressed sequence tag; RACE, rapid amplification of cDNA ends; aa, amino acid
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(00)00234-7

In the course of searching for proteins that interact with protein kinase B in 3T3-L1 adipocytes, we isolated a 105 kDa protein from 3T3-L1 adipocytes. Peptides sequenced from the protein were found to be present in several expressed sequence tags. A cDNA containing one of these expressed sequence tags was sequenced and appears to contain the entire coding region. Computer analysis revealed a potential protein kinase domain at the N-terminus; however, the first subdomain and several invariant residues characteristic of protein kinases are absent. An antibody was raised against a peptide from the 105 kDa protein. By immunoblotting, it was found that the protein was widely expressed in mouse tissues, and concentrated in the cytosol and low density microsome fractions of 3T3-L1 adipocytes.