Molecular Cloning and Identification of a Serine/Threonine Protein Kinase of the Second-Messenger Subfamily

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 88; no. 10; pp. 4171 - 4175
• Main Authors: Jones, Pamela F., Jakubowicz, Teresa, Pitossi, Fernando J., Maurer, Fransisca, Hemmings, Brian A.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 15.05.1991; National Acad Sciences
• Subjects: Amino Acid Sequence; Amino acids; Base Sequence; Binding Sites; Biological and medical sciences; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell lines; Cloning, Molecular; Complementary DNA; Cyclic AMP - pharmacology; DNA - genetics; DNA - isolation & purification; Fundamental and applied biological sciences. Psychology; Gene Expression; genes; Genes. Genome; HeLa cells; Histones; Humans; Immunoblotting; Immunoprecipitation; Immunosorbent Techniques; Methionine; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Molecular Weight; Open reading frames; Phosphorylation; Protein Biosynthesis; Protein Kinase C - chemistry; Protein Kinase C - genetics; Protein Kinases - chemistry; Protein Kinases - genetics; Protein Kinases - metabolism; Proteins; RNA; RNA, Messenger - genetics; Second Messenger Systems; second messengers; Sequence Homology, Nucleic Acid; serine-threonine protein kinase; Human; In vitro transcription; Protein kinase C; In vitro translation; Nucleotide sequence; Homology; Gene expression; Immunological method; Northern blotting; Signal transduction; Cell line; Enzymatic activity; Complementary DNA; Protein kinase; Regulatory sequence; Molecular cloning
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.10.4171

A partial cDNA was isolated that encoded a protein kinase, termed rac (related to the A and C kinases). This cDNA was subsequently used to screen libraries derived from the human cell lines MCF-7 and WI38 and led to the isolation of full-length cDNA clones. DNA sequence analysis identified an open reading frame of 1440 base pairs encoding a protein of 480 amino acids (Mr, 55,716). This result was supported by the synthesis of a Mr58,000 protein in an in vitro translation system that used RNA transcribed from cloned cDNAs with SP6 RNA polymerase. The predicted protein contains consensus sequences characteristic of a protein kinase catalytic domain and shows 73% and 68% similarity to protein kinase C and the cAMP-dependent protein kinase, respectively. Northern (RNA) analysis revealed a single mRNA transcript of 3.2 kilobases that varied up to 300-fold between different cell lines. Specific antisera directed towards the carboxyl terminal of the rac protein kinase were prepared and used to identify a protein of Mr59,000 by immunoblotting. A specific protein kinase activity was identified that phosphorylated several substrates in immunoprecipitates prepared with the rac-specific antisera.