MOLECULAR-CLONING AND IDENTIFICATION OF A SERINE THREONINE PROTEIN-KINASE OF THE 2ND-MESSENGER SUBFAMILY

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 WI28 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 (M(r), 55,716). This result was supported by the synthesis of a M(r) 58,000 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 M(r) 59,000 by immunoblotting. A specific protein kinase activity was identified that phosphorylated several substrates in immunoprecipitates prepared with the rac-specific antisera.