Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily

PAMELA F. JONES; TERESA JAKUBOWICZ; FERNANDO J. PITOSSI; FRANSISCA MAURER; ANO BRIAN A. HEMMIN

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA

NATL ACAD SCIENCES

Lugar: Washington DC, USA; Año: 1991 vol. 88 p. 4171 - 4175

0027-8424

A partialcDNA was isolatedthat encoded a protein kinase,termed rae (relatedto the A and !: kinases). ThiscDNA was subsequently used to screen libraries derived from the human cell Iines MCF-7 and WI38 and Ied to the isolation of full-length cDNA clones. DNA sequence analysis identified an open reading frame of 1440 base pairs encoding aprotein of 480 amino acids (M., 55,716). This resultwas supported by the synthesis of a Mr 58,000 protein in an in vitro translation  system that  usedRNA transcribed  from  cloned cDNAs with SP6  RNA polymerase. The  predicted  protein contains consensos sequences characteristic of a proteinkinase catalytic domain and shows 73% and 68% similarityto protein kinase C and the cAMP-dependent protein kinase, respec­ tively. Northern  (RNA) analysis revealed a single mRNA transcript  of 3.2 kilobases that varied up to 300-foldbetween different cell lines. Specific antisera  directed towards the carboxyl terminal of the rae protein kinasewere prepared and used to identify a protein of Mr 59,000 by immunoblotting. A specific proteinkinase activity was identified that phosphoryl­ ated severa) substrates in immunoprecipitates prepared  with the rac-specific antisera.