Characterization of two mutants of the LLC‐PK₁ porcine kidney cell line affected in the catalytic subunit of the cAMP‐dependent protein kinase

The catalytic (C) subunit activity of the cAMP‐dependent protein kinase (cAMP‐PK) from the mutant cell lines, FIB4 and FIB6, is only 10% compared with the parent cell line, LLC‐PK₁ [Jans and Hemmings (1986) FEBS Lett. 205, 127–131]. In order to understand the nature of the mutant phenotypes the cAMP‐PK from parent and mutant cell lines was studied in more detail. Analysis of mutant cAMP‐PK activity by ion‐exchange chromatography revealed that kinase activity associated with type I holoenzyme of both FIB4 and FIB6 was only 5% parental, and the activity of the type II holoenzyme was about 20% parental. The type I regulatory (R_I) subunits associated with the type I were also found to be reduced by 70–80% in both mutants, whereas the type II R subunit levels were similar to that of the parent. The residual kinase activity associated with the type I holoenzyme from FIB4 and FIB6 could not be activated by cAMP whereas the type II holoenzyme was activated by cAMP (K_a of 5.5 × 10⁻⁸M), and showed normal affinities for Kemptamide and ATP. A polyclonal antibody to the catalytic subunit was used to quantify the level of this protein in wild‐type and mutant cells. This analysis showed that FIB4 and FIB6 had nearly normal levels of C subunit, suggesting that the C subunit synthesized by the mutants was mostly inactive. As both type I and type II cAMP‐PK holoenzymes were abnormal, the most likely explanation of the mutant phenotype is a defect either in the structural gene for the C subunit or in an enzyme involved in its posttranslational processing. However, a second lesion affecting the R_I subunit cannot be ruled out at this moment.