Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase.

T J Singh,J Hochman,R Verna,M Chapman,I Abraham,I H Pastan,M M Gottesman

doi:10.1016/s0021-9258(17)38665-9

Abstract

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.

Highlights

Characterization of a Cyclic AMP-resistant Chinese Hamster Ovary Cell Mutant Containing Both Wild-typeand Mutant Species of Type I Regulatory Subunit of Cyclic AMP-dependentProtein Kinase*
Mato~aphy.This binding activity resembles the regulatory subunit of type I CAMP-dependent protein kinase (RI) and in some casesmay be formed by dissociation of this holoenzyme. in this work, we report the characterization
Protein assay extract is shifted to the right, requiring greater than 10-fold kits andall reagents for SDS-gel electrophoresis were from Bio-Rad. higher concentration of CAMP for maximal activation

Summary

RESULTS

~ PERIMENTA LPROCEDURES in Extra&-Extracts from the mutant and wild-type cells were analyzedfor their CAMP-dependentprotein kinase and f3H]cAMP-bmdingactivities. Type IIA histone, the protease inhibitor aprotinin, and all unla- the kinases by different concentrations of CAMP. It can be beled nucleotides were purchased from Sigma. [d"PJATP was from observed that the kinase activation curve from the mutant. Protein assay extract is shifted to the right, requiring greater than 10-fold kits andall reagents for SDS-gel electrophoresis were from Bio-Rad. higher concentration of CAMP for maximal activation.

Methods

B Dissociated Type I Kinase

DISCUSSION