UC San Diego

Regulatory subunits (R) of cAMP-dependent protein kinase (PKA) are differentially expressed and carry out various functions. Two general classes exist, RI and RII, as well as [alpha] and [Beta] isoforms within each class. Though the four isoforms share the same general domain organization, they are not functionally redundant. RI[alpha] is embryonically lethal in mice and cannot be compensated for by the other R isoforms. A change in RI[alpha] expression levels, higher or lower, is implicated in diseases, such as Carney complex (CNC). CNC is an autosomal dominant syndrome that is associated with RI[alpha] mutations, most of which are not expressed, resulting in a haploinsufficiency of RI[alpha]. I studied two mutations among the few that do get expressed, RI[alpha] R74C and RI[alpha](1-303). RI[alpha] R74C results in a mutation in the linker region, while RI[alpha](1-303) results in a truncation in the cAMP binding domain. My goals were to characterize these mutants in E. coli (Chapter 2) and in mammalian cells (Chapters 3 and 5) and to search for novel binding partners of RI[alpha] (Chapter 4). Purified RI[alpha](1- 303) showed a four-fold increase in PKA activation and RI[alpha] R74C dimers had a tendency to form disulfide- bonded tetramers. TAP-tagged constructs of RI[alpha] and RI[alpha] R74C were engineered and expressed in mammalian cells to make stable cell lines. Using diagonal gel electrophoresis, I showed that both constructs of TAP- tagged RI[alpha] formed heterodimers with endogenous RI[alpha]. No homodimers of endogenous RI[alpha] were observed when RI[alpha] R74C was expressed, which could explain the CNC phenotype. These stable cells were also used to study the RI[alpha] interactome. Using various affinity methods followed by mass spectrometry analysis, I discovered an A-kinase anchoring protein (AKAP), AKAP11, which binds with unusually high affinity to RI[alpha]. When RI[alpha] was overexpressed, it formed puncta, which were dependent on the disassociation from C-subunit and were reversible. In addition, RI-specific AKAPs abolished the puncta formation. Using indirect immunofluorescence and correlated light and electron microscopy, these puncta were found to localize to multivesicular bodies. In summary, I have identified an effect on the interchain disulfide bonding in RI[alpha], putative binding partners for RI[alpha], as well as a novel way of targeting RI[alpha]