The Forgotten PKAs: New Insights Revealed by the First Crystal Structure of a PKA C Beta Splice Variant, C beta 4ab

A major regulatory mechanism of enzyme function in cells is protein phosphorylation that is carried out by the large protein kinase family. While the catalytic (C) subunit of cAMP-dependent protein kinase A (PKA) has served as a prototype for this family, our enormous knowledge base is skewed towards Cα1, only one of the many tissue specific C subunit isoforms and splice variants expressed in cells. Accordingly, all published crystal structures are of Cα1. The other PKA splice variants, especially the Cβ splice variant gene products of PRKACB, have been obscure. In an effort to re-balance our PKA perspective we have solved a 2.6 A crystal structure of the PKA splice variant Cβ4ab, which not only crystallized in a new condition but also grew in a thus far for PKA not seen space group P2 with four molecules per asymmetric unit. Compared to Cα1, the isoform with the most deposited structures in the protein data bank, the protein sequence of Cβ4ab differs by 10% or 35 out of 350 amino acids. A majority of these amino acid differences cluster in the N-terminal and C-terminal tails. The rest are located mostly in the N-lobe. Some hot spots of amino acid variation nucleate two sets of C subunit dimers that interact in a novel way to create polymers within the crystal. In our structure, Cβ4ab is in a closed conformation with the inhibitor peptide IP20 bound but surprisingly no electron density is observed for either of the two Mg ions usually present with IP20 and nucleotide. SPR data for the protein kinase inhibitor provides further insights into the differences between Cβ4ab and Cα. Our structure and binding studies suggest that in addition to C beta being expressed in a tissue specific manner, they are also functionally non-redundant. The newly revealed inter-molecular interaction sites within the dimers highlight regions of the C subunit that have been less studied so far and are likely to have important regulatory functions.